INTRATUMORAL CHEMOTHERAPY FOR LIVER CANCER USING
BIODEGRADABLE POLYMER IMPLANTS
by
BRENT DEREK WEINBERG
Submitted in partial fulfillment of the requirements
For the degree of Doctor of Philosophy
Dissertation Advisors: Dr. Agata Exner and Dr. Jinming Gao
Department of Biomedical Engineering
CASE WESTERN RESERVE UNIVERSITY
August, 2007
Brent D. Weinberg
Gerald Saidel
James Anderson
Agata Exner
Jinming Gao
Ruth Keri
Horst von Recum
April 2, 2007
Copyright © 2007 by Brent Derek Weinberg All rights reserved
Brent D. Weinberg
To my parents, Glinda and Larry Weinberg, who have always been very supportive of my educational quests.
TABLE OF CONTENTS
List of Tables ...... 2
List of Figures ...... 3
Acknowledgements ...... 6
List of Abbreviations ...... 7
Chapter 1 Basis for locoregional cancer therapy and intratumoral drug delivery ...... 11
Chapter 2 Polymer millirods for treatment of small experimental liver tumors in
rabbits ...... 50
Chapter 3 Combined therapy using RF ablation and polymer millirods for liver cancer
in rabbits...... 87
Chapter 4 Model-based estimation of drug distribution properties in non-ablated and
ablated tumor tissue ...... 117
Chapter 5 Simulation of drug distribution in tumors to optimize complex tumor
treatments ...... 146
Chapter 6 Future prospects of polymer implants for the treatment of human tumors .....179
Appendix A VX2 tumor model...... 199
Appendix B Tumor treatment animations ...... 227
Appendix C Annotated publication list ...... 230
Bibliography ...... 248
1 LIST OF TABLES
Table 1-1. Doxorubicin transport properties in rat liver ...... 32
Table 2-1. Summary of doxorubicin release in vivo in VX2 tumors...... 66
Table 3-1. Summary of tumor treatment outcome after combined treatment with RF
ablation and a control or doxorubicin-containing millirod...... 100
Table 3-2. Summary of millirod implant and drug release properties...... 105
Table 5-1. Doxorubicin coverage of ablated rabbit liver...... 167
Table 5-2. Doxorubicin coverage of ablated liver tumors...... 169
Table A-1. Qualitative viability of VX2 cells using two suspension techniques...... 206
2 LIST OF FIGURES
Figure 1-1. Theoretical drug release and local drug concentration from three implant
types...... 18
Figure 1-2. Schematic of drug transport from an intratumoral implant...... 23
Figure 1-3. Computed tomography (CT) scan of a carboplatin-containing polymer
millirod placed in an RF ablated rat liver...... 30
Figure 1-4. CT-measured carboplatin distribution in liver tissue...... 31
Figure 1-5. In vivo relationship between drug release rate and local tissue concentrations.
...... 37
Figure 1-6. Local drug distributions resulting from sustained-release and dual-release
millirods over 8 days...... 38
Figure 1-7. Preventing fibrous capsule formation after RF ablation...... 39
Figure 2-1. Schematic of tumor treatment with polymer millirods...... 52
Figure 2-2. Qualitative fluorescence calibration in extracted tissue slices...... 60
Figure 2-3. Cumulative doxorubicin release from polymer millirods in vitro...... 63
Figure 2-4. Cumulative 5-FU release from polymer millirods in vitro...... 64
Figure 2-5. Gross appearance of doxorubicin implant treated VX2 tumors...... 65
Figure 2-6. Doxorubicin distribution in non-ablated tumors...... 67
Figure 2-7. Histology of DOX implant treated versus control tumors...... 70
Figure 2-8. H&E stained and fluorescent micrographs of a DOX implant treated tumor
on day 8...... 71
Figure 2-9. Tumor recurrence outside of DOX implant treated zone...... 72
Figure 2-10. Size change in 5-FU implant treated tumors...... 76
3 Figure 2-11. Histology of VX2 tumor treated with 5-FU-loaded implant...... 77
Figure 3-1. Schematic of combined tumor treatment with RF ablation followed by
polymer millirods...... 91
Figure 3-2. Schematic of background subtraction for ablated tissues...... 97
Figure 3-3. Tumor progression after ablation...... 101
Figure 3-4. Histology and doxorubicin fluorescence at the ablation boundary...... 102
Figure 3-5. Region quantification in two treated tumors with areas of viable tumor. ... 104
Figure 3-6. Doxorubicin concentration distribution maps in ablated tumors...... 107
Figure 3-7. Average doxorubicin concentration profiles in ablated tumor tissue...... 108
Figure 4-1. Modeling results from parameter estimation in non-ablated tumor...... 127
Figure 4-2. Parameter estimation in ablated tumors using constant parameters...... 128
Figure 4-3. Modeling results from parameter estimation in ablated tumor...... 129
Figure 4-4. Radial and time dependence of parameters in ablated tumor...... 130
Figure 4-5. Histology showing regional tissue variation in ablated tumor...... 134
Figure 4-6. Histology showing temporal tissue variation in ablated tumor...... 135
Figure 4-7. 3-D modeling of drug release in a 2.0 cm tumor...... 136
Figure 4-8. Comparison of transport parameter in different tissue types...... 140
Figure 5-1. Multiple implant scenarios simulated using 3-D model...... 150
Figure 5-2. Evaluation of simulated tumors ...... 154
Figure 5-3. One versus multiple implants in a single tumor, model output...... 159
Figure 5-4. Simulated drug concentrations in whole tumor and risk volume...... 160
Figure 5-5. Total time of and time to reach 100% coverage of risk volume...... 161
Figure 5-6. Simulated coverage from two configurations with a 13.5 mg DOX dose. . 162
4 Figure 5-7. Photographs of implantation of 4 polymer millirods...... 165
Figure 5-8. Model versus experimental drug concentrations in ablated liver...... 166
Figure 5-9. Model versus experimental drug concentrations in ablated tumor...... 168
Figure A-1. VX2 cell viability prior to implantation...... 205
Figure A-2. Gross appearance of VX2 tumors in the rabbit liver...... 210
Figure A-3. Computed tomography scans of VX2 tumors in liver...... 211
Figure A-4. Growth rate of VX2 tumors in rabbit liver...... 212
Figure A-5. Histology of untreated VX2 tumors in rabbit liver...... 213
Figure A-6. Ultrasound image of large VX2 tumor in rabbit liver...... 215
Figure B-1. Tumor recurrence after RF ablation...... 227
Figure B-2. Millirod only treatment of liver tumors...... 228
Figure B-3. Combined treatment with RF ablation and polymer millirods...... 229
5 ACKNOWLEDGEMENTS
I would like to first thank my two Ph.D. advisors, Dr. Jinming Gao and Dr. Agata
Exner. Dr. Gao provided me with extensive mentorship and guidance when I needed it most during my first few years as a graduate student and continued to challenge me throughout my course of study. Dr. Exner was gracious enough to incorporate me into her lab in the middle of my work, and her leadership was the ideal balance of oversight and freedom necessary for me to grow into an independent scientist. Additionally, I would like to thank them both for helping me choose a path of study that was both scientifically and personally beneficial.
I would also like to acknowledge my committee members: Dr. James Anderson,
Dr. Ruth Keri, Dr. Horst von Recum, and Dr. Gerald Saidel. Their continual support and helpful suggestions were always appreciated. My colleagues in lab were also instrumental to making this work possible. In Dr. Gao’s lab, Dr. Hua Ai, Elvin Blanco, and Scott Lempka were instrumental in performing the animal experiments in Chapters 2-
3. In Dr. Exner’s lab, Dr. Hanping Wu, Tianyi Krupka, and Ravi Patel also supported my work. Ravi Patel’s contributions to the modeling work in Chapters 4-5 were essential to the successful completion of this work.
Finally, this work would not have been possible without financial support.
Research described in this work was primarily funded by NIH grant R01 CA090696.
Throughout my training, I have been personally funded by NIH grant T32 GM07250 to the Case Medical Scientist Training Program (MSTP), NIH training grant T32
GM007535 for Systems and Integrative Biology, and Department of Defense predoctoral fellowship BC043453.
6 LIST OF ABBREVIATIONS
1-D one-dimensional
3-D three-dimensional
5-FU 5-fluorouracil
AAS atomic absorption spectroscopy
BCNU bis(chloroethyl)nitrosourea
CED convection enhanced delivery cGLP current good laboratory practice cGMP current good manufacturing practice
CT x-ray computed tomography
DEX dexamethasone
DMSO dimethyl sulfoxide
DOX doxorubicin hydrochloride
FBS fetal bovine serum
FDA Food and Drug Administration
FEM finite element method
FITC fluorescein isothiocyanate
H&E hematoxylin and eosin
HBSS Hank’s balanced salt solution
HCC hepatocellular carcinoma
HPLC high performance liquid chromatography
HPß-CD hydroxypropyl ß-cyclodextrin
IB inner boundary
7 IP intraperitoneal
ITP isolated thoracic perfusion
IV intravenous
MRI magnetic resonance imaging
MTC Masson’s trichrome
NFI net fluorescence intensity
NZW New Zealand white
OB outer boundary
PBS phosphate-buffered saline pCPP:SA 1,3-bis-(p-carboxyphenoxy) propane/poly(sebacic acid)
PEG poly(ethylene glycol)
PEO poly(ethylene oxide)
PET positron emission tomography
PLA poly(lactic acid)
PLGA poly(D,L-lactide-co-glycolide)
PVA poly(vinyl alcohol)
RF radiofrequency
SD standard deviation
SPECT single photon emission computed tomography
SPIO superparamagnetic iron oxide
TACE transarterial chemoembolization
TBS Tris-buffered saline
TN tumor/normal interface
8 Intratumoral Chemotherapy for Liver Cancer using Biodegradable Polymer Implants
Abstract
by
BRENT DEREK WEINBERG
Cancer is the second leading cause of death in the United States, behind only heart disease. Many cancers, particularly solid tumors, are inaccessible to surgery, and current systemic chemotherapy regimens are extremely limited. As a result, developing new treatments for unresectable tumors that minimize side effects is an active area of research. For example, image-guided radiofrequency (RF) ablation has had considerable success for treatment of liver tumors, but tumor recurrence at the periphery of the original tumor site is common. Recent work has proposed improving the efficacy of RF ablation by delivering a drug to the treatment area with a chemotherapeutic, biodegradable implant made of poly(D,L-lactide-co-glycolide) (PLGA). Previous studies have shown that these implants can successfully deliver antineoplastic drugs to normal and ablated liver in animal models. To establish the efficacy of these implants in treating tumors, drug-releasing PLGA implants were manufactured and tested in a rabbit liver tumor model both with and without radiofrequency ablation. Information gathered from the tumor drug distributions was used to develop a computer simulation for comparing alternative tumor treatment configurations. Optimal combined treatments were developed using the simulation and further validated with an animal model. The result of these studies is a comprehensive strategy for treating unresectable tumors with drug-
9 containing implants and RF ablation that may ultimately impact treatment of unresectable tumors in humans.
10 Chapter 1 Basis for locoregional cancer therapy and intratumoral drug delivery
1.1 Introduction
Cancer is an enormous health concern in the United States which in recent years has surpassed heart disease as the predominant cause of death for all but the most elderly
Americans.1 Currently, the most curative treatment option for solid tumors is surgical resection followed by adjuvant chemotherapy or radiation therapy to minimize the risk of recurrence. Some cancers respond well to this treatment strategy, but many patients are not eligible for surgical resection. For example, out of 70,000 newly diagnosed colon cancer metastases to the liver in the US per year, the number of patients who are actually candidates for surgery is disappointingly low at 2,500-4,500.2 Reasons limiting resection include tumor size, involvement of more than one liver lobe, or a coexisting liver condition (e.g. cirrhosis).2,3 Even for those patients eligible for resection, the overall survival rates after surgery are often low.4
Other abdominal cancers, such as those of the pancreas and stomach, also have low resection rates and poor overall patient survival.1 In addition, intravenously (IV) administered chemotherapy is also ineffective against many of these tumors. Since only a small amount of the systemic blood flow is directed to the tumor, a minute fraction of the total dose actually reaches the tumor site.5 The remainder of the dose is distributed throughout healthy organs and tissues, leading to a variety of undesirable side effects ranging from neutropenia to cardiomyopathy.6,7 Many chemotherapeutic drugs also have very rapid plasma clearance, leading to short tumor exposure times.8 To improve the outcome of these treatments, a new paradigm of minimally-invasive and locoregional
11 cancer therapy has rapidly evolved and received considerable attention in the recent years.9
Image-guided, minimally-invasive therapeutic interventions use regional tumor destruction as an alternative to surgical resection.10 Strategies for tumor ablation include thermal heating,11,12 cryosurgery,13,14 or chemical ablation.15,16 In each of these techniques, an interventional needle or electrode is inserted into the tumor with image guidance.17,18 Then, the ablation is applied to destroy the tumor and a surrounding margin of normal tissue. Since they can be administered percutaneously, these minimally-invasive treatments typically are viable outpatient alternatives to surgery that can be used in patients with poor overall health. Additionally, local administration of the treatment maximizes destruction to the tumor target while limiting damage to the surrounding normal tissue. Ablation has been used for the treatment of several unresectable cancers, including those in the liver,19 prostate,20 and lung.21
Other attempts to improve treatment of unresectable tumors have focused on means to increase the tumor specificity of chemotherapeutic drugs through locoregional delivery.22 Administering an anticancer drug either to the region that contains a tumor or directly within the tumor has the advantage of increasing tumor exposure to a drug while limiting systemic toxicity. One strategy for locoregional chemotherapy is to infuse a solution of a chemotherapeutic agent into the region of the malignancy. Intravesicular chemotherapy has become a common treatment for bladder tumors, and has been shown to be associated with a reduced tumor recurrence rate after surgery.23 Local chemotherapy infusion has also been used with some success in the case of advanced non small cell lung cancer when patients have a malignant pleural effusion.24 Additionally,
12 several studies have shown significant benefits in treating ovarian cancers with intraperitoneal infusions.25 All of these treatments require that malignant cells be in close contact with the surrounding space to which the chemotherapy is administered.
For tumors that are not externally accessible, localized perfusion, or the administration of a chemotherapeutic agent to a segment of the circulation that preferentially perfuses the tumor, is an alternative. Intra-arterial administration of drugs can maximize drug delivery to blood vessels supplying tumors. For example, transarterial chemoembolization (TACE) benefits from the fact that most hepatocellular carcinomas receive the vast majority of their blood supply from their hepatic artery while normal liver receives its blood supply largely from the portal vein.26 In this treatment, a catheter is selectively placed in the branches of the hepatic artery which feed the tumor.
Once arterial selection has been achieved, a solution of chemotherapeutic agents dissolved in an oily solvent followed by embolic agents is infused into the artery.26 This approach has been shown to increase concentrations of chemotherapy in the tumor by 10-
100 fold26 and to improve one-year survival of patients with unresectable hepatocellular carcinoma (HCC) by as much as 20%.27 As a result, TACE has become a commonly administered therapy for unresectable HCC.28 Another strategy to improve delivery is regional perfusion, in which the portion of the systemic circulation containing a tumor is isolated from the rest of the circulation.29 Isolated thoracic perfusion (ITP) is achievable by closing off the descending aorta and vena cava with balloon catheters, blocking blood flow to the arms with inflated cuffs, and introducing chemotherapy into the right atrium.
This approach has been used to increase the concentration of chemotherapy delivered to lung cancers by 6-10 fold,30 and an analogous approach exists for isolated abdominal
13 perfusion. Each of these locoregional chemotherapy methods have been shown to increase tumor exposure to drug while reducing systemic toxicity.
Intratumoral cancer treatments extend the locoregional treatment concept by attempting to further limit the scope of chemotherapy exposure. Treatments that have been studied extensively include intratumoral infusions, injections, and implantable devices that deliver either chemotherapeutic drugs or other therapeutic agents.22 Infusion of chemotherapeutic agents has been heavily studied in the area of brain tumors, where it has spawned a field known as convection-enhanced delivery (CED).31 In CED, a microcatheter is inserted into a tumor and the therapeutic agent is slowly administered to the surrounding tissue using positive pressure infusion. Major advantages of CED to brain tumors include bypassing the blood-brain barrier and delivering drugs further from the infusion site due to convection.32 CED has been used to deliver conventional chemotherapeutic drugs33 but has shown considerable promise for the delivery of targeted bacterial toxins31 and therapeutic antibodies.34 Intratumoral injections of therapeutic solutions have also shown success in treating tumors in locations other than the brain, such as the lung,35 pancreas36, and liver.37 Several studies have been performed in an attempt to determine optimal parameters for injection and to determine which tumor features, such as collagen content, contribute to the extent of drug delivery.38,39
Furthermore, recent studies have shown that using intratumoral injection to deliver viral gene therapy vectors minimizes non-specific expression of gene products.40,41 Since liquids injected in this manner may distribute irregularly and be cleared quickly, several investigators have introduced injectable drug depots to prolong the extent of drug release.
Examples of intratumoral depots include PLGA,42-44 alginate,45 and albumin46
14 microsphere formulations as well as injectable gels which solidify upon intratumoral injection.47-49 Injectable depots have the advantage of easy administration and prolonged tumor drug exposure.
Intratumoral, drug-releasing implants are a subset of the intratumoral drug delivery paradigm that have been pursued aggressively in recent years. Driven by developments for the treatment of prostate50,51 and brain cancers,52,53 implantable devices containing either radioactive elements or chemotherapeutic drugs have become viable treatment options. The only Food and Drug Administration (FDA) approved chemotherapeutic implant for cancer treatment is the Gliadel wafer, a carmustine(BCNU)-eluting implant fabricated from a polyanhydride copolymer, 1,3-bis-
(p-carboxyphenoxy) propane/poly(sebacic acid) (pCPP:SA).54 These implants were designed to treat glioblastoma multiforme, an aggressive brain cancer with extremely limited patient survival, through placement in the surgical cavity after primary surgical resection. After placement, the implants release their drug load over a period of approximately 5 days,55 and drug has been shown to penetrate several millimeters into the brain parenchyma.56 A recent long-term study showed that the Gliadel implant placement after surgery increases patient survival to 13.8 months versus 11.6 months for control and maintains this survival advantage for at least three years after initial treatment.57 Despite the clinical success of the Gliadel implant, the use of chemotherapeutic implants has yet to become widespread in the treatment of other cancers, such as those of the pancreas, liver, or lungs. However, it is likely that future chemotherapeutic implants can be optimized for use in a variety of different tumors to maximize patient comfort and survival.
15 This chapter describes the principles behind and development of a minimally invasive combination treatment strategy to treat unresectable liver tumors. The proposed approach consists of primary treatment of the tumor bulk with radiofrequency (RF) ablation followed by the placement of drug-eluting polymer implants in the ablated tumor region. These biodegradable polymer millirods have been fabricated from poly(D,L- lactide-co-glycolide) (PLGA) to deliver chemotherapeutic agents through and beyond the
RF ablated tumor, thus maximizing tumor destruction and reducing the risk of tumor recurrence. Section 1.2 describes the overall goals that must be considered when developing any local delivery device, including the use of models to predict local drug transport. Section 1.3 describes techniques for measuring local drug concentrations and the use of these measurements to customize drug release. Section 1.4 presents some previously developed methods for modulating drug distribution in ablated tissue. The final section of this chapter then describes the importance of the previous work to the experiments described in subsequent chapters as well as an overview of those chapters.
1.2 Overview of intratumoral drug delivery
1.2.1 Pharmacokinetic goals for intratumoral delivery
In considering the use of an intratumoral implant for tumor treatment, it is necessary to consider the generic characteristics that would benefit the device. First, the implant should be able to minimize shortcomings associated with systemically administered chemotherapy. Second, it should provide an optimal drug delivery profile to the tumor, which is to say that it should be able to provide effective drug
16 concentrations to the targeted region over a prolonged period of time. Third, the device should be part of a comprehensive and complete treatment strategy that is versatile and applicable in a wide range of realistic situations. Achieving these goals should maximize the treatment success of these intratumoral implants.
When delivering their drug cargo to tumors, intratumoral implants must provide an optimal drug release profile that is characterized by the ability to deliver drug to a large volume, to rapidly reach the therapeutic concentration, and to maintain the therapeutic concentration for an extended time. Previous studies have shown that limited penetration distance is one of the major restrictions on the efficacy of intratumoral treatments.55,56 Any successful implant must be designed in such a way that takes into consideration ways to maximize the drug delivery distance. Additionally, the implant must provide drug to the surrounding tissue at an appropriate rate.58 A schematic of ideal drug release rates is shown in Figure 1-1. Consider implant A, an implant which releases drug at a constant rate somewhere above the elimination rate. While local drug concentration will slowly rise, it may take too long to reach tissue concentrations that are toxic to the surrounding cancer cells. Alternatively, implant B provides a rapid dose of chemotherapy that will quickly surpass the effective concentration. However, the release rate after the initial burst is insufficient to maintain this concentration for any extended length of time. Such a release rate is undesirable, as it could allow cancer cells to recover, perhaps even with newly acquired drug resistance.59 The ideal implant, implant
C, combines the best characteristics of both implants: rapid ascent to the effective concentration followed by a maintenance dose to remain at a useful drug level. While this explanation is a simplification (e.g. elimination is almost certainly not constant, etc.),
17
Figure 1-1. Theoretical drug release and local drug concentration from three implant types.
A zero-order release implant (A) releases drug at a constant rate, but it may take a long period of time to reach the therapeutic concentration. A burst-release implant (B) releases large amounts of drug early, but may not have sufficient extended release to maintain a therapeutic concentration. A dual-release implant (C) combines an early burst of drug to accelerate the rise to therapeutic concentrations with sustained release to maintain therapeutic concentrations.
18 it serves as an example of how different drug release rates might affect local drug concentrations. Additionally, it offers some insight on how the situation can be changed by modifying the elimination rate or therapeutic concentration. For the most part, however, local drug concentrations surrounding implants must be determined experimentally.
The success of chemotherapeutic implants for cancer treatment also depends on their inclusion in a comprehensive tumor treatment strategy. As an example, with the previously mentioned Gliadel treatment, the tumor is first surgically resected
(“debulked”) followed by the placement of multiple BCNU-impregnated implants in the surgical cavity.52 The design of a liver cancer treatment using polymer millirods proposes a similar strategy to Gliadel treatment: using radiofrequency (RF) ablation to destroy the majority of the tumor mass followed by placement of polymer implants in the tumor to kill any residual cancer cells and limit tumor recurrence. RF ablation is already used clinically to treat liver tumors, but tumor recurrence, particularly around the ablation boundary, has greatly limited the clinical success of tumor ablation.40,60 Using chemotherapeutic implants with ablation may maximize the benefit compared to using
RF alone. The ablation destroys the majority of the tumor cells, leaving the implants to kill only the remaining cells and thereby to reduce the risk of recurrence. Furthermore, tumor ablation may facilitate drug delivery from the implants by changing the fundamental rates governing drug transport in the tumors.58 To develop this strategy, drug-impregnated, PLGA polymer millirods with different release rates were developed and tested in animal models in non-ablated and ablated liver tissue.58,61-63 Results from these studies are described in Sections 1.3 and 1.4.
19 1.2.2 Interstitial drug transport models in tumor and surrounding tissues
In addition to generic considerations on drug release from implants, the mechanisms of drug transport and elimination in the surrounding tumor tissue have a major effect on how an implant delivers drug to the tumor.64 Drug released locally into the tumor has several possible fates that will ultimately affect the outcome of the treatment. Drug molecules generally face two possible outcomes: they can either be transported to another location or be eliminated such that they no longer exert their desired effect.
Drug can typically be transported by two mechanisms: diffusion and convection.55,65,66 In diffusion, free drug moves from a region of higher drug concentration to an adjacent region of lower drug concentration at a rate proportional to the concentration gradient. Diffusion is a primary mode of transport, particularly when drug is being released from a local implant. Convection, on the other hand, is the transport of drug along with the bulk flow of a fluid. In organs that have a high rate of interstitial fluid flow, convection is especially important. Convection also has a significant role in the flow of systemically administered chemotherapeutic agents from the vascular space to the tumor, where it travels along the same flow that delivers nutrients to the tumor.64 The relative importance of diffusion or convection in drug transport depends on the delivery system and tissue type. For instance, in the brain, where interstitial fluid constantly flows from the ventricles to the surrounding parenchyma, convection can have a significant effect on the extent of drug penetration.67
In situations with small molecular drugs where flow is more limited, diffusion is the predominant mode of drug transport.
20 Drug elimination can occur through several different mechanisms. One route of drug elimination is through metabolism. Once in a cell, drug can be altered or bound in a variety of ways. Some drug molecules, such as 5-fluorouracil, bind irreversibly to their therapeutic target, after which they are no longer in the population of available drug.68
More generally, cells have a variety of nonspecific methods for detoxification, such as organelles for breaking down foreign molecules through enzymatic or pH-mediated degradation. Additionally, cells contain protective molecules, such as glutathione, which are designed specifically to bind foreign molecules and render them more hydrophilic and less potent.69 Either of these metabolic pathways essentially inactivates the drug. When considering implantable drug delivery systems, another mechanism of drug loss is perfusion away from the target region.58 In this situation, drug is transported by either diffusion or convection into one of two systemic circulations, the blood or the lymph.
The vasculature is a fast-moving circulation which rapidly moves drugs away from the target region and into other parts of the body. Since most chemotherapeutic agents have short plasma half-lives, once the drug reaches the plasma it is unlikely to return to the target tumor, and for practical purposes can be considered eliminated. While the lymph is a slower-moving body of fluid that can contribute to drug convection, its effects are probably less influential than those of blood because tumors are known to have limited and poorly organized lymphatic drainage.66 Any drug contained in lymphatic fluid eventually moves into the venous circulation, where it undergoes the same fate as drug that directly diffuses into blood vessels.
Consider the example of drug transport shown in Figure 1-2, in which drug is being delivered to a liver tumor from a cylindrical implant in the center of the tumor.
21 Previous work has shown that transport in liver can be reasonably approximated without including convection.58 Drug leaving the implant is transported away from the implant into the tumor tissue based on a tumor diffusion rate, Dtumor. Once in the tumor, drug can be eliminated in one of two ways, through blood flow and metabolism, proportional to two different rates which sum to the total elimination, γtumor. Once drug reaches the outer boundary of the tumor, it can diffuse into the surrounding normal liver tissue, where its fate is again governed by new diffusion and elimination rates. If elimination can be considered approximately first order, the drug transport in each tissue is governed by the following equation:
C DCC 2 (1.1) t where C is the drug concentration, t is time, is the gradient operator, and D and γ are the tissue-specific rates of diffusion and elimination, respectively. Drug transport properties in each tissue can be estimated from experimentally measured concentrations by solving this equation computationally and minimizing the error between model output and experimentally collected data.
The use of such a model provides insight into factors that can facilitate or impede drug transport from a local implant. First, any factor that increases the rate of drug transport or lowers the rate of drug elimination will increase the permeation of drug within the tissue. For instance, some work in ex vivo tumors has shown that prolonged drug exposure raises drug diffusion coefficients, presumably by killing cells and destroying overall structure.70 Other work by Strasser et al. has shown that including high molecular weight molecules, such as dextrans, can increase transport away from
22
Figure 1-2. Schematic of drug transport from an intratumoral implant.
Local transport of drug in the tumor tissue is governed by the diffusion constant of drug in tumor (Dtumor), and two simultaneous modes of elimination: metabolism to inactive forms in tumor cells (tumor metabolism) or transport into nearby blood vessels which wash drug out of the region (tumor perfusion). Once drug reaches the surrounding normal tissue, it continues to diffuse outward into liver tissue (Dliver), where it has different rates of elimination by metabolism ( liver metabolism) or perfusion ( liver perfusion).
23 implants by increasing the convective fluid flow contribution while decreasing elimination due to blood perfusion.56 Similarly, it can be expected that any factor that reduces elimination will also have a beneficial effect, facilitating deeper drug penetration into the tumor. On the other hand, any action that decreases transport or increases elimination will act as a barrier to drug delivery, reducing the distance over which an implant can be effective. Inflammation, such as that occurring after RF ablation, may raise blood flow and decrease drug diffusion rates as a result of collagen deposition around the wound.71 These side effects could certainly impede successful drug transport from a local tumor treatment. The distance at which an implant can have an effect on a tumor depends on the drug release rate from the implant as well as the balance between local transport and elimination. Unfortunately, several studies have indicated that antitumor implants are likely only effective for a few millimeters away from the implant surface.72 Studies into local drug concentration and local transport mechanisms, however, have provided useful information on ways to overcome these limitations.
1.3 Measuring local drug distributions
In developing an intratumoral chemotherapy device, tools for monitoring local drug concentrations are necessary to optimize implant design. Measuring drug concentration as a function of time provides a quantitative method to compare multiple treatments. Many different methods can be used to monitor drug release from intratumoral implants. While certain techniques require extraction of tissue and measurement of drug concentration ex vivo, alternate, noninvasive imaging based- techniques can be used to measure concentrations in vivo. New implant designs or
24 treatment conditions can be tested by creating an implant that has the ideal characteristics described in Section 1.2: a rapid ascent to and prolonged stay above the therapeutic concentration. Additionally, drug concentration information can then be used as input to estimate tissue transport properties. The ideal implants can then be created through a combination of empirical testing and engineering design.
1.3.1 Measuring drug concentrations in extracted tissues
Considerable information has been obtained by monitoring local drug concentrations using ex vivo analysis of extracted tissues. Two main categories of ex vivo analysis exist: bulk tissue analysis by conventional spectroscopic methods or tissue section analysis by imaging-based methods. The key principle of bulk tissue measurements is the removal of a sizeable piece of tissue followed by the use of a spectroscopic method to determine the average drug concentration in that tissue.73 For targeted drug delivery to tumors, spatial variation in drug concentrations is important, so tissues are often removed in different sections. To determine tissue drug concentrations, tissues are weighed and either mechanically or chemically homogenized according to the desired detection mechanism. Examples of techniques to measure drug concentration in the extracted tissues include fluorescence detection, high performance liquid chromatography (HPLC),73,74 mass spectrometry, and atomic absorption spectroscopy
(AAS).61 If the drug target is radiolabeled, drug concentrations can also be measured using liquid scintigraphy. Key advantages of measuring drug concentrations in removed tissues include definitive drug detection, high sensitivity, and the ability to detect low drug concentrations. However, these techniques are restricted by low spatial resolution
25 and accuracy, as measurements are an average over an entire piece of tissue. Achieving spatial measurements depends on the size of pieces cut from the tissue, which usually limits spatial resolution from these techniques to the millimeter range.
Imaging of ex vivo tissues can help overcome the spatial resolution limitations of bulk tissue analysis methods. For imaging-based methods, the tissue is removed and sliced into a thin piece followed by drug detection through imaging the slice. At least two techniques have been used for imaging drug detection: autoradiography and fluorescence.70 For autoradiography, the drug target is radiolabeled and then detected by exposing the tissue section to a flat panel detector or x-ray film.56,70 Advantages of this technique include high sensitivity, very low detection limits, and high resolution, while the major limitation is working with a radiolabeled drug. Fluorescent detection of drug concentration in tissues is an alternate strategy. In this method, tissue slices are analyzed using a fluorescence scanner or fluorescent microscope to detect drug.74 To use this technique, the drug must either be intrinsically fluorescent or labeled with a fluorescent tag, such as fluorescein isothiocyanate (FITC).75 While also offering low detection limits, reasonable sensitivity, and good resolution, only a few small molecule drugs are inherently fluorescent, and labeling of other drugs inevitably modifies their transport and efficacy, unlike radiolabeling methods. For large molecules, such as protein drugs or antibodies, fluorescent labeling may have only a minimal effect on the overall drug properties and may not adversely affect the delivery system, making the approach more tenable. Ex vivo drug detection is a major tool in developing local drug delivery methods, but temporal information is limited because every time point requires animal euthanasia and removal of tissue.
26 1.3.2 Imaging based methods for measuring local drug concentrations
Noninvasive imaging methods for measuring local drug concentrations represent a growing trend in attempts to address the temporal limitations of ex vivo approaches.
Driven by advances in imaging technology as well as proliferation of scanner availability, noninvasive imaging methods likely hold the future for monitoring drug concentrations from local delivery strategies. With noninvasive imaging, a single subject can be imaged several times throughout the study period, greatly increasing the data available from a smaller number of animal subjects. The most straightforward extension of previous detection technologies is continued use of radiolabeled drugs coupled with positron emission tomography (PET)76 or single photon emission tomography (SPECT) for drug detection.5 These detection methods have existed clinically for several years, but recent development of specialized small animal scanners, often coupled with x-ray computed tomography (CT) for anatomical information, has improved resolution and usability, making nuclear medicine techniques key for development of targeted therapies.
Additionally, these techniques can be easily translated to clinical use for monitoring of clinical trials of newly developed devices or treatment strategies. Other imaging techniques, such as in vivo fluorescence imaging, have been specifically developed for use in small animals and can contribute primarily to small animal studies. With in vivo fluorescence imaging, fluorescently labeled molecules are imaged directly in the animal.77 Most fluorescent imaging suffers from greater background noise than radiographic imaging and limitation to two dimensions, but developments in tomographic fluorescence offer the potential to reduce noise and provide 3-D localization of drug.78
Beyond radiolabeling and fluorescence, magnetic resonance imaging (MRI) detection of
27 drugs or drug carriers labeled with an MRI contrast agent, such as gadolinium or superparamagnetic iron oxide (SPIO), also offers the potential to noninvasively image anatomical detail and drug concentrations simultaneously.79-81 Recent advances and the benefits afforded by noninvasive imaging make it likely that these techniques will dominate the future landscape of monitoring local drug delivery strategies.
A novel noninvasive method used in the development of polymer millirods for liver cancer treatment is drug detection using CT.82-84 Polymer implants were loaded with the anticancer drug carboplatin and tested in both normal and ablated rat liver tissue.
Carboplatin has a unique property among cancer drugs in that it contains the heavy metal platinum (Z = 78), which has high x-ray attenuation and provides inherent CT contrast.
Polymer millirods containing 10% carboplatin and 90% PLGA were implanted in non- ablated or RF ablated rat livers.84 For carboplatin detection, the rats were scanned with
CT at multiple time points after the implantation. A representative CT scan of one of these rats is shown in Figure 1-3. Slices perpendicular to the long axis of the implant clearly show the higher absorption of the implant compared to the surrounding tissue. By comparing the intensity of the implant to premeasured implants with known concentrations, the remaining carboplatin in the implant was determined. The drug concentration in the surrounding tissue was determined after subtracting the background signal due to ablated liver tissue and accounting for partial volume effects. Tissue concentrations of carboplatin determined from CT images are shown in Figure 1-4 along with validation measurements determined using AAS of platinum concentration in extracted tissues. CT provided higher spatial resolution than AAS and revealed differences in drug distribution in non-ablated and ablated tissues not appreciated by
28 AAS. Ablated tissue retained carboplatin for longer times and at greater distances from the implant than non-ablated tissue, illustrating a fundamental difference between drug transport in these tissue environments. Additionally, these results validate the use of a noninvasive imaging strategy to monitor local drug release from implants.
Further studies of local drug concentration around implants using fluorescent imaging allowed for greater quantification of the differences between ablated and normal tissue.85 Doxorubicin, a topoisomerase II inhibitor commonly used in liver cancer treatment,3 also has the fortuitous property of natural fluorescence with an excitation of
488 nm and an emission of 595 nm. Polymer millirods containing doxorubicin were implanted in non-ablated and RF ablated rat livers.86 Liver tissues were removed at various times after implantation, and tissues were sliced into 100 µm sections using a cryostat microtome. Sections were thawed and imaged for fluorescence intensity using a fluorescent gel scanner. Drug distribution profiles were determined by converting net fluorescence intensity (NFI) to tissue drug concentrations, and average doxorubicin concentrations at each distance from the implant boundary were calculated. These tissue drug concentrations at time points ranging from 1 hour to 4 days after implant placement were used to estimate the transport properties of liver tissues within the framework of a theoretical model of drug distribution as described in Section 1.2. The resulting estimates are shown in Table 1-1. These studies established the baseline transport properties of doxorubicin in normal rat liver as well as how ablation modifies them. Ablation reduced the diffusion coefficient, perhaps by destroying cell structure and making more sites
29
Figure 1-3. Computed tomography (CT) scan of a carboplatin-containing polymer millirod placed in an RF ablated rat liver.
(A) Oblique slice through the rat showing the general location of the polymer implant in the liver (black square). (B) Enlargement of the implant region showing the ablated region and implant. Drug concentrations can be approximated by measuring the image intensity arising from carboplatin as a function of distance, R, from the implant surface.
Adapted from Ref. 84.
30
Figure 1-4. CT-measured carboplatin distribution in liver tissue.
Normal liver () and ablated () liver tissue concentrations are shown as the mean
SD between animals. Atomic absorption spectroscopy (AAS) measurements confirm drug concentrations in tissue sections 2 mm wide from both ablated (white rectangle) and normal (gray rectangle) liver tissue. AAS data represent the average drug concentration determined over a tissue sample the width of the rectangle. Adapted from Ref. 84.
31
Table 1-1. Doxorubicin transport properties in rat liver
normal liver ablated liver
apparent diffusion, D*, (cm2·s-1) 6.7 x 10-7 1.1 x 10-7
apparent elimination, *, (cm2·s-1) 9.6 x 10-4 n/a
32 available for drug binding. Even more notably, ablation virtually abolished elimination, which is sensible since RF ablation might be expected to reduce elimination both due to metabolism in cells (by killing them) and perfusion related losses (by coagulating and destroying blood vessels). This reduction in drug elimination can largely explain why drug penetration distances and retention were higher in ablated liver tissue.
In summary, techniques for measuring local drug concentrations surrounding implants are fundamentally important for the development of a local drug delivery system for tumors. Many methods exist for measuring drug concentrations, each with advantages and disadvantages, and it is likely that a combination of methods provide the best overall information about drug delivery. After obtaining local drug concentrations, they can be compared empirically to determine qualitative differences in delivery or interpreted through the use of a model to obtain quantitative transport information. Both sets of data can then be used to modify implant properties to provide the best drug coverage to the tumor.
1.4 Modulating local drug pharmacokinetics from polymer implants
1.4.1 Controlling rate of drug release from implants
Development of techniques to monitor local drug pharmacokinetics allows for the design and assessment of different implant types. As described in Section 1.2, an ideal implant should provide a rapid ascent to the therapeutic concentration and maintenance of this dose for as long as possible. The first generation polymer millirod provided rapid release of a drug mimic, largely within the first few days.63 However, through
33 modification of the implant design it is possible to customize the delivery profile of the implants. Consequently, local drug concentrations arising around the implants were compared and evaluated based on overall tissue drug exposure.
Several modifications to the initial compression-heat molded millirods have been used to prolong drug release or change the timing of the released dose. The first such modification was the addition of a semi-permeable membrane around the outside of the implant. Physically, this modification can be made either by wrapping the cylindrical device with a membrane87 or by dip-coating the implant.62 To wrap the implant with a membrane, thin films of PLGA containing NaCl (10-50% w/w) were solvent cast onto a
Teflon dish and then wrapped around the implant. When placed in an aqueous environment, the NaCl component of the outer membrane rapidly dissolved, leaving a semi-permeable membrane that could be controlled by modulating the amount of NaCl included in the implant. The addition of such a barrier slowed the overall rate of drug release, allowing for drug release over a period as long as five weeks.87 Dip-coating the implants followed a similar strategy but allowed for a more uniform coating process. To dip-coat the implants, a solution of poly(lactic acid) (PLA) and poly(ethylene oxide)
(PEO) was created by dissolving the polymers in methylene chloride. The cylindrical implants were then dipped in the polymer solution and allowed to dry, creating a membrane around the original implant.62 Once the implant was exposed to water, the
PEO fraction rapidly dissolved, leaving a semi-permeable membrane. Again, the release rate could be modified by changing the PEO content (5-20% w/w) of the layer.58 Either wrapping or dip-coating the implant to create a membrane around it substantially prolonged the drug release from the resulting implants.
34 By further modifying the polymer millirods, it was possible to create an implant that adds an additional burst dose to the prolonged drug release. As discussed in Section
1.2, the tissue surrounding a sustained release implant may not reach the therapeutic concentration for some time, delaying the onset of action of the drug. To accelerate the rise to the therapeutic concentration, a burst dose can be added to the implant to act as a loading dose. Dual-release implants combining the benefits of a drug burst followed by sustained release were created by supplementing the implant with two drug coatings.58
Monolithic millirod implants were first created by compression molding of PLGA (60%),
NaCl (24%), and doxorubicin (16%). To sustain the release of drug from this implant, it was dip-coated with a layer of PLA/PEG as described above. Then, this coated implant underwent further dip-coating with a suspension of doxorubicin (75%) and PEO (25%) in methylene chloride. The total burst amount of drug could be controlled by applying multiple coatings to increase the thickness of the burst layer. The resulting implants, termed dual-release millirods, released a burst dose of doxorubicin followed by a sustained dose of doxorubicin for as long as 10 days.58 In this manner, polymer millirods that could release doxorubicin into tumors with different dose timings were created.
To evaluate the differences in local drug distribution generated by different implant types, these burst, sustained, and dual-release millirod formulations were tested in vivo.62 In the rat model, liver tissue was ablated for 2 min at 90ºC to create an ablation region 8-10 mm in diameter. Subsequently, polymer millirods of each type were placed in the ablation needle tract and sutured into place. At specified time points, the rats were euthanized, and the polymer implants and surrounding liver tissue were extracted.
Doxorubicin remaining in the implant was quantified by an extraction procedure and used
35 to calculate average release rates, which are shown in Figure 1-5A. As expected, the dual-release implants released a higher amount of drug in the first 24 hours, but after this time the drug release rates were not statistically different. Tissue doxorubicin concentrations were determined using fluorescence scanning of sliced tissues, and the doxorubicin concentrations at the outer ablation boundary are shown in Figure 1-5B. The dual-release implants provided a more rapid ascent to therapeutically relevant concentrations that was statistically different from the sustained-release implants. The similarity between the experimental results and the desired theoretical profiles shown in
Figure 1-1 (panel 2, curves A and C) is notable. More detailed fluorescent images of tissues that confirm this finding are shown in Figure 1-6. Dual-release implants led to local doxorubicin concentrations as high as 1000 µg/g within one day, while it took nearly 4 days for the drug distributions around the sustained implant to reach this extent.
This study established that differences in implant formulation could have a substantial impact on local drug concentrations.
1.4.2 Controlling host tissue response in local drug therapy
Histology studies of treated tissue from ablated livers were performed to provide a more detailed understanding of the effect of changing tissue properties on drug transport.71 Ablated rat livers were treated with doxorubicin-containing polymer implants, and tissues were subsequently removed at time points ranging from 1 hour to 8 days after ablation. Throughout the first four days after ablation, an area of coagulation necrosis surrounding the implant was gradually infiltrated by inflammatory cells, particularly neutrophils and monocytes. By 8 days after ablation, fibroblasts and the
36
Figure 1-5. In vivo relationship between drug release rate and local tissue concentrations.
(A) Average rate of drug release from two implant formulations in vivo in ablated rat livers. (B) Doxorubicin concentration at the outer edge of the ablated region for the same two implant formulations. Adapted from Ref. 62.
37
Figure 1-6. Local drug distributions resulting from sustained-release and dual- release millirods over 8 days.
Concentration were determined by fluorescent imaging of extracted tissue slices. The dotted line is the ablation boundary, and the scale bar is 5 mm. Adapted from Ref. 62.
38
Figure 1-7. Preventing fibrous capsule formation after RF ablation.
Masson’s trichrome stained histology images of ablated liver samples 8 days after RF ablation and millirod implantation show: (A) Ablated liver receiving control PLGA millirod, (B) Sample that received a control PLGA millirod and intraperitoneal (IP) injection of DEX, and (C) Ablated liver receiving DEX-loaded millirod. The arrows point to the center of the liver ablation/millirod implantation site. All scale bars are 0.5 mm. Adapted from Ref. 88.
39 formation of a dense, collagenous fibrous capsule were evident at the ablation boundary.
Furthermore, it was found that doxorubicin concentrations leading up to the fibrous capsule were high but dropped precipitously in the nascent fibrous capsule. From these results, it appeared that the wound healing response after ablation could have a major role in drug diffusion, acting as a barrier to transport outside the ablation region.71 This finding reiterates the importance of considering the tissue surrounding the implant not as a static environment, but instead as a dynamic milieu that can ultimately affect the success of the treatment itself.
Since the tissue surrounding the implant has a large impact on the efficacy of drug therapy, modifying the response of the surrounding tissue can be used to favor effective drug dispersion. One way to modify the properties of ablated tissue is to moderate the ensuing inflammatory response with an anti-inflammatory agent. To test this hypothesis, the potent corticosteroid dexamethasone (DEX) was loaded into PLGA millirod implants.88 Given that dexamethasone is a very hydrophobic molecule, a DEX formulation complexed with hydroxypropyl ß-cyclodextrin (HPß-CD) to increase the water solubility was incorporated in the implants. The final formulation of the implants contained 1.7% dexamethasone, 38.3% HPß-CD, and 60% PLGA. Implants containing the complexed dexamethasone released 95% of their drug over 4 days, compared to 14% of dexamethasone released in implants containing 1.7% uncomplexed dexamethasone.88
These implants were tested for their ability to reduce fibrous capsule formation following liver ablation in rats. Masson’s trichrome stained histology showed that the dexamethasone-impregnated implants drastically suppressed the thickness of the collagen fibrous boundary compared to a control ablation treatment (Figure 1-7). The average
40 thickness of the fibrous boundary was 0.04 ± 0.01 mm in subjects receiving a DEX implant, reduced both compared to a control ablation (0.29 ± 0.08 mm) or ablation followed by an intraperitoneal (IP) DEX injection (0.26 ± 0.07 mm).88 In addition to reducing fibrous capsule formation, dexamethasone may have other beneficial effects when administered after ablation. Chemokine and growth factor production resulting from inflammation as well as angiogenic processes during wound healing have been implicated in tumor growth and recurrence,89-91 and at least one report has suggested that liver tumor recurrence after ablation is potentiated by inflammation.60 For this reason, including dexamethasone in the implants may improve the primary outcome of ablation while facilitating drug delivery. Future polymer implants which concomitantly release dexamethasone and an anticancer agent may maximize the therapeutic benefits of using polymer millirod devices after RF ablation.
1.5 Goals of this work
The primary goal of this work is to build on the previous expertise with polymer millirods by using a modeling and experimental approach to develop a comprehensive strategy for the treatment of solid tumors. Previously published work, as described in this chapter, has developed the means for implant manufacture, techniques for measuring local drug concentrations, and strategies for modifying local drug release and distribution. While they provide an essential foundation, these studies deal with implants in non-ablated or ablated normal liver tissue, giving little cancer relevance to the work.
No studies have been published investigating drug distributions arising from these implants and their subsequent treatment effects in tumors. Thus, the next step in polymer
41 millirod development and the focus of this work is to determine the antitumor efficacy and local drug distribution after implant placement in experimental tumors.
Developing a comprehensive treatment strategy for experimental tumors using RF ablation and polymer implants is described here in four steps. First, two types of polymer millirods were tested for the treatment of small experimental liver carcinomas (Chapter
II). These experiments investigated the antitumor efficacy of polymer millirods alone in treating an experimental liver tumor model. Second, the implants were tested as part of a combined treatment: RF ablation followed by implant placement (Chapter III). These experiments revealed key differences in drug distribution and anti-tumor efficacy between non-ablated and ablated liver tumors. Third, experimentally measured drug distributions were used to determine doxorubicin transport properties in tumor through the use of a transport model (Chapter IV). Fitting dynamics of drug transport to a model allowed for explicit quantification of differences in doxorubicin diffusion and elimination in tumors before and after ablation. Fourth, the estimated doxorubicin transport properties in tumors were used to simulate combined treatments in larger tumors (Chapter
V). These simulated results allowed for the optimization of a multiple implant strategy for treating realistically sized tumors. Finally, Chapter VI summarizes the results, describes some of the challenges of using intratumoral implants for cancer treatment, and anticipates the future directions of using polymer implants as part of a treatment for human tumors.
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48 81. Nasongkla N, Bey E, Ren J, Ai H, Khemtong C, Guthi JS, Chin SF, Sherry AD, Boothman DA, Gao J 2006. Multifunctional polymeric micelles as cancer-targeted, MRI- ultrasensitive drug delivery systems. Nano Lett 6(11):2427-2430.
82. Szymanski-Exner A, Stowe NT, Lazebnik RS, Salem K, Wilson DL, Haaga JR, Gao J 2002. Noninvasive monitoring of local drug release in a rabbit radiofrequency (RF) ablation model using X-ray computed tomography. J Control Release 83(3):415-425.
83. Szymanski-Exner A, Stowe NT, Salem K, Lazebnik R, Haaga JR, Wilson DL, Gao J 2003. Noninvasive monitoring of local drug release using X-ray computed tomography: optimization and in vitro/in vivo validation. J Pharm Sci 92(2):289-296.
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49 Chapter 2 Polymer millirods for treatment of small experimental liver tumors in rabbits
2.1 Introduction
As discussed in Chapter 1, cancer is a major health problem in the United States for which the current treatment armamentarium is inadequate. Surgery, radiation therapy, and systemic chemotherapy are viable treatment options either alone or in some combination, but these choices leave much to be desired in the way of limited effectiveness and serious side effects. Moreover, many cancer patients, such as those with liver cancer, are not good candidates for any of these treatments. Several minimally invasive ablative techniques have emerged to address this need and have shown considerable initial success.1-4 However, these treatments are limited by the ability to treat only small tumors and the potential for tumor recurrence around the ablated tumor periphery. Locoregional5-8 and intratumoral9,10 chemotherapy have also been successful in some instances but have limitations of their own, such as inadequate drug penetration into tissue and rapid clearance from the tumor site.11,12 While all of these treatments have proven useful in individual situations, none of them has emerged as a universally successful treatment strategy. The overall goal of this project is to use the biodegradable polymer millirod platform to develop a new, comprehensive tumor treatment strategy that improves on previous minimally invasive cancer treatments.
In light of this overall goal, this chapter describes the use of polymer implants without RF ablation to treat small experimental carcinomas. This initial study is of fundamental importance for several reasons. First, while polymer millirods have been
50 studied extensively in normal and ablated liver tissue, no studies have thus far been carried out to describe the effects of these implants in tumors. Previous studies have established the release properties of these implants in both normal and ablated liver tissues,13-15 modeled the transport of drug through these tissues,16 and established techniques for customizing the release rate of these millirods.17,18 However, the drug distribution from and efficacy of these implants in tumor tissue, which is considerably different from normal tissue in vascularity and transport properties,19 have not been examined. Second, this chapter describes the tumor model and experimental techniques necessary to evaluate the success of tumor treatment with polymer millirod implants.
Third, this study establishes the baseline for local drug distribution and antitumor efficacy resulting from the use of drug-containing PLGA implants alone. Further studies using combined treatment can then use these results to establish the benefits of combined treatment using the polymer millirod implants.
In this chapter, polymer millirods were tested for the treatment of small experimental liver tumors. The implants had the same formulation as the previously described millirods (diameter 1.6 mm, length 8.0 mm), which were composed of poly(D,L-lactide-co-glycolide) (PLGA) and designed to be administered with image- guidance through the bore of a 14-gauge biopsy needle. Polymer implants were placed directly into tumors in rabbit livers, and treatment effects were assessed either 4, 8, or 16 days after implant placement. The tumor model used was an aggressive, realistic allograft model of hepatic cancer (rabbit VX2 tumor) that has been widely used in studies of minimally invasive tumor treatments.20,21 Details of the tumor model are described more extensively in Appendix A.
51
Figure 2-1. Schematic of tumor treatment with polymer millirods.
Polymer millirod implants were placed in the center of rabbit VX2 liver tumors on day 0.
Tumors were then extracted and assessed on day 4 and 8 (for DOX treated tumors) and day 16 (for 5-FU treated tumors).
52 Two different types of millirods were used in two different arms of this study. In the first section, doxorubicin was incorporated into millirods designed to quickly release their drug contents. Doxorubicin (DOX) was used as the therapeutic agent because of its previous use in the treatment of primary and metastatic liver cancers22,23 as well as its natural fluorescence that permits quantitative measurement of drug concentration in tissue slices. DOX is an anthracycline antibiotic that acts primarily by inhibition of topoisomerase II,24 a key enzyme in DNA replication that is primarily responsible for resealing DNA strand breaks.25 DOX also causes secondary damage by intercalating
DNA, hindering DNA strand separation, and forming free radical adducts.24 A rapid delivery implant was chosen to provide the highest possible drug concentrations early in the study to maximize the initial tumor kill from the treatment and minimize the amount of time in which tumor cells might become doxorubicin resistant. Treatment effects in the DOX arm were gauged 4 and 8 days after treatment by monitoring tumor size through gross tissue measurements, drug distribution through fluorescent imaging of tissue slices, and cell morphology through histology. Using adjacent tumor sections for histology and fluorescent drug measurement allowed for direct correlation of drug concentrations with drug effects. While other studies have established the initial proof of concept for minimally invasive, intratumoral chemotherapy,26 few studies of intratumoral therapies have simultaneously measured local drug concentration and tumor histology to correlate tumor response to drug exposure. The main goals of the DOX arm of the study were to determine local drug concentrations and antitumor efficacy of the polymer implants a short time after implantation.
In the second arm of the study, 5-fluorouracil (5-FU) was incorporated into
53 implants, which were designed to release their contents over a much longer period of time. 5-FU is a widely used anticancer drug which works through inhibition of the thymidylate synthase enzyme, preventing DNA synthesis.27,28 The main goal of this arm of the study was to determine if millirod implants containing an alternative drug with a different release profile have distinctly different effects on the tumor and to corroborate the results obtained from doxorubicin implants. Primary outcomes of this the 5-FU millirod studies were tumor size and histology. Since the 5-FU containing implants released their drug loading over a much longer period of time (half-time of 16.3 days) treatment effects were assessed 16 days after millirod implantation.
2.2 Methods
2.2.1 Materials
Poly(D,L-lactide-co-glycolide) (PLGA, 1:1 lactide:glycolide, inherent viscosity
0.65 dL/g) was obtained from Birmingham Polymers (Birmingham, AL). Tris-buffered saline (TBS, pH 7.4), phosphate-buffered saline (PBS, pH 7.4), hydrochloric acid, sodium hydroxide, acetonitrile, and dimethyl sulfoxide (DMSO) were purchased from
Fisher Scientific (Pittsburg, PA). Ammonium formate, methylene chloride, poly(vinyl alcohol) (PVA, MW 13,000–23,000 Da, 87–89% hydrolyzed), 5-fluorouracil (5-FU), and
Hank’s balanced salt solution (HBSS) were acquired from Sigma-Aldrich (St. Louis,
MO). Doxorubicin HCl (DOX) (2 mg/mL) in saline (9 mg/mL) was acquired from
Bedford Laboratories (Bedford, OH). Teflon tubes (i.d. 1.6 mm) and stainless steel plungers (o.d. 1.6 mm) were purchased from McMaster-Carr Supply Company
54 (Cleveland, OH). Fetal bovine serum was obtained from Cambrex (East Rutherford, NJ).
2.2.2 Implant fabrication
Polymer millirod implants were produced as reported previously29 and described briefly below. PLGA microspheres (approximately 4 µm in diameter) were produced using a single-emulsion procedure. Three different sets of implants were produced: doxorubicin-containing implants, 5-FU-containing implants, and control implants.
Doxorubicin millirods were manufactured by incorporating doxorubicin and sodium chloride into the polymer matrix. Doxorubicin/NaCl powder mixture was prepared as follows. The acidic doxorubicin solution (pH ~ 3.0) was basified to pH 9.0 by adding sodium hydroxide, leading to doxorubicin precipitation. The precipitated drug was washed, resuspended, and lowered to pH 3.0 using hydrochloric acid. The resulting concentrated solution was combined with doxorubicin in saline and lyophilized to yield a final powder containing 38.5% doxorubicin and 61.5% NaCl (w/w). To produce the implants, 65% PLGA microspheres and 35% doxorubicin/NaCl powder were mixed with a mortar and pestle, packed into a Teflon tube, and compressed with steel plungers at 90º
C for 2 hours. The resulting millirods had a composition of 65% PLGA, 21.5% NaCl, and 13.5% doxorubicin (w/w). Implants containing 5-fluorouracil (5-FU) were produced in a similar fashion. PLGA microspheres (60% w/w) were combined with powdered 5-
FU (40%) and mixed in a mortar and pestle before packing into a Teflon tube and heating to a temperature of 90ºC under compression for two hours. Control implants were produced through compression molding of 100% PLGA microspheres, also for 2 hours at
90ºC.
55 2.2.3 Drug release measurement
In vitro release of drug from doxorubicin-containing implants was performed in
Tris-buffered saline (TBS, pH 7.4) at 37ºC. Tris-buffered saline was used for doxorubicin release studies because doxorubicin has limited solubility in saline and the organic component of TBS allows it to more accurately approximate DOX solubility in tissue. Millirods (n = 3) were placed in vials containing 5 mL of TBS in an incubator/shaker rotating at 100 rpm. To maintain sink conditions, the implants were moved into a fresh vial of TBS at each sampling point, and the doxorubicin concentration of the solutions was determined by measuring the absorbance at 480 nm (Perkin Elmer
Lambda 20 Spectrophotometer) and determining doxorubicin concentration using an extinction coefficient of 17.62 mL/(cm·mg). Similarly, in vitro release of 5-FU from implants was performed in PBS (pH 7.4) at 37ºC. 5-FU-containing millirods were placed in vials containing 10 mL, with vials changed periodically to sample the solution. 5-FU concentrations in the PBS were determined by measuring the absorbance at 266 nm with an 5-FU extinction coefficient of 44.0 mL/(cm·mg).
2.2.4 Animals and tumor model
Adult New Zealand White rabbits (n = 20; Covance, Princeton, NJ) weighing 2.8-
3.2 kg were used. All animal studies were approved by the Institutional Animal Care and
Use Committee at Case Western Reserve University and carried out according to its guidelines. For surgical procedures, animals were anesthetized with intramuscular ketamine (40 mg/kg), acetylpromazine (5 mg/kg), and xylazine (5 mg/kg).
The tumor model used in this study was the VX2 carcinoma in rabbit liver. To
56 implant the VX2 cells into the liver, the tumor was first grown for 4 weeks on the hind limb of a donor rabbit. The donor rabbit was euthanized, and the tumor was removed and dissected into small pieces of approximately 2-3 mm3. Tumor pieces were stored in fetal bovine serum with 10% DMSO in a liquid nitrogen storage tank. Prior to liver tumor implantation, the tumor pieces were rapidly thawed and washed three times with HBSS.
The implantation surgery was modified from a published procedure30 and was performed on 12 days before implant treatment. The abdomens of the recipient rabbits were shaved and prepped with betadine, after which a midline subxyphoid incision was made. The anterior surface of the middle liver lobe was perforated to a depth of 5 mm with the outer cannula of a 22-gauge angiocatheter, and a piece of tumor measuring approximately 1 mm3 was placed into the puncture. A small piece of cotton followed by a small piece of abdominal fat was secured over the puncture site using a single biodegradable suture. This method allowed for the growth of a single, well demarcated tumor in the liver of each rabbit. The tumors were grown in the liver for 12 days until day 0, when they reached an approximate diameter of 8 mm. Pretreatment tumor sizes were assessed by surgical palpation before the implant treatment.
2.2.5 Tumor treatment procedure
For testing of doxorubicin-containing implants, 16 rabbits were randomly divided into two groups, a treatment group (n = 8) and a control group (n = 8), receiving 13.5%
(average dose 1.0 mg/kg) and 0% (w/w) doxorubicin implants, respectively. On day 0, the rabbits’ abdomens were reopened as described above, and the tumor was located by palpation. The tumor was punctured through the center and perpendicular to the liver
57 surface with an 18-gauge needle. The implant was inserted into the center of the tumor and sutured in place with a piece of cotton and fat on top of the liver. Using sodium pentobarbital euthanasia, half of the animals from each group were then euthanized on day 4, and the remaining half were euthanized on day 8.
To test the 5-FU loaded implants, 4 rabbits were divided into control (n = 2) and treatment (n = 2) groups. Treatment was performed on day 0 in an identical fashion to the doxorubicin-containing implants, with treatment animals receiving implants that contained 40% 5-FU (average dose 3.9 mg/kg) and control animals receiving 100%
PLGA implants. One notable difference in the 5-FU study was that animals were all euthanized on day 16 after tumor treatment.
Explanted doxorubicin-containing rods were dissolved in acetonitrile to extract the remaining doxorubicin, and the concentrations of the resulting solutions were measured using HPLC on a C-18 column (150 x 4.6 mm, 5.0 µm particle size) with a mobile phase consisting of 35% acetonitrile and 65% ammonium formate buffer (0.1% w/w) at pH 4.0.
2.2.6 Tumor analysis
After tissue removal, tumors were hemisected and photographed. One half of the tumor was placed in 10% buffered formalin solution and subsequently embedded in paraffin. Alternating slices of paraffin embedded tissue were stained with hematoxylin and eosin (H&E) or left unstained and observed qualitatively with fluorescence microscopy. For the doxorubicin arm of the study, the other half of the tumor was frozen at -20ºC for use in fluorescent imaging analysis.
58 To measure tumor size, a cross sectional area was calculated using the area of an elliptical cross section through the center of the tumor. This area is given by the formula
A = ·Rl·Rs, where Rl and Rs are the long and short radii of the ellipse, respectively. For the animals implanted with doxorubicin millirods, tumor area was measured only at the endpoint of the study using photographs of the sliced tumors. In 5-FU treated tumors, computed tomography (CT) scans were taken the day before the treatment and the day before animal euthanasia using a Philips MX8000 CT (120 kVp, 250 mAs, and voxel size
0.57 x 0.57 x 5 mm). Tumor size was assessed by measuring the long and short dimension of the tumor in the CT slice containing the largest tumor area. Statistical comparison of tumor sizes was performed using a two-tailed, unpaired t-test with a significance level of 0.05.
2.2.7 Quantitative fluorescence analysis
To determine the amount of doxorubicin present in the tissues surrounding the implants, a previously established fluorescence imaging technique that takes advantage of doxorubicin’s natural fluorescence was used.31 Frozen liver sections 100 µm thick were sliced from each tumor using a cryostat microtome (Microm 505E) and then scanned with a fluorescent imager (Molecular Dynamics Fluorimager SI) using the following conditions: pixel size, 100 µm; bit depth, 16; photomultiplier gain, 850; and sensitivity,
59
Figure 2-2. Qualitative fluorescence calibration in extracted tissue slices.
Fluorescent measurement of doxorubicin concentration was calibrated by imaging tissue slices with known doxorubicin concentrations (A) and then fitting the net fluorescence intensity (NFI) to a function of doxorubicin concentration (B).
60 high. A calibration between net fluorescence intensity (NFI), the fluorescence minus the liver background fluorescence, and doxorubicin concentration was established by imaging weighed slices of normal liver which had known amounts of doxorubicin. This calibration is shown in Figure 2-2. The derived empirical relationship, NFI =
194·[Dox]0.67, where [Dox] is the doxorubicin concentration in µg/g, was then used to convert fluorescence intensities to doxorubicin concentrations. Mean drug distribution profiles were calculated by averaging 8 fluorescence profiles evenly spaced by 45º around the millirod location. The drug penetration distance was calculated as the average distance between the implant boundary and the point where the drug concentration dropped below 64 µg/g, which is 10 times the therapeutic drug concentration.32,33 To estimate the total mass of drug remaining in the tumor, we determined the average drug concentration within 4 mm of the implant surface (the approximate size of the original tumor) and multiplied this value by the tissue volume.
2.3 Results
2.3.1 Implant properties
The doxorubicin-containing implants used in this study had an average length of
8.0 ± 0.3 mm and an average diameter of 1.49 ± 0.04 mm. In vitro, the doxorubicin- containing implants were found to release a total of 2.07 ± 0.05 mg of drug over the 8 day period, corresponding to 71.3 ± 1.7% of the total drug loading. The release half-time was approximately 4 hours, and the vast majority of doxorubicin, 1.98 ± 0.03 mg, was released in the first 24 hours. The cumulative doxorubicin release normalized by the
61 length of the implant is shown in Figure 2-3.
In contrast, the 5-FU containing implants used in this study had a much slower rate of in vitro drug release, as is shown in Figure 2-4. By day 16, the implants released an average of 2.8 ± 0.8 mg 5-FU, corresponding to 55 ± 14% of the total drug loading.
The release half-time for 5-FU was 16.3 days. After approximately four and a half weeks, 77 ± 12% of the total drug loading had been released, at which point the polymer began to quickly degrade. The remaining drug contents were quickly released over the next week, providing total release from the implants. The 5-FU containing implants had greater variation in their release rate than the doxorubicin implants. One explanation for this finding is that at high drug loadings 5-FU may dissolve in the PLGA, leading to disparity in the release rates depending on the quantity of 5-FU dissolved in the polymer.
2.3.2 Size of doxorubicin implant treated tumors
After tumor removal and sectioning, the VX2 tumors were observed qualitatively and photographed. The tumors were largely spherical, but most of the cross sections were found to be slightly elliptical. Photographs of control and DOX implant treated tumors taken on day 8 are shown in Figure 2-5A and B, respectively. The control tumor seen in Figure 2-5A was a large and well-circumscribed tumor. The boundary of the tumor was a solid mass of dense tumor tissue, while the center of the tumor was necrotic and consisted of a mixture of necrotic cells, inflammatory debris, red blood cells, and edema. Because the center of the tumor was liquid filled, the original location of the implant which was placed in the center of the tumor is not visible in the photograph. The
DOX-implant treated tumor seen in Figure 2-5B was also well-circumscribed but
62
Figure 2-3. Cumulative doxorubicin release from polymer millirods in vitro.
Cumulative release of doxorubicin from the polymer millirod implants in a TBS buffer solution over 4 days is shown. Error bars indicate the standard deviation of each measurement (n = 3).
63
Figure 2-4. Cumulative 5-FU release from polymer millirods in vitro.
Cumulative release of 5-FU from the polymer millirod implants in a PBS buffer solution over 4 weeks is shown. 100% release was reached in just longer than 5 weeks. Error bars indicate the standard deviation of each measurement (n = 3).
64 Figure 2-5. Gross appearance of doxorubicin implant treated VX2 tumors.
Photographs of a control (A) and a DOX implant treated (B) tumor cross section on day
8. The boundary between the tumor and normal liver tissue is indicated with a white dotted outline, and the arrows indicate two directions along which the tumor dimensions would be measured to calculate the cross sectional area. The scale bars are 1 cm. Bar graph showing the mean cross sectional area of the implant treated and control tumors after
4 and 8 days (C). The error bars indicate the standard deviation of each measurement (n =
4).
65 Table 2-1. Summary of doxorubicin release in vivo in VX2 tumors.
Day 4 Day 8
Original DOX loading (µg) 3150 ± 110 3000 ± 190
DOX remaining in extracted implant (µg) 400 ± 40 390 ± 90
DOX released (µg) 2750 ± 130 2610 ± 120
DOX released (%) 87.2 ± 1.5% 87.2 ± 2.3%
DOX in tissue (µg) 210 ± 120 160 ± 70
DOX in tissue (%) 6.7 ± 3.7% 5.5 ± 2.8%
DOX penetration distance (mm)* 2.8 ± 0.5 1.3 ± 0.4
Percentages shown are based on the original DOX loading. All values are shown ± standard deviation, and (*) indicates a statistically significant difference between days.
66 Figure 2-6. Doxorubicin distribution in non-ablated tumors.
Doxorubicin concentration distribution maps derived from fluorescent imaging of representative tumor sections from the treated group on day 4 (A) and day 8 (B). Before removal, the implant was located in the small clearing found in the center of the image, and the scale bars are 2 mm. From these two distributions, concentration profiles plotting the average doxorubicin concentration against the distance from the implant boundary are shown for both times (C).
67 considerably smaller than the DOX-treated tumor. The doxorubicin-containing millirod was removed from the cavity seen in the center of the tumor, and the remaining tumor is seen as a small, hard shell only a few millimeters in thickness surrounding the implant location.
Tumor measurements taken from the photographs were used to assess the effect of the implant on the growth of the tumor. An example of how the measurements were taken is indicated with the white arrows in Figure 2-5A. The resulting measurements of cross-sectional area were then averaged for all the animals in each group, and the resulting data is shown in Figure 2-5C. On day 4, the DOX implant treated tumors (0.17
± 0.06 cm2) were approximately half the size (p = 0.048) of the comparable controls (0.31
± 0.08 cm2). The control tumors were also slightly smaller than the 8 mm diameter on day 0, which may be attributed to a response to placement of the drug free implant. By day 8, the size difference was dramatic, as the DOX implant treated tumors (0.14 ± 0.04 cm2) were more than 10 times smaller (p = 0.025) than the controls (1.77 ± 0.78 cm2).
Notable variation in tumor size, especially in the untreated tumors on day 8, was evident, but there was a statistically significant difference in treated and control tumors on both day 4 and day 8.
2.3.3 Doxorubicin release in vivo
A summary of the in vivo drug release properties of the doxorubicin-containing implants is shown in Table 2-1. DOX masses in the original implant, the extracted implant, and in the surrounding tissue as measured by fluorescence are shown along with the values as a percentage of the original doxorubicin loading. For implants extracted on
68 both day 4 and day 8, approximately 87% of the original drug was released, suggesting that little additional drug was released after day 4. A greater fraction of the drug was released in vivo than in vitro. Two notable differences are present between the two time points: the tissue at day 4 retained 50 µg more doxorubicin and the doxorubicin penetration distance at day 4 is 1.5 mm further from the implant boundary. Of these differences, however, only the increase in the drug penetration distance was statistically significant (p = 0.004). From these measurements, it was also possible to estimate the apparent elimination rate from the tumor tissue by fitting a decaying exponential of the form D(t) = D0·exp(-kt) to the known concentrations of drug present in tissue at each time. Using this method, the elimination rate constant from the tumor area was calculated to be k = 0.42 ± 0.06 day-1, which corresponds to an apparent elimination half time from tumor of t1/2 = 1.6 ± 0.2 days.
2.3.4 Fluorescent doxorubicin distribution
Local DOX distributions from the treated tumors on both day 4 and day 8 were used to evaluate the overall drug exposure to each tumor region. Representative doxorubicin distributions from tumor sections on day 4 and day 8 are shown in Figure
2-6A and Figure 2-6B, respectively. At both 4 days and 8 days after the millirod implantation, concentrations of drug higher than 1000 µg/g were found in a band surrounding the implant location. These concentrations were considerably higher than the previously reported effective concentration for doxorubicin, 6.4 µg/g.32,33
Additionally, it was observed that the band of drug around the implant location appears to be both more intense and thicker in the day 4 distribution as compared to the day 8
69
Figure 2-7. Histology of DOX implant treated versus control tumors.
H&E stained sections of control (A, C, E) and DOX treated (B, D, F) tumor sections on day 8. Low magnification images of the control (A) and treated (B) tumors indicate with lower case letters the regions from which the high magnification images are taken. The scale bars are 1 cm. High magnification images of the tumor core for control (C) and treated (D) tumors as well as the tumor/normal liver interface for control (E) and treated
(F) tumors are also shown. The clearing on the right side of slide (D) is the original implant location. In (E) and (F), normal liver is found to the left of the panel while tumor is found to the right. All high magnification images (C-F) have scale bars of 100 µm.
70 Figure 2-8. H&E stained and fluorescent micrographs of a DOX implant treated tumor on day 8.
The H&E section (A) illustrates the different tissue regions moving outward from the implant location
(*): (1) a dense necrotic region, (2) a sparse necrotic region, and (3) the inflammatory boundary of the tumor.
An aligned fluorescence micrograph
(B) illustrates the pattern of doxorubicin distribution throughout the tumor regions. A higher magnification image (C) of the area indicated with a white box in (B) shows significant cellular uptake of doxorubicin in the inflammatory boundary of the tumor. All scale bars are 100 µm.
71
Figure 2-9. Tumor recurrence outside of DOX implant treated zone.
H&E stained micrograph of a DOX implant treated day 8 tumor showing potential spread of the tumor beyond its circumscribed boundary. A low magnification image (A) indicates the implant location (*) and viable tumor cells (black box), which are magnified in (B). The two arrows note cords of viable tumor cells that appear to be spreading beyond the treated zone. The scale bars are 100 µm.
72 distribution. Another characteristic of interest was the large degree of asymmetry in the distributions, which is more pronounced than in previous studies in normal and RF ablated liver tissues.13 This asymmetry likely reflected the inhomogeneity of the doxorubicin transport properties inside VX2 tumor tissues. Average drug concentration profiles for the distributions in Figure 2-6A and B are shown in Figure 2-6C. These profiles quantitatively reaffirm that average concentrations near the implant are higher and the drug penetrates to a greater distance in the day 4 distribution. The average doxorubicin concentration within 0.5 mm of the implant boundary in these slices was
1870 ± 60 and 1380 ± 110 µg/g for day 4 and day 8, respectively. In contrast, little to no fluorescence was seen in the control tissue (data not shown).
2.3.5 Histological comparison of doxorubicin-treated tumors
Representative H&E sections of a control and DOX implant treated tumor on day
8 are shown in Figure 2-7. The control tumor (Figure 2-7A) was large and has a boundary of darkly staining viable tumor cells surrounding a core comprised of viable tumor cells mixed with necrotic debris, while the treated tumor (Figure 2-7B) had a smaller boundary of largely necrotic tumor cells. The higher magnification image from the core of the control tumor (Figure 2-7C) revealed clusters of large, irregularly shaped tumor cells with darkly staining nuclei interspersed with lighter regions of necrosis and cellular debris. In contrast, the treated tumor core (Figure 2-7D) was heavily necrotic and contains few viable cells. Cells lacked discernible boundaries and have no distinct nuclei, and basophilic remains have aggregated. Similar differences were visible in the high magnification images of the tumor boundaries (Figure 2-7 E and F). Again, the
73 control tumor was filled with viable tumor cells while the DOX treated tumor is largely necrotic. Both tumors were separated from normal tissue by a lighter staining fibrous region containing a mixture of tumor and inflammatory cells. Histological comparison of the tumors revealed marked differences between the regions exposed to doxorubicin from the implant and the untreated controls.
H&E histology (Figure 2-8A) and fluorescent microscopy (Figure 2-8B) from the same tissue area after 8 days allowed for explicit localization of the doxorubicin into three main regions of the treated tumor. Region 1, closest to the implant location, contained dense basophilic and eosinophilic necrotic debris accompanied by high doxorubicin concentrations. Region 2 was filled with less dense necrotic debris characterized by a lack of nuclei and has detectable but considerably smaller amounts of fluorescence. Finally, Region 3 contained a fibrous band separating tumor from normal tissue, which was marked by a dotted band of fluorescent cells skirting the outer tumor boundary. Enlargement of this region (Figure 2-8C) showed an area of enhanced cellular uptake of doxorubicin. Particularly, the nuclear detail visible in the image indicates the presence of drug in cell nuclei, the primary location of action for doxorubicin. These images reveal that even those cells at the tumor/normal tissue interface have significant doxorubicin exposure over the 8 day period of treatment. Fluorescence in this region was not observed in drug-free controls (data not shown).
While the main tumor mass in the treated animals was necrotic and exposed to high drug concentrations, there was histological evidence that residual viable tumor cells still exist on day 8. An H&E stained section shows cords of tumor cells extending approximately 1-2 mm from the tumor/normal tissue boundary (Figure 2-9). These cells
74 appear to be viable and are far enough from the implant location that they are unlikely to be exposed to therapeutic drug concentrations.
2.3.6 Size of 5-FU implant treated tumors
Tumors treated with 5-FU-impregnated implants were assessed for therapeutic efficacy by monitoring tumor size. Size was measured using CT scans one day prior to treatment as well as one day prior to animal euthanasia, and the results are displayed in
Figure 2-10. The average pretreatment tumor area for all of the tumors was 0.5 ± 0.3 cm2, corresponding to a tumor diameter of 0.8 cm. There was no statistically significant difference in the pretreatment sizes of the groups. After treatment, the average area measured 2.9 ± 0.6 cm2 for the control tumors compared with 2.1 ± 2.0 cm2 for the treatment tumors. The variation was particularly large in the day 15 treatment measurements because the two tumors responded very differently to the treatment. One tumor, largely unaffected by the treatment, increased in size to 3.5 cm2 while the other, more responsive tumor, was measured at 0.7 cm2. This disparity highlights the importance of considering individual differences in response to tumor treatments.
2.3.7 Histology of 5-FU implant treated tumors
Tumors treated with 5-FU millirods were also assessed using tumor histology.
Results from the tumor which had the smaller measured volume are shown in Figure
2-11. The cellular response seen surrounding the implant was similar to that found using
DOX-containing implants. The region immediately surrounding the implant showed extensive necrosis, presumably due to 5-FU exposure from the implant. Cells in this area
75 4 Control 3.5
Treatment ) 2 3
2.5
2
1.5
1 Cross section area (cm area section Cross
0.5
0 Pretreatment Day 15
Figure 2-10. Size change in 5-FU implant treated tumors.
Cross sectional areas of 5-FU implant treated tumors are shown one day prior to treatment (pretreatment) and one day prior to euthanasia (day 15) as measured with CT scans. Bars indicate the mean tumor area (n = 2) and the error bars denote the range of the two measurements.
76
Figure 2-11. Histology of VX2 tumor treated with 5-FU-loaded implant.
(A) Photograph of sliced tumor denoting implant (arrow) and untreated tumor nodule (*)
16 days after treatment. (B) Corresponding H&E stained histology indicating location of high magnification panels (c-f). (C) Necrotic area directly adjacent to the implant. (D)
Necrotic area adjacent to normal liver. (E) Cells in a viable tumor nodule. (F) Boundary between 5-FU treated region and viable tumor. Scale bars are 5 mm (A-B) and 100 µm
(C-F).
77 appeared glassy with few intact cell membranes or visible nuclei (Figure 2-11C). This 5-
FU treated region extended several millimeters from the implant and featured other regions with denser debris and a more irregular structure (Figure 2-11D). Despite effective treatment to this region, a small nodule of viable tumor measuring approximately 5 mm in diameter was present. This nodule featured characteristic VX2 morphology: dense packing, poor differentiation, a high nuclear/cytoplasmic ratio, and mitotic figures (Figure 2-11E). This viable tumor was relatively close to the implant location, and was only separated from necrotic tissue by an abrupt transition (Figure
2-11F). Like the DOX-containing implants, the 5-FU implants show some degree of volume control but did not treat the entire tumor.
2.4 Discussion
In this study, two different formulations of PLGA implants were used to treat
VX2 carcinomas in rabbit liver, and the response to the treatments was evaluated using tumor size, fluorescence drug distribution, and histology. Tumors treated with doxorubicin implants were significantly smaller than untreated controls at both day 4 and day 8. Histological analysis confirmed the tumor response to drug treatment at a cellular level, where regions of viable tumor cells in untreated tumor were replaced by broad regions of necrosis in the tumor surrounding the doxorubicin millirods. However, viable tumor cells appeared just beyond the doxorubicin treated radius, where they were unlikely to receive sufficient drug coverage to be completely killed. Tumors treated with
5-FU containing implants were less affected by the treatment, but histology confirmed the pattern of tumor destruction seen after DOX treatment. Near the implant location,
78 tumor cells were highly necrotic and retained none of the morphological characteristics of viable VX2 cells. Several millimeters from the implant, however, nodules of tumor that appeared relatively unchanged by drug exposure were found. In both DOX and 5-FU treated tumors, the cells in the immediate vicinity of the implant were killed, but regions of tumor slightly further away were found relatively undamaged.
Doxorubicin concentration measurements in tumor slices provided information about the localization of the drug released from the implants. Doxorubicin concentrations exceeding 100 times the therapeutic level were observed in tumor tissues surrounding the implant even after 8 days. In comparison, intravenously administered doxorubicin is typically eliminated from the plasma with a half-life of approximately 10 minutes.34 Cellular uptake, drug binding to extracellular proteins, and limited perfusion in the necrotic tumor core may each contribute to prolonged drug retention from these intratumoral implants. Qualitatively, the pattern of distributions revealed a greater degree of asymmetry than previously observed in either normal or ablated liver.16 The irregular drug distribution patterns may be attributed to the inhomogeneity of tumor vasculature19 and greater drug clearance in highly perfused areas. Overall, the doxorubicin-containing implants provided high drug concentrations for a prolonged exposure time, albeit to only a limited region surrounding the implant. Little drug was found more than 5 mm from the implant surface.
In the doxorubicin treated tumors, drug spread to the tumor boundary despite the limited drug penetration distance. While the initial tumors had a radius of approximately
4 mm, the maximum drug penetration distance was less than 3 mm on both day 4 and day
8. These suggest that the tumor periphery would not receive adequate doxorubicin to kill
79 the VX2 cells. To the contrary, microscopic examination of histology slides showed that doxorubicin was present at the tumor boundary and necrotic tumor abutted the surrounding normal liver tissue, demonstrating the treatment of entire main tumor volume. Two plausible mechanisms could explain this contradiction. In one scenario, doxorubicin may successfully treat the tumor mass without reaching 4 mm from the implant. Rapid release of high drug concentrations eliminates the VX2 cells closest to the implant, causing the tumor shrinkage observed in the study. The collapse of the tumor core subsequently brings the outer tumor boundary closer to the implant interface.
Continued diffusion of drug away from the implant simultaneously moves the front of drug penetration outward, where it eventually meets the inwardly moving tumor boundary by day 4. Alternatively, doxorubicin may reach the therapeutic level at 4 mm but be undetected by this study for various reasons. First, the time points in this study were chosen to maximize the information available from drug distribution, tumor pathology, and histology. Peak drug concentrations likely occur earlier, as soon as 24 hours after implantation, when doxorubicin may reach the 4 mm tumor boundary.16 By day 4, drug penetration distances decrease through clearance and tumor shrinkage as described above. Second, doxorubicin may have been present at undetectable and yet therapeutic levels beyond the measured penetration distances in this study. While previous studies have established an effective doxorubicin concentration at 6.4 µg/g,32,33 this number was calculated after a single intrahepatic injection. For a prolonged exposure such as that provided by this implant, the effective drug concentration is expected to be even lower. Future studies that differentiate the above two mechanisms will provide significant insights to fundamentally understand drug transport in tumor tissues and their
80 effects on tumor treatment.
While this study illustrates considerable promise of polymer millirods for treating local regions of VX2 tumors, these implants demonstrated an inability to treat the entire tumor. In DOX treated tumors, small clusters of viable tumor cells were observed advancing beyond the main front of the tumor (Figure 2-9), potentially by lymphatic spread, which is a known mode of metastasis for the VX2 tumor cells35 as well as many human tumors.36-38 Areas of untreated tumor were larger in the 5-FU treated tumors on day 16 (Figure 2-11), suggesting that the short penetration distance can eventually lead to sizeable remnants of tumor. Several conceivable explanations could exist for why the residual tumor volume was more significant in the 5-FU treated tumors: 1) VX2 tumor may be less sensitive to 5-FU exposure, 2) the slow release of 5-FU may not be sufficient to treat the tumor, or 3) the spread of 5-FU covered a smaller therapeutic region, or 4) the tumor simply had more time to expand after the treatment. In subsequent chapters, burst- release doxorubicin-containing implants are used exclusively because they appear to provide better tumor treatment and the drug can detected using fluorescence imaging, facilitating a mechanistic understanding of implant success. Because of the risk of tumor recurrence beyond the implant treated region, future development of intratumoral treatments should actively focus on successfully delivering drug to a margin of safety beyond the main tumor mass to minimize the risk of recurrence.39
On the other hand, combined treatment strategies with these implants could improve drug delivery in a way that allows for complete tumor eradication. For example, radiofrequency (RF) ablation has been shown to facilitate drug distribution and retention in normal tissues through destruction of vasculature,16 which makes it a particularly
81 attractive candidate for use with these implants. In a combined therapy, RF ablation could destroy the vast majority of the tumor volume and surrounding vasculature, allowing greater delivery of doxorubicin to the tumor periphery. This combined therapy is the subject of Chapter 3. Another approach to improving tumor treatment with these implants is using multiple implants placed throughout the tumor periphery, which is covered in Chapter 5. Other strategies, such as systemically administered targeted nanoparticles,40-42 that have demonstrated effective targeting to the well-perfused tumor periphery43 could also prove synergistic to the millirod therapy by treating regions that are ineffectively covered by drug spread from the implants. Alternatively, these implants could also be used as a neoadjuvant treatment prior to resection. Tissue conserving surgical removal of the smaller, post-treatment tumor could then be performed, minimizing the risk of local recurrence from residual cancer cells. Irrespective of the combined methods, the described polymer implants provide a versatile platform for minimally invasive, intratumoral chemotherapy, and this study provides detailed insight about drug distribution and antitumor efficacy that can be used in their further development.
In conclusion, millirods containing doxorubicin or 5-FU successfully treated small regions of aggressive liver tumors in rabbits but were unable to destroy all of the viable tumor cells. However, successful treatment of tumor cells in the immediate vicinity of the implants opens the door for investigation of combined treatment approaches to improve the scope of tumor eradication. The next chapter describes the use of RF ablation along doxorubicin-containing implants to improve tumor treatment.
82 2.5 References
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83 13. Qian F, Stowe N, Saidel GM, Gao J. 2004. Comparison of doxorubicin concentration profiles in radiofrequency-ablated rat livers from sustained- and dual- release PLGA millirods. Pharm Res 21(3):394-399.
14. Szymanski-Exner A, Gallacher A, Stowe NT, Weinberg B, Haaga JR, Gao J. 2003. Local carboplatin delivery and tissue distribution in livers after radiofrequency ablation. J Biomed Mater Res 67A(2):510-516.
15. Szymanski-Exner A, Stowe NT, Lazebnik RS, Salem K, Wilson DL, Haaga JR, Gao J. 2002. Noninvasive monitoring of local drug release in a rabbit radiofrequency (RF) ablation model using X-ray computed tomography. J Control Release 83(3):415- 425.
16. Qian F, Stowe N, Liu EH, Saidel GM, Gao J. 2003. Quantification of in vivo doxorubicin transport from PLGA millirods in thermoablated rat livers. J Control Release 91(1-2):157-166.
17. Qian F, Nasongkla N, Gao J. 2002. Membrane-encased polymer millirods for sustained release of 5-fluorouracil. J Biomed Mater Res 61(2):203-211.
18. Qian F, Saidel GM, Sutton DM, Exner A, Gao J. 2002. Combined modeling and experimental approach for the development of dual-release polymer millirods. J Control Release 83(3):427-435.
19. Jain RK. 2001. Delivery of molecular and cellular medicine to solid tumors. Adv Drug Deliv Rev 46(1-3):149-168.
20. Ramirez LH, Zhao Z, Rougier P, Bognel C, Dzodic R, Vassal G, Ardouin P, Gouyette A, Munck JN. 1996. Pharmacokinetics and antitumor effects of mitoxantrone after intratumoral or intraarterial hepatic administration in rabbits. Cancer Chemother Pharmacol 37(4):371-376.
21. Yoon CJ, Chung JW, Park JH, Yoon YH, Lee JW, Jeong SY, Chung H. 2003. Transcatheter arterial chemoembolization with paclitaxel-lipiodol solution in rabbit VX2 liver tumor. Radiology 229(1):126-131.
22. Clavien PA, Selzner N, Morse M, Selzner M, Paulson E. 2002. Downstaging of hepatocellular carcinoma and liver metastases from colorectal cancer by selective intra- arterial chemotherapy. Surgery 131(4):433-442.
23. Leung TW, Patt YZ, Lau WY, Ho SK, Yu SC, Chan AT, Mok TS, Yeo W, Liew CT, Leung NW, Tang AM, Johnson PJ. 1999. Complete pathological remission is possible with systemic combination chemotherapy for inoperable hepatocellular carcinoma. Clin Cancer Res 5(7):1676-1681.
24. Hande KR. 1998. Clinical applications of anticancer drugs targeted to topoisomerase II. Biochimica et biophysica acta 1400(1-3):173-184.
84 25. Binaschi M, Bigioni M, Cipollone A, Rossi C, Goso C, Maggi CA, Capranico G, Animati F. 2001. Anthracyclines: selected new developments. Current medicinal chemistry 1(2):113-130.
26. Jackson JK, Gleave ME, Yago V, Beraldi E, Hunter WL, Burt HM. 2000. The suppression of human prostate tumor growth in mice by the intratumoral injection of a slow-release polymeric paste formulation of paclitaxel. Cancer Res 60(15):4146-4151.
27. Sergeeva OA, Khambatta HG, Cathers BE, Sergeeva MV. 2003. Kinetic properties of human thymidylate synthase, an anticancer drug target. Biochemical and biophysical research communications 307(2):297-300.
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29. Qian F, Szymanski A, Gao J. 2001. Fabrication and characterization of controlled release poly(D,L-lactide-co-glycolide) millirods. J Biomed Mater Res 55(4):512-522.
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33. Swistel AJ, Bading JR, Raaf JH. 1984. Intraarterial versus intravenous adriamycin in the rabbit Vx-2 tumor system. Cancer 53(6):1397-1404.
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86 Chapter 3 Combined therapy using RF ablation and polymer millirods for liver cancer in rabbits
3.1 Introduction
One method to improve the success of tumor treatment with polymer millirod implants is to pursue a combined treatment strategy. In Chapter 2, millirods demonstrated the potential to treat small VX2 tumors in rabbits. However, only tumor tissue within a few millimeters of the implant was eliminated because the drug only penetrated a small distance away from the implant surface. Combined treatment has the potential to improve this situation by killing a fraction of the tumor cells as well as improving the penetration and retention of drug released from the implants.
Cancer biology and clinical precedent make a strong case for the pursuit of combined treatments. Tumors consist of a large number of cells (109-1012), many of which are rapidly dividing and capable of growing into a new tumor. Even if a high proportion of the tumor cells, e.g. 99.9%, are killed by a single treatment, many viable cells may remain and ultimately lead to tumor recurrence.1 Because it is very difficult to completely eradicate a tumor with a single treatment, most clinical treatments are already used in a combinatorial fashion. In the case of hepatocellular carcinoma (HCC) treated with systemic chemotherapy, using multiple administrations combining several drugs with synergistic activity has been shown to provide improved probability of complete remission.2 Resection of HCC is also often followed by adjuvant chemotherapy to kill any residual microscopic disease.3 Because surgery has removed a large portion of the cancer cell load, chemotherapy can further reduce the number of viable cancer cells and
87 limit the chance of recurrence or metastasis. As mentioned in Chapter 1, the only clinically applicable drug implant for cancer treatment, the Gliadel wafer, is used following bulk surgical resection of the main tumor mass,4 analogous to surgery followed by adjuvant chemotherapy. Previous success of combined treatment regimens is a compelling argument for the development of a multimodal treatment using polymer millirods.
Several treatments could potentially be combined with polymer millirods for liver cancer treatment, including surgery or percutaneous treatments. With surgery, the main tumor mass could be removed via resection, and millirods could be placed around the resection site to limit the chance of local recurrence. The main drawback of this approach is that the use of millirods as a post-surgical adjuvant offers no improvement for the 70-80% of liver cancer patients who are not candidates for surgery.5,6
Percutaneous approaches that have been developed in recent years may be a better choice because they can be used in patients with poor overall health or advanced disease. In recent years, a variety of ablative methods such ethanol7 or acetic acid injection;8 heating with radiofrequency,9 laser,10 or microwave11 energy; and cryoablation12 have been developed. All of these treatments can be applied with image guidance, maximizing the number of patients who are candidates to receive treatment. Of the ablation treatments, radiofrequency (RF) ablation has shown particular promise in treating liver disease because of the ease of application, high response rates, and relatively low incidence of major side effects.13 However, tumor recurrence, particularly at the boundary of RF ablation, has been found as a major limitation of this treatment.14 Recent research has attempted to improve the outcomes of RF ablation by improving the technology of
88 ablation equipment.15,16 However, an alternative means of improving RF ablation is by combining it with chemotherapy.
Merging tumor treatment with RF ablation and polymer millirods could improve the quality of both treatments. Recent studies have shown that concomitant liposomal doxorubicin administered intravenously or intratumorally can increase the size of the RF coagulated region in liver tumors.14,17,18 Two other studies have demonstrated improved tumor treatment response using RF ablation combined with intratumoral implants. In one study, VX2 tumors in rabbits responded more favorably when treated with RF ablation followed by 5-FU impregnated polyanhydride implants than when treated with RF ablation alone.19 In the other study, in situ forming carboplatin-containing PLGA depots combined with ablation improved treatment of subcutaneous tumors in rats.
Additionally, drug delivery from polymer millirods may be substantially improved by ablating the tumors beforehand. Previous studies using polymer millirods in rat and rabbit liver demonstrated that RF ablation drastically increased drug penetration and retention in tissue.20,21 Together, mutual improvement of RF ablation outcomes and drug delivery underscores the value of developing a tumor treatment combining RF ablation with polymer millirods.
The goal of the current chapter was to determine whether combined treatment with RF ablation and drug implants improves treatment outcome over RF ablation alone in the VX2 liver carcinoma model. Additionally, local drug distributions in ablated tumors were compared to drug distributions using polymer millirods alone as established in Chapter 2. A schematic of the treatment tested is shown in Figure 3-1. The tumors were first treated with purposely insufficient RF ablation to simulate incomplete
89 treatment followed by the implantation of doxorubicin-impregnated implants.
Doxorubicin implants were used for several reasons. First, doxorubicin-containing implants showed superior treatment of tumors compared to 5-FU-containing implants
(Chapter 2). Second, doxorubicin’s natural fluorescence allows for imaging based measurement of local drug distributions tissues. Third, the high burst release of doxorubicin may be well-suited for combination with ablation because tumor cells should be most vulnerable to chemotherapy just after the heat exposure. Previous studies have shown that intratumorally administered drugs have a maximum effect on enlarging the treatment area when administered within 30 minutes after ablation, with much of the effect lost after 48 hours.14 The burst-release implants maximize tumor drug exposure during this narrow window of drug sensitivity. Furthermore, high drug concentrations around the implant should minimize the risk of drug resistance, which could occur if tumor cells were treated with drug concentrations below the therapeutic level for long periods of time. Four and eight days after the treatment procedure, tumors were assessed by measuring gross tumor size, local drug distribution, and histology. The hypothesis was that the doxorubicin-containing implants with RF ablation would facilitate drug retention and penetration inside tumor tissues, thereby increasing treatment efficacy over
RF ablation or millirod therapy alone.
90
Figure 3-1. Schematic of combined tumor treatment with RF ablation followed by polymer millirods.
Rabbit VX2 liver tumors were first treated by RF ablation insufficient to ablate the entire tumor. After ablation, PLGA control or doxorubicin-containing polymer millirods were placed in the center of the tumor along the ablation track. Tumors were then extracted and assessed on day 4 and day 8.
91 3.2 Methods
3.2.1 Materials
Poly(D,L-lactide-co-glycolide) (PLGA, 1:1 lactide:glycolide, inherent viscosity 0.65 dL/g) was obtained from Birmingham Polymers (Birmingham, AL). Tris-buffered saline
(TBS, pH 7.4), hydrochloric acid, sodium hydroxide, N,N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO) were purchased from Fisher Scientific (Pittsburg, PA).
Methylene chloride, poly(vinyl alcohol) (PVA, MW 13,000–23,000 Da, 87–89% hydrolyzed), and Hank’s balanced salt solution (HBSS) were acquired from Sigma-
Aldrich (St. Louis, MO). Doxorubicin HCl (DOX) (2 mg/mL) in saline (9 mg/mL) was acquired from Bedford Laboratories (Bedford, OH). Teflon tubes (i.d. 1.6 mm) and stainless steel plungers (o.d. 1.6 mm) were purchased from McMaster-Carr Supply
Company (Cleveland, OH). Fetal bovine serum (FBS) was obtained from Cambrex (East
Rutherford, NJ).
3.2.2 Implant fabrication
Only doxorubicin implants were used in this chapter. Implants were produced using the previously published compression molding procedure described in greater detail in Chapter 2.22,23 Briefly, PLGA microspheres (approximately 4 µm in diameter) were produced using a single-emulsion procedure. DOX in solution was concentrated by raising the pH to 9.0 with sodium hydroxide, washing with water, resuspending, and returning the solution pH to 3.0 with hydrochloric acid. The resulting concentrated solution was combined with doxorubicin in saline and lyophilized to yield a final powder
92 containing 38.5% doxorubicin and 61.5% NaCl (w/w). To produce the implants, 65%
PLGA microspheres, 21.5% NaCl, and 13.5% doxorubicin (w/w) were mixed with a mortar and pestle, packed into a Teflon tube, and compressed with steel plungers at 90º C for 2 hours. Previous studies have shown that these implants release the majority of their doxorubicin loading in vitro within 24 hours.23 Control implants were produced by compression molding 100% PLGA.
3.2.3 Animals and tumor model
Adult New Zealand White rabbits (n = 16; Covance, Princeton, NJ) weighing 2.8-
3.2 kg were used. Studies with these animals were approved by the Institutional Animal
Care and Use Committee at Case Western Reserve University and carried out according to its guidelines. All surgical procedures were performed under intramuscular anesthesia with ketamine (40 mg/kg), acetylpromazine (5 mg/kg), and xylazine (5 mg/kg)
The tumor model used in this study was the VX2 carcinoma in rabbit liver, which has been widely used in studies of minimally-invasive and image-guided procedures.24,25
Greater detail about this tumor model is provided in Appendix A. VX2 cells were first grown for 4 weeks in the thigh muscle of a donor rabbit. This tumor was harvested, cut into small pieces 1.5 mm on each side, and frozen in FBS containing 10% DMSO in a liquid nitrogen storage tank. Before implantation, the tumor pieces were thawed and washed 3 times in HBSS. Liver tumors were generated in the livers of the study rabbits by implanting a small piece of frozen tumor tissue in the rabbit liver. The abdomen of the recipient rabbit was shaved and a midline incision just distal to the sternum was made. The anterior surface of the middle liver lobe was perforated to a depth of 5 mm
93 with the outer cannula of a 22-gauge angiocatheter, and a piece of tumor measuring approximately 1 mm3 was placed into the puncture. The tumor piece was secured in place with a small piece of gelatin foam and a single biodegradable suture. The tumors were then allowed to grow in the liver for 18 days until they reached an approximate diameter of 11 mm.
3.2.4 Tumor treatment procedure
All tumors were treated with RF ablation, and rabbits were randomly assigned to two groups. Control group subjects (n = 8) received a drug-free implant, and treatment group subjects (n = 8) received a doxorubicin-containing implant after RF ablation was performed. For treatment, the abdomen was opened and the liver tumor located by palpation. A 17-gauge, 1 cm exposed tip ablation probe (Radionics, Burlington, MA) was inserted into the center of the tumor perpendicular to the surface of the liver. The tumor tissue was then ablated to a temperature of 80ºC for 2 minutes, a condition sufficient to create a coagulated region approximately 8 mm in diameter. Following ablation, a control or doxorubicin-containing millirod was placed into the electrode tract and secured again using a small piece of gelatin foam and a single suture. Half of the animals in each group were euthanized with a barbiturate overdose at 4 and 8 days after the treatment. Doxorubicin content remaining in the implants was measured by dissolving the implant in 2 mL N,N-dimethylformamide and extracting the remaining doxorubicin.26 The solution was diluted to 20 mL with Tris-buffered saline and centrifuged to remove precipitated polymer. The doxorubicin concentration of the
94 solution was then determined by measuring the absorption at 480 nm on a spectrophotometer (Perkin Elmer Lambda 20).
3.2.5 Tumor analysis
Tumors were removed from the surrounding liver tissue and sliced in half at an orientation perpendicular to the implant track. The halves of the tumor were photographed, and one half of the tumor was placed in 10% buffered formalin solution while the other half of the tumor was frozen at -20ºC. The fixed tissue was embedded in paraffin, sliced, and stained with hematoxylin and eosin (H&E) or Masson’s trichrome
(MTC). Unstained sections were analyzed qualitatively with fluorescence microscopy.
Maps of the entire stained histology slices at 40x magnification were generated using a video microscopy system consisting of a light microscope (Olympus BX60), video camera (Sony DXC-390), position encoded motorized stage (Prior Scientific ProScan), and software (Media Cybernetics Image-Pro).27 Ablated tissue, viable tumor, and inflammatory regions on these image maps were identified and manually segmented using the ImageJ (NIH) software.
3.2.6 Quantitative fluorescence analysis
Fluorescent doxorubicin in tissue slices was measured by the fluorescent imaging technique described in Chapter 2 to include subtraction of an ablated tissue background.
An overview of the background subtraction technique is shown in Figure 3-2. Frozen tissue samples were sliced to 100 µm thick sections on a cryostat microtome (Microm
505E) and scanned with a fluorescent imager (Molecular Dynamics Fluorimager SI) with
95 the following conditions: pixel size, 100 µm; bit depth, 16; photomultiplier gain, 850; and high sensitivity mode. On each fluorescent image, the ablated area was manually segmented using the software ImageJ. Background fluorescence of the control ablated tissue varied with distance from the ablation probe (Figure 3-2B), so an ablation background profile was generated by averaging the fluorescence as a function of distance from the ablation center. To allow the technique to be scaled to the appropriate size of each ablation, points within the ablated region were assigned a relative distance based on the fraction of the distance between the ablation center, 0, and the ablated/normal tissue interface, 1 (Figure 3-2C). For each treated tissue slice, the segmentation of the ablated region together with the average background profile was then used to generate a custom mask of the expected background fluorescence in the ablated region (Figure 3-2D). Net fluorescence intensity (NFI) was calculated by subtracting the mask from the original fluorescence image. NFI images were converted to doxorubicin concentration maps
(Figure 3-2F) using the relationship determined in Chapter 2, NFI = 194·[Dox]0.67, where
[Dox] is the doxorubicin concentration in µg/g (Figure 3-2E).23 Average drug distribution profiles were calculated by finding the mean of 4 profiles evenly spaced by
90º around the fluorescence image. Values were binned in increments of 0.2 mm to decrease noise. The drug penetration distance was calculated as the average distance between the implant boundary and the point where the drug concentration dropped below
64 µg/g, which is 10 times the therapeutic drug concentration.28,29 To estimate the total mass of drug remaining in the tumor, the average drug concentration within 4 mm of the implant surface (the approximate size of the ablated area) was multiplied by the tissue volume.
96 Figure 3-2. Schematic of background subtraction for ablated tissues.
Treated tumor fluorescence (a) required subtraction of background fluorescence from an ablated control (b). Background fluorescence was averaged based on relative distance between the ablation center and the ablation boundary (c). This average profile was subsequently used to create a custom ablation mask for each tumor slice (d), which was subtracted from the treated tumor fluorescence. Background subtracted fluorescence was subsequently converted using an empirical fluorescence relationship derived in Chapter 2
(e) to yield a DOX concentration map for each slice (f).
97 3.3 Results
3.3.1 Tumor treatment results
VX2 tumors were implanted in the middle liver lobe of 16 rabbits, 8 each in the control and treatment categories. At the time of treatment, one animal in the day 4 treatment group did not appear to have a tumor. In a second animal in the day 8 treatment group, the tumor was significantly adhered to the lateral peritoneal wall such that the tumor could not be safely ablated. These subjects were excluded from further study. The remaining 14 rabbits had tumors in the middle lobe that were treated with RF ablation followed by the implantation of a drug-free or doxorubicin-containing implant.
A summary of the treatment outcomes is shown in Table 3-1. Livers with viable tumor were found in 3 out of the 4 treatment groups. The presence of residual tumor cells in these livers was confirmed by histology. Viable tumor was not detected in any of the rabbits in the day 8 treatment group. Grossly, the treated tissues were all characterized by a single, spherical region of ablated tissue. Livers with residual cancer contained tumor nodules outside the region of ablation induced necrosis.
3.3.2 Tumor histology
Tissue histology was used to further assess the outcome of different treatment groups. Typical images of H&E stained tissue section from an RF ablated liver on day 8 are shown in Figure 3-3. Figure 3-3A is a low magnification overview of the entire treated region, showing the ablation treated region (dashed line) outside of which were two nodes of recurrent tumor (asterisks). A lighter staining region characterized by a
98 moderate inflammatory response, infiltrating fibroblasts, and mild collagen deposition was found circumscribing the ablation treated region. The ablated region demonstrated typical coagulative necrosis from RF ablation, and a greater magnification of this region is illustrated in Figure 3-3B. The outlines of pretreated cells were somewhat visible, but most cytoplasmic and nuclear details were lost. Necrotic debris and partially staining cells were contained throughout the area. In contrast, the tumor region outside the ablated zone shown in Figure 3-3C was filled with viable tumor cells which appeared relatively unaffected by the nearby ablation 8 days before. The cells in this region had all of the common characteristics of VX2 cells: dense packing, low differentiation, high nuclear to cytoplasm ratio, and the presence of mitotic figures. This pattern of recurrence, a treated region bounded by untreated areas of tumor, was found in all of the liver samples with incompletely treated tumors.
Closer inspection of the doxorubicin-treated slides is shown in Figure 3-4.
Masson’s trichrome (MTC) stained images are shown alongside fluorescent microcopy images from the same region. In the MTC stain on day 4 (Figure 3-4A), the boundary between ablated and normal tissue was subtle and characterized by the presence of inflammatory cells. Fluorescence from that region (Figure 3-4B) demonstrated doxorubicin fluorescence that gradually tapered across the ablation boundary. In contrast, by day 8 (Figure 3-4C) the boundary became much more evident and was clearly demarcated by a blue-staining boundary approximately 200 µm thick showing moderate collagen deposition. The corresponding fluorescent image (Figure 3-4D) shows doxorubicin in the ablated region dropping off abruptly at the ablation boundary.
99 Table 3-1. Summary of tumor treatment outcome after combined treatment with
RF ablation and a control or doxorubicin-containing millirod.
Livers with Ablation area Inflammatory Residual tumor viable tumor (cm2) area (cm2) area (cm2)
Control 1/3 0.89 ± 0.54 0.29 ± 0.04 0.89 Day 4 Treatment 2/4 0.71 ± 0.30 0.15 ± 0.14 2.11, 0.75
Control 1/4 0.81 ± 0.31 0.23 ± 0.10 0.57 Day 8 Treatment 0/3 0.82 ± 0.28 0.21 ± 0.07 n/a
100 Figure 3-3. Tumor progression after ablation.
Representative H&E histology of tumor progression after RF ablation in a day 8 control subject. (A) Overview of tumor region showing ablation treatment boundary (dashed line), two nodules of viable tumor (*), and regions magnified below. (B) High magnification image of ablation treated necrotic region. (C)
High magnification region of viable tumor cells untreated by the ablation.
Scale bars are 2 mm in (A) and 200 µm in (B) and (C).
101
Figure 3-4. Histology and doxorubicin fluorescence at the ablation boundary.
Masson’s trichrome stained images of the ablation boundary on day 4 (A) and day 8 (C).
Fluorescent microscopy images of the matching regions on day 4 (B) and day 8 (D).
Ablated tissue is on the right and normal tissue on the left of each slide, with the boundary marked by the black arrows. Scale bars are 200 µm.
102 3.3.3 Region quantification
To further quantify the effects of ablation alone and the combined treatment, areas of ablation necrosis, inflammatory tissue, and, when present, viable tumor, were calculated using regions of interest manually segmented on histology images. The results are shown in Table 3-1. The mean sizes of all 14 ablated regions and inflammatory regions were 0.80 ± 0.32 and 0.22 ± 0.10 cm2, respectively. There were no statistically significant differences found between any of the groups. The individual areas of recurrent tumors are also shown in the table. The day 8 treatment group did not have any samples with residual tumors, but drawing conclusions from such a small sample size may be very premature. From slides containing tumor, distances from the ablation center were calculated. In the day 4 tumors receiving RF ablation followed by doxorubicin- containing implants, the regions of viable tumor were identified circumscribing the ablation treated region as seen in Figure 3-5. In these slides, the two residual tumors began on average 4.1 mm from the ablation electrode location, 50% of tumor was found within 7.9 mm, and 100% of the tumor area was within 12.0 mm. These distances provided valuable information about what drug distribution would be necessary to limit tumor regrowth.
3.3.4 Drug release in vivo
Several values quantifying drug release in vivo are shown in Table 3-2.
Doxorubicin masses in the original implant, the extracted implant, and in the surrounding tissue as measured by fluorescence are shown along with the values as a percentage of the original doxorubicin loading. After 4 and 8 days, an insignificant amount of drug
103
Figure 3-5. Region quantification in two treated tumors with areas of viable tumor.
H&E histology of the doxorubicin implant treated tumors (day 4) which had areas of viable tumor (A, C). Segmented regions of ablation coagulated tissue (green), fibrous capsule formation (blue), and viable tumor (orange) are shown on corresponding morphological maps of the region (B,D). Scale bars are 5 mm.
104 Table 3-2. Summary of millirod implant and drug release properties.
Drug release characteristics Day 4 Day 8
Original drug loading (µg) 2990 ± 200 3080 ± 120
Drug remaining in extracted implant (µg) 190 ± 50 200 ± 30
Drug released (µg) 2800 ± 200 2880 ± 90
Drug released (% of original loading) 93.7 ± 1.6 93.6 ± 0.8
Drug in tissue (µg)* 590 ± 300 120 ± 100
Drug in tissue (% of original loading)* 20.4 ± 11.6 3.8 ± 3.0
Drug penetration distance (mm)* 3.7 ± 1.3 2.1 ± 0.3
Ablation region with [DOX] > 64 µg/g (% of area) 81.4 ± 13.8 39.9 ± 28.1
Percentages shown are based on the original drug loading. All values are shown ± standard deviation, and (*) indicates a p-value of < 0.1.
105 remained in the implants. The drug penetration distances, defined as the average distance from the implant at which the drug concentration dropped below 64 µg/g, were 3.7 mm on day 4 and 2.1 mm on day 8. In addition to critical drug concentrations being found
1.6 mm further from the implant on day 4, the tissue on day 4 retained significantly more of the original drug (20.4% of loading) than tissue on day 8 (3.8%). Apparent elimination rate from the tumor tissue and local half life were determined by fitting a decaying exponential of the form D(t) = D0·exp(-kt) to the known drug masses in tissue.
The elimination rate from the tumor area was k = 0.35 ± 0.02 day-1 and the apparent elimination half-time from tumor was of t1/2 = 2.0 ± 0.1 days.
3.3.5 Local drug distribution
Fluorescent imaging of tissue slices from the tumors revealed local drug concentrations in the extracted tissue. Two-dimensional doxorubicin distribution maps from these tissue sections are shown in Figure 3-6. Slices perpendicular to the long axis of the implant as well as parallel to the implant are shown on day 4 and day 8. Drug concentrations on day 4 exceeded 2000 µg/g and extended completely to the ablation boundary (Figure 3-6A). Day 8 tissue drug concentrations were characterized by lower drug concentrations (approximately 1000 µg/g) and a different distribution pattern
(Figure 3-6C). This pattern consisted of two regions of high drug concentration, within 2 mm of the implant boundary and a ring at the periphery of the ablated region, separated by a cleared zone containing much less drug. Slices parallel to the long axis of the implant show drug concentrations radiating from the cap of the cylindrical implant on
106
Figure 3-6. Doxorubicin concentration distribution maps in ablated tumors.
Day 4 slices through the tumor center perpendicular to the implant axis (A) and slices through the end of the tumor parallel to the implant (B). Corresponding slices through the center of the tumor (C) and the end of the tumor (D) on day 8. The white cylindrical outlines indicate the orientation of the implants in the parallel slices before removal.
Scale bars are 5 mm.
107
Figure 3-7. Average doxorubicin concentration profiles in ablated tumor tissue.
Doxorubicin concentrations are plotted against distance from the implant boundary for day 4 and day 8. Error bars represent the 95% confidence interval.
108 day 4 (Figure 3-6B) and day 8 (Figure 3-6D). These distributions had similar properties but featured slightly lower drug concentrations and a less conspicuous clearing between the implant and the ablation boundary. The drug did not accumulate substantially beyond the ablation boundary.
3.3.6 Drug distribution profiles
To quantitatively display the drug distribution data, drug concentrations were binned and averaged in 0.2 mm increments for all animals from each time point. The resulting radial distribution profiles are shown as a function of the implant boundary in
Figure 3-7. The figure numerically corroborated the observations from the 2D drug distributions. Drug concentrations were higher on day 4, when significant amounts of drug extended roughly to the ablation boundary found at approximately 4 mm. On day 8, average concentrations dipped to near 0 by 2 mm, but rose again slightly from drug observed at the ablation boundary. On average, significant amounts of doxorubicin did not extend beyond the ablation boundary.
3.4 Discussion
In this chapter, experimental tumors were treated by RF ablation supplemented with polymer millirods. To simulate a clinical situation in which ablation incompletely kills cells at the tumor periphery, VX2 tumors in rabbit liver were treated with an ablation intensity not expected to completely eradicate the tumors. Subsequently, doxorubicin millirods were placed into the center of the ablated tumor. Local drug distributions were
109 monitored, and therapeutic efficacy was compared against tumor treatment with RF ablation alone.
Measurements of local doxorubicin concentrations around the implants revealed elevated drug concentrations in the ablation treated region throughout the study. On day
4, more than 80% of the ablation coagulated zone contained drug at concentrations greater than 64 µg/g, or 10 times higher than the accepted therapeutic concentration.
Although this coverage dropped to 40% by day 8, drug concentrations as high as 1000
µg/g were still seen adjacent to the implant and just inside the ablation boundary.
Ablation of the tumor tissue facilitated the spread of drug from the implants, as the doxorubicin penetration distance (3.7 mm) and total doxorubicin contained in the tumor
(20.4%) at day 4 were substantially higher than the values reported for non-ablated tumors (2.8 mm, 6.3%) in Chapter 2. Furthermore, the apparent elimination half-life from ablated tumor (2.0 ± 0.1 days) was longer than previously found in nonablated tumors (1.6 ± 0.2 days), indicating that drug was eliminated more slowly from ablated tumor than from normal tumor.23 Slower elimination may have occurred because ablation is known to destroy tumor vasculature, halt mechanisms for drug metabolism, and increase drug binding to macromolecules.20 This data confirmed the expected advantage of using RF ablation to improve drug delivery efficiency from millirod implants inside VX2 tumors. Meanwhile, despite high drug concentrations within the ablated area, concentrations dropped steeply to undetectable levels outside the ablation boundary. Viable tumor cells were found beyond the ablation boundary in both groups on day 4 (1/3 control vs. 2/4 treatment) but only in the control group on day 8 (1/4 control vs. 0/3 treatment). While the results represent a possible improvement in the day
110 8 treatment group, concluding that the treatment is better than the control is limited by small group sizes and experimental variability among rabbits.
Detailed results from histological analysis of the treated tissue sections provided insight about possible limitations of this treatment strategy. Tumors recurred outside the ablation area, where doxorubicin was not seen in substantial amounts at either time point.
Doxorubicin transport to the cells outside the ablated region was limited by the formation of an inflammatory region around the ablated tissue. At day 4 this boundary consisted largely of neutrophils and monocytes typical of the chronic stages of inflammation, but by day 8 the boundary contained predominantly fibroblasts and moderate collagen deposition characteristic of fibrous capsule formation. Formation of this boundary was previously noted in ablated normal liver tissue, where it was noted as a potential barrier to drug transport.30 The inflammatory tissue could have impeded transport barrier two ways. First, fibrous capsule formation with collagen deposition may have created boundary that is denser and more tortuous than ablated tumor or normal liver, which could result in slower diffusion through this area. Second, the inflammatory boundary may have exhibited a higher rate of drug clearance and metabolism due to a high fraction of vascular tissue and large number of cells that may take up and metabolize drugs and debris. A combination of these two effects limited drug exposure outside the ablation boundary, where tumor cells were found at distances ranging from 4.0 to 12.0 mm away from the millirod location.
Overall, the potential benefit of combined therapy using RF ablation and chemotherapeutic implants was high despite some challenges. Doxorubicin infiltrated the tumor tissue to a greater distance and was retained in the tissue to a greater extent
111 than when these implants were used in tumors without ablation.23 Drug concentrations throughout the ablated region exceeded therapeutic values throughout the eight day study period. On the other hand, untreated tumor cells persisted outside the ablated area, where drug was not found in substantial quantities.
Identifying the role of fibrous capsule development in restricting doxorubicin delivery to tumor cells beyond the periphery of the ablated region was a key finding of this chapter. Future attempts at combining RF ablation with drug releasing implants must consider this challenge in their design. One way to address the fibrous capsule formation is to create implants incorporating an anti-inflammatory drug such as dexamethasone.
Dexamethasone has been shown to markedly reduce the formation of the fibrous capsule when released from implants after ablation31 and could be incorporated along with an anticancer drug such as doxorubicin.
The other factor limiting the effects of the combined treatment was the distance of the peripheral residual tumor from the implants. Improving drug coverage at this area tumor periphery can also be addressed through changes in treatment design. Previous work found that implants that have sustained drug release over several days provide greater drug coverage and penetration distances.26 However, since the fibrous boundary became progressively denser and less permeable to drug released at a later times, sustained drug release might not improve the treatment outcomes. Treatments targeted to the tumor periphery, such as nanoparticles targeted to tumor vasculature,32 may more effectively deliver drugs to the tumor cells most likely to survive ablation. One other option is to use peripheral placement of multiple polymer millirods to reduce the drug
112 transport distance to the viable tumor cells. Each of these approaches could maximize the therapeutic impact of local implants for tumor treatment in future studies.
Optimizing drug delivery to RF ablated tumors is the subject of the remaining chapters. In Chapter 4, a model of drug transport is used to estimate doxorubicin transport parameters in non-ablated and ablated tumor tissue and thereby gain a mechanistic understanding of rates limiting drug penetration. Then, this model is used to simulate the placement of multiple implants around the periphery of a tumor (Chapter 5).
Ultimately, the goal of these chapters is to improve on the current combined treatment to develop a more ideal treatment that can have an impact on treatment of human tumors.
3.5 References
1. Hande KR 1998. Clinical applications of anticancer drugs targeted to topoisomerase II. Biochimica et biophysica acta 1400(1-3):173-184.
2. Leung TW, Patt YZ, Lau WY, Ho SK, Yu SC, Chan AT, Mok TS, Yeo W, Liew CT, Leung NW, Tang AM, Johnson PJ 1999. Complete pathological remission is possible with systemic combination chemotherapy for inoperable hepatocellular carcinoma. Clin Cancer Res 5(7):1676-1681.
3. Little SA, Fong Y 2001. Hepatocellular carcinoma: current surgical management. Semin Oncol 28(5):474-486.
4. Guerin C, Olivi A, Weingart JD, Lawson HC, Brem H 2004. Recent advances in brain tumor therapy: local intracerebral drug delivery by polymers. Invest New Drugs 22(1):27-37.
5. Bentrem DJ, Dematteo RP, Blumgart LH 2005. Surgical therapy for metastatic disease to the liver. Annu Rev Med 56:139-156.
6. Leung TW, Johnson PJ 2001. Systemic therapy for hepatocellular carcinoma. Semin Oncol 28(5):514-520.
7. Livraghi T, Benedini V, Lazzaroni S, Meloni F, Torzilli G, Vettori C 1998. Long term results of single session percutaneous ethanol injection in patients with large hepatocellular carcinoma. Cancer 83(1):48-57.
113 8. Shah SS, Jacobs DL, Krasinkas AM, Furth EE, Itkin M, Clark TW 2004. Percutaneous ablation of VX2 carcinoma-induced liver tumors with use of ethanol versus acetic acid: pilot study in a rabbit model. J Vasc Interv Radiol 15(1 Pt 1):63-67.
9. Goldberg SN 2001. Radiofrequency tumor ablation: principles and techniques. Eur J Ultrasound 13(2):129-147.
10. Vogl TJ, Mack MG, Muller PK, Straub R, Engelmann K, Eichler K 1999. Interventional MR: interstitial therapy. Eur Radiol 9(8):1479-1487.
11. Ohmoto K, Tsuduki M, Shibata N, Takesue M, Kunieda T, Yamamoto S 1999. Percutaneous microwave coagulation therapy for hepatocellular carcinoma located on the surface of the liver. AJR Am J Roentgenol 173(5):1231-1233.
12. Garcea G, Lloyd TD, Aylott C, Maddern G, Berry DP 2003. The emergent role of focal liver ablation techniques in the treatment of primary and secondary liver tumours. Eur J Cancer 39(15):2150-2164.
13. Curley SA, Izzo F, Delrio P, Ellis LM, Granchi J, Vallone P, Fiore F, Pignata S, Daniele B, Cremona F 1999. Radiofrequency ablation of unresectable primary and metastatic hepatic malignancies: results in 123 patients. Ann Surg 230(1):1-8.
14. Goldberg SN, Saldinger PF, Gazelle GS, Huertas JC, Stuart KE, Jacobs T, Kruskal JB 2001. Percutaneous tumor ablation: increased necrosis with combined radio- frequency ablation and intratumoral doxorubicin injection in a rat breast tumor model. Radiology 220(2):420-427.
15. Buscarini E, Buscarini L 2004. Radiofrequency thermal ablation with expandable needle of focal liver malignancies: complication report. European Radiology 14(1):31 - 37.
16. Livraghi T, Goldberg SN, Monti F, Bizzini A, Lazzaroni S, Meloni F, Pellicano S, Solbiati L, Gazelle GS 1997. Saline-enhanced radio-frequency tissue ablation in the treatment of liver metastases. Radiology 202(1):205-210.
17. Ahmed M, Liu Z, Lukyanov AN, Signoretti S, Horkan C, Monsky WL, Torchilin VP, Goldberg SN 2005. Combination radiofrequency ablation with intratumoral liposomal doxorubicin: effect on drug accumulation and coagulation in multiple tissues and tumor types in animals. Radiology 235(2):469-477.
18. Ahmed M, Monsky WE, Girnun G, Lukyanov A, D'Ippolito G, Kruskal JB, Stuart KE, Torchilin VP, Goldberg SN 2003. Radiofrequency thermal ablation sharply increases intratumoral liposomal doxorubicin accumulation and tumor coagulation. Cancer Res 63(19):6327-6333.
19. Haaga JR, Exner AA, Wang Y, Stowe NT, Tarcha PJ 2005. Combined tumor therapy by using radiofrequency ablation and 5-FU-laden polymer implants: evaluation in rats and rabbits. Radiology 237(3):911-918.
114 20. Qian F, Stowe N, Liu EH, Saidel GM, Gao J 2003. Quantification of in vivo doxorubicin transport from PLGA millirods in thermoablated rat livers. J Control Release 91(1-2):157-166.
21. Szymanski-Exner A, Gallacher A, Stowe NT, Weinberg B, Haaga JR, Gao J 2003. Local carboplatin delivery and tissue distribution in livers after radiofrequency ablation. J Biomed Mater Res 67A(2):510-516.
22. Qian F, Szymanski A, Gao J 2001. Fabrication and characterization of controlled release poly(D,L-lactide-co-glycolide) millirods. J Biomed Mater Res 55(4):512-522.
23. Weinberg BD, Ai H, Blanco E, Anderson JM, Gao J 2007. Antitumor efficacy and local distribution of doxorubicin via intratumoral delivery from polymer millirods. J Biomed Mater Res A 81(1):161-170.
24. Hazle JD, Stafford RJ, Price RE 2002. Magnetic resonance imaging-guided focused ultrasound thermal therapy in experimental animal models: correlation of ablation volumes with pathology in rabbit muscle and VX2 tumors. J Magn Reson Imaging 15(2):185-194.
25. Miao Y, Ni Y, Mulier S, Yu J, De Wever I, Penninckx F, Baert AL, Marchal G 2000. Treatment of VX2 liver tumor in rabbits with "wet" electrode mediated radio- frequency ablation. Eur Radiol 10(1):188-194.
26. Qian F, Stowe N, Saidel GM, Gao J 2004. Comparison of doxorubicin concentration profiles in radiofrequency-ablated rat livers from sustained- and dual- release PLGA millirods. Pharm Res 21(3):394-399.
27. Breen MS, Lazebnik RS, Wilson DL 2005. Three-dimensional registration of magnetic resonance image data to histological sections with model-based evaluation. Ann Biomed Eng 33(8):1100-1112.
28. Ridge JA, Collin C, Bading JR, Hancock C, Conti PS, Daly JM, Raaf JH 1988. Increased adriamycin levels in hepatic implants of rabbit Vx-2 carcinoma from regional infusion. Cancer Res 48(16):4584-4587.
29. Swistel AJ, Bading JR, Raaf JH 1984. Intraarterial versus intravenous adriamycin in the rabbit Vx-2 tumor system. Cancer 53(6):1397-1404.
30. Blanco E, Qian F, Weinberg B, Stowe N, Anderson JM, Gao J 2004. Effect of fibrous capsule formation on doxorubicin distribution in radiofrequency ablated rat livers. J Biomed Mater Res A 69(3):398-406.
31. Blanco E, Weinberg BD, Stowe NT, Anderson JM, Gao J 2006. Local release of dexamethasone from polymer millirods effectively prevents fibrosis after radiofrequency ablation. J Biomed Mater Res A 76(1):174-182.
115 32. Nasongkla N, Shuai X, Ai H, Weinberg BD, Pink J, Boothman DA, Gao J 2004. cRGD-Functionalized Polymer Micelles for Targeted Doxorubicin Delivery. Angew Chem Int Ed Engl 43(46):6323-6327.
116 Chapter 4 Model-based estimation of drug distribution properties in non-ablated and ablated tumor tissue
4.1 Introduction
Optimization of drug delivery efficiency with polymer millirods has the potential to greatly improve the success of the intratumoral treatment regimen discussed in this thesis. While the previous chapters describe the therapeutic potential of the implants, they also show several limitations. The main challenge of the millirod treatment was the limited drug penetration distance into the surrounding tissue. Ablation partially alleviated this situation, but the increased drug penetration was still insufficient to completely treat the periphery of the experimental tumors larger than 1 cm in diameter.
Further improvements to the polymer millirods may be able to increase the therapeutic volume of tissue treated by the implants, thereby increasing the antitumor efficacy.
Although several changes to implant design have already been employed,1,2 how these changes would affect the anti-tumor efficacy of the implants is not obvious. One way to test improvements is to change implant formulations or treatment approaches and then test them in an animal model. This approach comes with a high cost in terms of time, money, and animal experiments. A more strategic method for improving millirod therapy is to develop a mechanistic understanding of the treatment process and its shortcomings.
Then, improvements to the treatment design can be made in a way that intelligently addresses the limitations.
One strategy to develop such a mechanistic understanding of millirod treatment is to implement a mathematical model of drug transport in the tissue surrounding the
117 implant. A precedent for using modeling to design drug delivery systems has already been established, particularly in the development of brain cancer therapies. Strasser et al. estimated an elimination/diffusion modulus to explain differences in transport of several different drugs away from polymers implanted in the brain.3 Wang et al. used model simulation to compare BCNU delivery in brain tumors by systemic injection or intratumoral ethylene-vinyl acetate implants.4 From model simulations, these authors concluded that local delivery provided higher tumor drug exposures than systemically administered drug and that surgical debulking of the tumor was a critical first step in local treatment. Computational modeling and imaging validation has been particularly important in understanding convection enhanced delivery (CED), in which a microcatheter is used to instill drugs into the center of a brain tumor.5,6 In CED, modeling has been used to optimize treatment parameters, such as infusion rate and pressure, to maximize the area treated by drug infusion. These insights might have been unavailable without the use of the model. Transport modeling has also been used to describe other local cancer treatments, such as intravesicular infusion, where a model established the value of using a drug pretreatment to increase the drug diffusion rate, and hence drug penetration, into bladder tumors.7 More generally, models have been used to predict drug accumulation in tumors after systemic injections, where they demonstrated the value of using nanoparticles over free drug.8 Valuable conclusions drawn from these works emphasize the importance of modeling to optimize drug delivery to solid tumors, where drug distribution is often limited by poor perfusion and limited diffusion distances.
Using a mathematical model to aid in polymer millirod design has several advantages over empirical experimental testing. First, measurement of doxorubicin
118 distribution rates in tumor tissue provides insight into differences between tissue types and cellular changes that result from RF ablation. This information allows for a better understanding of why a particular treatment may be effective and allows for better treatment design. Second, model simulation can be used to test a wide variety of treatments and minimize the number of animal subjects. Different drug release profiles, implant locations, or number of implants can be modeled, and only the best available treatments can be tested in animals. Third, drug distributions can be predicted in larger tumors that are a better approximation of clinical cases. Tumors in rat and rabbit models typically range from 0.5 – 2.0 cm in diameter, but human tumors that are difficult to ablate can have a diameter greater than 3.0 cm.9 By simulating treatments in these larger tumors, problems in treatment scale-up can be anticipated and addressed. These advantages make a distinct argument for using mathematical modeling to improve post- ablation drug delivery.
A model to describe local drug transport in the tissue surrounding polymer millirods has already been introduced in previous publications. Qian et al. introduced a mass transport model that described the radially outward transport of drug away from the polymer millirod surface using diffusion and elimination terms.2 This model could be analytically solved to determine drug release rates necessary to create a desired drug concentrations at the boundary of an ablated liver region.2 In a subsequent application of this model, doxorubicin diffusion and elimination parameters in non-ablated and ablated liver tissue were estimated, providing initial insights about how ablation influences local drug transport by dramatically decreasing drug elimination.10 Together, these studies demonstrated the feasibility of using a model to predict local drug delivery from polymer
119 millirods in non-ablated and ablated liver. However, the applicability of this model to transport in tumors has not been investigated.
The goal of this chapter is to extend the previously developed doxorubicin transport model to describe transport in non-ablated and ablated liver tumors. To accomplish this goal, two different implementations of a mass transport model incorporating doxorubicin diffusion and elimination in tumors was developed. First, a one-dimensional (1-D), cylindrically symmetric transport model was used to simulate intratumoral doxorubicin release from polymer millirods. Parameters of this doxorubicin transport model were estimated using nonlinear least-squares estimation to minimize the error between the model output and experimentally measured DOX concentration distributions. Second, a three-dimensional (3-D) implementation of the drug transport model was used to simulate treatment of larger tumors with RF ablation and polymer millirods. This implementation was used demonstrate how this model can be effective in predicting doxorubicin distributions in different tumor treatment scenarios. Results from this chapter provide essential information about doxorubicin transport and elimination in tumors and will ultimately be used to improve future intratumoral treatment with polymer millirods.
4.2 Methods
4.2.1 Implant fabrication
Implants were produced by a compression molding procedure described in more detail in Chapter 2.11 Briefly, a mixture of 65% poly(D,L-lactide-co-glycolide) (PLGA)
120 microspheres, 13.5% doxorubicin (DOX), and 21.5% NaCl (w/w) was mixed with a mortar and pestle, packed into a Teflon tube (1.6 mm outside diameter), and compressed with steel plungers (1.6 mm inside diameter) at 90º C for 2 hours. Final implants were cylindrical with an approximate diameter of 1.5 mm and length of 8.0 mm.
4.2.2 Non-ablated tumor treatment
Treatment of liver tumors in rabbits using doxorubicin-containing PLGA implants was described in Chapter 2 and reported in a previous publication.12 VX2 liver carcinomas (n = 16) were generated in rabbit livers by surgically implanting a small piece
(1 mm3) of frozen tumor tissue in the rabbit liver. The tumors were grown in the liver for
12 days until they reached an approximate diameter of 8 mm. After 12 days, the abdomen was surgically opened and a control or doxorubicin-containing implant was inserted into the center of the tumor and secured with a suture. The rabbits were euthanized 4 or 8 days after the onset of treatment, and the tumors were removed.
4.2.3 Ablated tumor treatment
Combined treatment of liver tumors in rabbits with RF ablation followed by doxorubicin-containing polymer implants was described in Chapter 3 and also reported in a previous publication.13 VX2 carcinomas (n = 16) were implanted as above but were allowed to grow for 18 days until they reached an approximate diameter of 11 mm. A
17-gauge ablation probe with a 1-cm exposed tip (Radionics, Burlington, MA) was placed into the center of the tumor. The tissue in contact with the tip was heated to 80ºC for 2 min, which created a coagulated region with a diameter of approximately 8 mm.
121 After ablation, a control or doxorubicin-containing millirod was placed into the electrode tract and secured with a suture. The rabbits were euthanized 4 or 8 days after the onset of treatment, and the tumors were removed.
4.2.4 Tumor assessment
Tumors removed from the surrounding liver tissue were sliced in half perpendicular to the major axis of the implant. Half of the tumor was fixed in formalin solution and the other half of the tumor was frozen at -20ºC. The fixed tissue was embedded in paraffin, sliced, and stained with hematoxylin and eosin (H&E) or Masson’s trichrome (MTC). Frozen liver sections 100 µm thick were sliced from each section using a cryostat microtome (Microm 505E) and then scanned with a fluorescent imager
(Molecular Dynamics Fluorimager SI). For ablated tissue sections, the ablation background was adjusted for using a background subtraction algorithm described in more detail in Chapter 3.13 Then, tumor net fluorescence intensity (NFI) was converted to doxorubicin concentration using a previously established relationship, NFI =
194·[DOX]0.67, where [DOX] is the doxorubicin concentration in µg/g.12 Radial drug distribution profiles as a function of distance from the implant center were determined by averaging four evenly spaced samples from the fluorescence image.
4.2.5 Drug transport model
Doxorubicin transport into tumor tissue from an implant device was analyzed by a dynamic mass balance transport model incorporating diffusion and elimination,10
C DCC 2 (4.1) t
122 where D and represent apparent diffusion and elimination coefficients, respectively.10
Drug elimination results from both perfusion and metabolism.
4.2.6 Boundary and initial conditions
The boundary conditions were chosen to approximate experimental measurements. At the inner implant boundary,
r RIB :() C f t (4.2) where f(t) is the time dependent concentration. At the outer normal tissue boundary,
r ROB :0 C (4.3) where ROB is the maximum extent of normal liver tissue included in the model. The concentration at this boundary was assumed to go to zero because of the elimination process. Initially, the tissue drug concentration was zero,
tC0: 0 (4.4) because there was no doxorubicin present in the tissue before the implants were placed.
Drug flux and concentration were assumed continuous at all interior boundaries.
4.2.7 One-dimensional (1-D) simulation strategy
Doxorubicin diffusion and elimination parameters were estimated for non-ablated and ablated tumor by implementing a 1-D, cylindrically symmetric simulation. For spherical tumors with a single cylindrical implant placed in the center, the dominant transport process occurs in the radial (r) direction, which can be described by a one- dimensional (1-D) model of the drug concentration distribution with time C(r,t),
123 CDC rC (4.5) t r r r
In both non-ablated and ablated tissue models, a single polymer implant (r = 0.8 mm) was located at the tumor center. In experimental studies of non-ablated VX2 liver tumors treated with a doxorubicin implant, the tumor radius began as 4 mm but decreased to 2.4 and 2.1 mm after 4 and 8 days, respectively, responding to the chemotherapeutic treatment. To account for the tumor shrinkage in the theoretical model, the boundary between tumor and normal liver, RTN, tissue was placed at a radius of 2.3 mm. In the ablated model, drug transport from a centrally located implant in ablated tumor was simulated using an ablated tissue radius, RAB, of 4.3 mm, corresponding to measurements in fresh tissue slices. The outer boundary of normal tissue, ROB, was placed at 10 mm in both non-ablated and ablated models.
The drug distributions from the 1-D transport models in non-ablated and ablated tumor were then solved. With this model, previously estimated values for the diffusion
-11 2 -1 -4 -1 coefficient (Dliver = 6.7 x 10 m s ) and elimination coefficient (γliver = 9.6 x 10 s ) in non-ablated, normal liver tissue were used.10 The cylindrically symmetric model was spatially discretized with a uniform mesh spacing of 0.1 mm to match the spacing of the experimental data. This system was then solved using a finite element method (FEM) implemented by COMSOL 3.3 (Burlington, MA). The simulated model output consisted of the doxorubicin concentration as a function of radius at the experimentally measured times, 4 and 8 days. Values for D and γ in non-ablated and ablated tumor tissue were determined by least-squares fitting of the model simulated drug concentration distributions to the experimental data. This step was implemented using the lsqcurvefit function of MATLAB 7.1 (Mathworks, Natick, MA). Initially, D and γ were assumed to
124 be independent of position and time. When the model did not yield a good fit to the experimental data, however, these coefficients were represented as functions of r and/or t based on an understanding of the underlying mechanisms. Since various functional forms were consistent with changes in these coefficients, the final choices were those for which the model output most closely fit the data and involved the fewest number of model parameters.
4.2.8 Three-dimensional (3-D) simulation strategy
To demonstrate that the estimated tissue parameters could be used more generally to determine drug concentrations throughout a tumor, treatment of tumors with a diameter of 2.0 cm were simulated in 3-D. This simulation phase considered two polymer millirods implant treatment strategies: (A) treatment of a non-ablated tumor (2.0 cm diam.) with a central implant; (B) treatment of a tumor (2.0 cm diam.) with ablation of 75% of the volume (1.8 cm diam.) and a central implant. Because these cases did not assume that transport was cylindrically symmetric, simulations were based on a 3-D drug transport model,
C 2C 2C 2C D C (4.6) t x2 y2 z2
Boundary and initial conditions were set as described above with the exception that RNT=
4 cm to allow for placement of larger tumors. Diffusion in the ablated region was scaled to the larger size by setting RAB at 0.9 cm and linearly scaling the spatial dependence of the diffusion parameter. Drug concentrations within the tumor were obtained using a 3-D
FEM solution implemented by COMSOL. The two drug distributions were compared by calculating average drug concentrations in the entire tumor and an outer tumor region
125 measuring 25% of the total tumor volume, which corresponds to the non-ablated region of geometry B. Therapies were further compared by determining the fractions of the tumor and risk volumes that were covered with greater than 2 times the known therapeutic value of doxorubicin in VX2 tumor, 6.4 µg/g.14,15
4.3 Results
4.3.1 Drug transport in non-ablated tumors
Rates of drug diffusion and elimination in non-ablated tumor were estimated from drug distribution profiles measured 4 and 8 days after implant placement. Drug transport was approximated using transport coefficients that did not vary as a function of time or position. Experimental drug measurements compared to model generated output are shown in Figure 4-1. The estimated value of doxorubicin diffusion throughout non-
-11 2 -1 ablated tumor, Dtumor, was calculated as 5.01 ± 0.32 x 10 m s . Doxorubicin
-4 -1 elimination, tumor, was calculated as 0.58 ± 0.04 x 10 s .
4.3.2 Drug transport in ablated tumors
Drug diffusion and elimination coefficients were estimated from ablated tumor drug distributions obtained 4 and 8 days after tumor ablation and implant placement. In ablated tumors, constant diffusion and elimination coefficients provided model output that varied considerably from experimental measurements (Figure 4-2). Using only constant parameters, the predicted concentration distribution did not yield a close fit to the experimental data; the model substantially overpredicted drug concentrations in the
126
Figure 4-1. Modeling results from parameter estimation in non-ablated tumor.
Experimental radial drug distributions are shown compared to model output on day 4 (A) and day 8 (B).
127
Figure 4-2. Parameter estimation in ablated tumors using constant parameters.
Experimental drug distributions are shown compared to model output on day 4 (A) and day 8 (B). Model simulations were performed using constant values for diffusion and elimination that did not change with time or radius. The model substantially overpredicted concentrations in the ablated tumor on day 8 (black arrow).
128
Figure 4-3. Modeling results from parameter estimation in ablated tumor.
Experimental radial drug distributions are shown compared to model output on day 4 (A) and day 8 (B).
129
Figure 4-4. Radial and time dependence of parameters in ablated tumor.
Graphs showing radial dependence of the doxorubicin diffusion coefficient, D (A), and time dependence of the elimination coefficient, (B), in ablated tumor.
130 outer region of the ablated tumor on day 8. These two findings suggest that constant model parameters were inadequate to simulate ablated tumor tissue. To more accurately predict drug transport in ablated tumors, diffusion and elimination coefficients that were a function of distance from the implant radius (r), time (t), or both were tested. Where possible, assumptions about the parameters were made based on a priori information, such as the physiology of ablation-induced damage and histological changes occurring after ablation, to minimize the number of parameters estimated.
Optimal model fits in ablated tumor (Figure 4-2) were obtained by using diffusion that varied as a function of r and elimination that varied as a function of t. The diffusion rate of doxorubicin in ablated tissue was obtained by setting the diffusion rate in the
center center of the ablated tumor (0.8 r < 2.0 mm) to a constant parameter, Dablated tumor , and allowing the parameter to vary linearly between and Dtumor in the outer portion of the ablated tumor (2.0 r < 4.3 mm),
rD 2 mm : center ablated tumor D (4.7) centerr 2 mm center r2 mm : Dablated tumor D ablated tumor D tumor 2 mm
In this equation, 2 mm was chosen as the distance for the change in the diffusion parameter function by testing several different locations and determining which model output most closely approximated the experimental data. Additionally, 4.3 mm was chosen as the outer boundary of the ablated region because this was the experimentally measured size of the ablation coagulated region. The piecewise linear function of D was constant for the 8 day period of the study. In contrast, the elimination coefficient gave the best model fit when it varied as a function of t and not r. The elimination coefficient
131 was set to a constant value of 0 for the first four days of the study, after which it was
day 8 allowed to vary linearly between 0 and elimination on day 8, ablated tumor ,
t 4 days : 0 t 4 days day8 (4.8) t 4 days : ablated tumor 4 days
This pattern of elimination was chosen based on the mechanism of tissue destruction due to RF ablation. Previous modeling work has shown that after ablation of normal tissue, the elimination rate is reduced to zero, a sensible finding because ablation destroys the living tissue and stops blood flow to the area. However, because ablation also induces a wound healing response that includes the formation of new blood vessels in the injured region, the elimination may begin to return several days after ablation. In this case, 4 days was chosen as the inflection point because experimental data showed that this was about the time when new blood vessel was formed after tissue injury.16
center Using this parameter structure, which is shown in Figure 4-4, Dablated tumor was estimated to be 8.76 ± 0.41 x 10-11 m2s-1, and was determined to be 0.57 ±
0.04 x 10-4 s-1. Parameter structures with greater parameter complexity, such as power and exponential growth, did not improve the quality of the model fits. Furthermore, functions that allowed each parameter to vary simultaneously as a function of t and r increased the number of parameters required and did not improve the model approximation.
132 4.3.3 Histology of ablated tumors
Histology of tumor tissues treated with RF ablation followed by polymer implants provided mechanistic information about the pattern of tissue destruction for comparison with quantitative transport information. H&E stained sections at the center and periphery of the ablated region on day 8 indicated some essential differences between tissues in these regions (Figure 4-5). Tissue approximately 1-2 mm from the ablation probe tip showed extensive necrosis and protein denaturation, with few visible nuclei or intact cell membranes. Damage to this region was extensive and indicative of high heat exposure.
At a distance of 3-4 mm from the ablation probe, a different structural pattern was seen.
Cells showed pallor, pyknotic nuclei, and shrunken cytoplasm while retaining much of their underlying morphology. This finding was consistent with tissues receiving a lower heat dose but loss of blood supply. This pattern provided a rationale for the choice of a diffusion parameter which varied with radius in the model. The structure of tissue in the center of the ablated region was more extensively destroyed, and this region corresponded to the location of ablated tissue which had a higher estimated diffusion rate in the model. Tumor around the periphery that was exposed to less heat was less severely damaged, and exhibited a diffusion rate that deviated less from normal tissue.
Comparative histology between 4 and 8 days also revealed time-dependent processes that take place in the ablated tissue (Figure 4-6). Ablated tumor after 4 days showed significant signs of coagulative necrosis, featuring numerous blood vessels filled with coagulated red blood cells, particularly around the ablation periphery. Lack of patent blood vessels in the day 4 tissue was compatible with the initial segment of the elimination parameter, which had no elimination. However, by day 8 the region revealed
133
Figure 4-5. Histology showing regional tissue variation in ablated tumor.
(A) H&E stained overview slide of ablated tumor tissue showing the relative location of the high magnification regions (b, c) with respect to the ablation probe (*). Tissue near the probe (B) has lost its cellular structure and cell membranes, while tissue 4 mm from the probe (C) contains dead cells with no nuclei but overall structure largely intact. Scale bars are 1 mm (A) and 100 µm (B, C). 134
Figure 4-6. Histology showing temporal tissue variation in ablated tumor.
Masson’s trichrome histology showing coagulated blood vessels (white arrows) in the ablated tumor region 4 days after ablation (A). By day 8 (B), new blood vessels (black arrow) are forming in the ablated region. Scale bars are 100 µm.
135 Figure 4-7. 3-D modeling of drug release in a 2.0 cm tumor.
Finite element mesh used to simulate the drug release (A). Simulated drug distributions 4 days after implant placement in a non-ablated (B) and ablated (C) tumor.
136 progression of the ablation-induced injury. In addition to granulation tissue found around the boundary, moderate amounts of new blood vessels were seen throughout the ablated region. This day 8 tissue section provided evidence supporting the assumption that elimination returns gradually between 4 and 8 days after ablation.
4.3.4 Three-dimensional (3-D) simulation of tumor transport
With the diffusion and elimination coefficients determined from the 1-D model analysis, a 3-D version of the model was applied to simulate treatment scenarios for a larger tumor. Drug distribution was simulated from 0 to 8 days after a doxorubicin millirod was centrally placed in a 2.0 cm diameter tumor: (A) without thermal ablation
(B) following RF ablation of 75% of the tumor volume (1.8 cm diameter). A sample finite element mesh and corresponding results for the two scenarios are shown in Figure
4-7. Over 8 days, the average drug concentrations in the entire tumor were 32 and 119
µg/g in the non-ablated and ablated case, respectively. When considering the outer rim of tumor within 1 mm of normal liver, average concentrations dropped to 0.2 µg/g in the non-ablated scenario or 17 µg/g in the ablated scenario. None of the tissue in the outer rim reached the therapeutic margin (13 µg/g, or 2x the known therapeutic value) without ablation, but ablation pretreatment increased coverage of the outer tumor to 80% of the outer rim volume. Finally, total drug released from the simulated implants in ablated tumor was 2.6 mg compared with 3.6 mg in non-ablated tumor.
4.4 Discussion
In this chapter, mass transport modeling was used to estimate doxorubicin transport in tumor tissues and then to simulate drug distribution in a larger tumor. In
137 addition to providing doxorubicin transport parameters, this work represents an advancement in methodology that can be used in future work. The finite element method
(FEM) of solving the model provided a flexible platform that had some advantages over an analytically solution, such as the ability to use arbitrary geometries or functions that are not easily expressed analytically. This flexibility allowed transport modeling with space and time variant parameters, representing a significant advancement in modeling drug transport in ablated tissues that had not been accomplished previously.
Additionally, future uses of this model can simulate drug transport in scenarios that are not symmetric or incorporate spatial data about a tumor into the model.
4.4.1 Estimation of doxorubicin transport properties in tumors
In the ablated tumor tissue, diffusion was best represented by a function of radius, while elimination was best represented by a function of time. Diffusion was a bulk property depending largely on the overall composition and structure of the tissue, which probably depended heavily on the initial ablation tissue damage that resolved slowly over time. In contrast, because the heat exposure was sufficient to coagulate all of the blood vessels, the initial elimination rate was negligible throughout the ablation region. In the days following ablation, the inflammatory response led to the formation of new blood vessels in and around the ablated tumor, in turn leading to a return of elimination as a function of time. These few small blood vessels exerted a sizeable effect on drug elimination without affecting the bulk tissue properties because they comprised a small fraction of the ablated volume. Choosing the diffusion rate coefficient as a function of
138 position and elimination coefficient as a function of time allowed for effective simulation of drug distribution in ablated tumors.
Simulation of drug transport using a mass balance model allowed for reasonable approximation of drug release from doxorubicin millirods placed in tumors. Simulation combined with nonlinear parameter estimation was used to estimate doxorubicin transport rates in these tissues, which are shown compared to previous estimates from liver tissue in Figure 4-8. Parameter values for non-ablated and normal liver tissue were taken from a previous study by Qian et al. in which parameters were calculated from drug distribution data measured from 1 hour to 4 days.10 The estimated value for diffusion in non-ablated liver tumors was 25% lower than diffusion in non-ablated normal liver tissue. This may have occurred because tumors are often comprised of dense tissue with high cellularity, elevated collagen content, and a tortuous extracellular matrix, all of which can act as a barrier to drug transport.17,18 The elimination rate in tumor was 6% of the value in normal liver, indicating that doxorubicin was removed from tumor at a drastically lower rate. Normal liver parenchyma is heavily populated with blood vessels, and hepatocytes may have high enzymatic activity for drug elimination. Additionally, the
VX2 tumors used in this study may have a lower blood vessel density and may metabolize drug more slowly than normal liver.
After ablation, diffusion at the center of the tumor region was increased by 75% over non-ablated tumor. This finding is in contrast to the previous results showing that ablation decreased the diffusion coefficient in normal liver.10 However, at least one study
139
Figure 4-8. Comparison of transport parameter in different tissue types.
Diffusion, D, (top) and elimination, , (bottom) coefficients of liver and VX2 tumor tissue before and after ablation. Error bars show the standard error of the estimated value. (*) denotes that the values for normal liver tissue were taken from previous work by Qian, et al.10
140 has demonstrated that drug diffusion rates could be increased by using an apoptosis- inducing drug pretreatment to reduce barriers to drug transit.7 Analogously, RF ablation may increase the rate of doxorubicin diffusion by disrupting tumor morphology and cellular structure. Changes in the diffusion value did not extend to the ablation periphery, which had the same diffusion value as non-ablated tumor. This finding suggests that RF- induced structural damage to the tumor may not be uniform throughout the tumor, with higher damage found at the center of the coagulated region. Histology corroborated the pattern of more extensive cell damage at the center of the ablation region. The temperature distribution during ablation, with high temperatures near the ablation probe that gradually decrease to normal at the ablation periphery,19 can explain this fundamental difference. Other published reports of ablation-induced damage support this finding that such damage is not homogeneous throughout the ablated region.20,21 RF ablation also reduced tumor drug elimination to zero for the first four days after treatment, consistent with previous results in ablated liver tissue. However, elimination in the ablated tumor increased as a function of time between days 4 and 8, which was not observed in the previous study that stopped at day 4. The increase in elimination between days 4 and 8 could result from the host inflammatory response to the ablation, which resulted in infiltration of inflammatory cells and formation of new blood vessels in the ablated region.
Three-dimensional simulation of a larger, more clinically relevant tumor treated locally with a doxorubicin-containing implant without or following RF thermal ablation demonstrated the ability to evaluate 3-D scenarios based on their expected drug distributions. First, the feasibility of using parameters from a 1-D model simulation for
141 application in a 3-D simulation of a larger tumor was established. Because it was solved using a finite element method, this 3-D simulation strategy could be implemented in situations that are not symmetric, such as when multiple implants are placed around the periphery of a tumor. Second, the simulation reaffirmed previous findings about using ablation before implant placement. Ablation drastically decreased drug elimination in the tumor region, which had the most pronounced effects on drug retention in the peripheral tumor region. While experimentally observed previously, this effect appeared more pronounced in the larger simulated tumors. Furthermore, RF ablation allowed for significantly slower drug release from the implant, depleting the implant more slowly and maintaining a therapeutic level, especially at the tumor periphery, for a longer period.
These simulations provide the basis for designing future animal experiments to confirm these conclusions and validate the extension of this model into a larger tumor.
This computational model provided a feasible means of estimating drug distribution dynamics following placement of an intratumoral chemotherapeutic implant.
RF ablation was shown to facilitate intratumoral drug delivery in tissue not only by reducing normal elimination processes but also by increasing diffusion. Extension of parameter estimates from a 1-D model into a 3-D simulation further demonstrated the benefits of ablation in conjunction with drug delivery. The computational modeling approach indicates the advantages of using simulation to design and rapidly prototype new implant treatment strategies.
The model and parameters estimated in this chapter can be used to optimize treatments using ablation and polymer implants. In the next chapter, this model is used to predict how multiple polymer implants placed in a single tumor can be used to maximize
142 drug delivery to the tumor periphery. Additionally, the model could be used in the future to anticipate how other changes to implant design, such as modifying the drug release rate or including dexamethasone to moderate the inflammatory response after ablation,22 can affect drug distribution in the tumors. This computational strategy should allow for rapid development and prototyping of different implant designs that can optimally treat larger, more clinically relevant tumor models. Ultimately, this drug transport model may be used as part of a comprehensive treatment planning tool. After acquiring imaging data about tumor geometry, ablation treatment could be planned using a thermal damage model, and drug coverage in the ablated tumor could be predicted using this 3-D finite element model.
4.5 References
1. Qian F, Nasongkla N, Gao J. 2002. Membrane-encased polymer millirods for sustained release of 5-fluorouracil. J Biomed Mater Res 61(2):203-211.
2. Qian F, Saidel GM, Sutton DM, Exner A, Gao J. 2002. Combined modeling and experimental approach for the development of dual-release polymer millirods. J Control Release 83(3):427-435.
3. Strasser JF, Fung LK, Eller S, Grossman SA, Saltzman WM. 1995. Distribution of 1,3-bis(2-chloroethyl)-1-nitrosourea and tracers in the rabbit brain after interstitial delivery by biodegradable polymer implants. J Pharmacol Exp Ther 275(3):1647-1655.
4. Wang CC, Li J, Teo CS, Lee T. 1999. The delivery of BCNU to brain tumors. J Control Release 61(1-2):21-41.
5. Kalyanasundaram S, Calhoun VD, Leong KW. 1997. A finite element model for predicting the distribution of drugs delivered intracranially to the brain. Am J Physiol 273(5 Pt 2):R1810-1821.
6. Raghavan R, Brady ML, Rodriguez-Ponce MI, Hartlep A, Pedain C, Sampson JH. 2006. Convection-enhanced delivery of therapeutics for brain disease, and its optimization. Neurosurg Focus 20(4):E12.
143 7. Au JL, Jang SH, Wientjes MG. 2002. Clinical aspects of drug delivery to tumors. J Control Release 78(1-3):81-95.
8. Sinek J, Frieboes H, Zheng X, Cristini V. 2004. Two-dimensional chemotherapy simulations demonstrate fundamental transport and tumor response limitations involving nanoparticles. Biomed Microdevices 6(4):297-309.
9. Solbiati L, Ierace T, Tonolini M, Osti V, Cova L. 2001. Radiofrequency thermal ablation of hepatic metastases. Eur J Ultrasound 13(2):149-158.
10. Qian F, Stowe N, Liu EH, Saidel GM, Gao J. 2003. Quantification of in vivo doxorubicin transport from PLGA millirods in thermoablated rat livers. J Control Release 91(1-2):157-166.
11. Qian F, Szymanski A, Gao J. 2001. Fabrication and characterization of controlled release poly(D,L-lactide-co-glycolide) millirods. J Biomed Mater Res 55(4):512-522.
12. Weinberg BD, Ai H, Blanco E, Anderson JM, Gao J. 2007. Antitumor efficacy and local distribution of doxorubicin via intratumoral delivery from polymer millirods. J Biomed Mater Res A 81(1):161-170.
13. Weinberg BD, Blanco E, Lempka SF, Anderson JM, Exner AA, Gao J. 2007. Combined radiofrequency ablation and doxorubicin-eluting polymer implants for liver cancer treatment. J Biomed Mater Res A 81(1):205-213.
14. Ridge JA, Collin C, Bading JR, Hancock C, Conti PS, Daly JM, Raaf JH. 1988. Increased adriamycin levels in hepatic implants of rabbit Vx-2 carcinoma from regional infusion. Cancer Res 48(16):4584-4587.
15. Swistel AJ, Bading JR, Raaf JH. 1984. Intraarterial versus intravenous adriamycin in the rabbit Vx-2 tumor system. Cancer 53(6):1397-1404.
16. Anderson JM. 1993. Mechanisms of inflammation and infection with implanted devices. Cardiovascular Pathology 2(3, Supplement 1):33-41.
17. McGuire S, Zaharoff D, Yuan F. 2006. Nonlinear dependence of hydraulic conductivity on tissue deformation during intratumoral infusion. Ann Biomed Eng 34(7):1173-1181.
18. Netti PA, Berk DA, Swartz MA, Grodzinsky AJ, Jain RK. 2000. Role of extracellular matrix assembly in interstitial transport in solid tumors. Cancer Res 60(9):2497-2503.
19. Johnson PC, Saidel GM. 2002. Thermal model for fast simulation during magnetic resonance imaging guidance of radio frequency tumor ablation. Ann Biomed Eng 30(9):1152-1161.
144 20. Coad JE, Kosari K, Humar A, Sielaff TD. 2003. Radiofrequency ablation causes 'thermal fixation' of hepatocellular carcinoma: a post-liver transplant histopathologic study. Clinical transplantation 17(4):377-384.
21. Nikfarjam M, Muralidharan V, Christophi C. 2005. Mechanisms of focal heat destruction of liver tumors. The Journal of surgical research 127(2):208-223.
22. Blanco E, Weinberg BD, Stowe NT, Anderson JM, Gao J. 2006. Local release of dexamethasone from polymer millirods effectively prevents fibrosis after radiofrequency ablation. J Biomed Mater Res A 76(1):174-182.
145 Chapter 5 Simulation of drug distribution in tumors to optimize complex tumor treatments
5.1 Introduction
In order to develop polymer millirods into a treatment strategy that is applicable to a wide range of tumors, the volume of tissue which can be successfully treated by the implants must be improved. In Chapter 2, it was demonstrated that the implants could treat a region with a radius of 2-3 mm, a distance too small to be applicable to human hepatocellular carcinomas, 80% of which are greater than 2 cm in diameter at the time of treatment.1 Combining polymer millirods with radiofrequency (RF) ablation improved the extent of the treatment to 4 mm (Chapter 3), a significant improvement that nonetheless falls short of the target. In order for polymer millirods to be relevant to treatment of human tumors, an effective approach to enlarging the treatment radius must be devised. Chapter 4 introduced a drug transport model that could be used not only to estimate doxorubicin transport rates in tissue but also to simulate drug distributions in different scenarios. Using this tool, changes to the millirod treatment strategy can be planned and tested.
One strategy to increase the volume of tumor covered by drug is to use multiple polymer millirods placed in a single ablated tumor. Using multiple implants to increase the volume of treated tissue has a number of potential advantages. First, using multiple implants increases the total amount of drug delivered to the tumor without drastically changing the systemic drug exposure. Second, multiple implants in a single tumor decrease the average distance between implants and the target tissue at the ablated region
146 periphery. Unlike increasing the drug loading of a single implant, segmenting a dose into several implants offers a targeting advantage by delivering more drug to regions that need a greater dose. These advantages make a multiple implant strategy a desirable direction of research.
Another important consideration for a multiple implant strategy is practicality and ease of administration. Previous work suggests that using multiple polymer millirods in a single tumor is reasonable. Large liver tumors are already often treated by multiple percutaneous RF applications in which the RF probe is repositioned after each application.2 In these cases, an implant could be inserted after each RF application. In other instances, RF is applied laparoscopically3 or as part of an open surgical procedure,4 providing ample opportunity for placement of multiple implants. Use of multiple drug administrations has also been used in several other intratumoral treatment designs.
Gliadel wafers, the BCNU-impregnated polyanhydride implant used for treating gliomas, is used by surgically placing several wafers into the resection cavity.5 In France, clinical trials have tested stereotactic injection of 5-fluorouracil (5-FU) containing microspheres into brain tumors, using up to 5 injection tracks per patient.6,7 Finally, placement of multiple radioactive seeds (brachytherapy) is now the standard of care in prostate cancer.8
These precedents indicate that placing multiple polymer millirods in a single tumor is feasible.
The first step in designing a multiple implant strategy for using polymer millirods is to simulate drug distributions using the drug transport model developed in Chapter 4.
The drug transport model can be used to predict drug exposure throughout the tumor, analogous to using computational tools to plan tumor radiation doses during prostate
147 brachytherapy.9,10 The main advantage of using the model is that drug exposures can be rapidly evaluated without animal experiments. Using the predicted drug concentrations, implant configurations can be compared by different measurements of treatment quality.
One measure of treatment quality is the average drug concentration during the evaluation period. This value provides an overall measure of drug exposure to the entire tumor over time. Another valuable measure is the fraction of the tumor covered with a therapeutic concentration of drug, as well as how long the tumor is covered completely with drug.
These measures provide information about what fraction of the tumor is likely to be killed by the drug, as well as if the drug is likely to eradicate the entire tumor.
Additionally, the model can be used to evaluate drug exposures in different regions of the tumor. During RF ablation, not all areas of the tumor are exposed to the same thermal dose, and parts of the tumor near the periphery of the ablated region may be exposed to temperatures at the low end of the lethal dose.11 For this reason, RF ablated tumors are more likely to recur around the tumor periphery if an adequate margin of tissue around the tumor is not ablated.12 Drug concentrations in this region are likely to be more important than those in the central core of the tumor. To account for this in model predictions, the periphery of the tumor can be defined as a risk volume where tumors are more likely to recur. Then, drug concentrations generated by a treatment can be assessed by comparing average drug concentrations and therapeutic coverage in the risk volume only. Comparing treatments in this way emphasizes the importance of delivering drug to the tumor periphery more than the tumor core.
This chapter describes the development of a multiple polymer millirod treatment strategy using a drug transport model and its subsequent animal validation. The first goal
148 of the chapter is to establish a 3-D finite element method for simulating local drug concentrations in ablated tumors. Then, 3-D modeling results are used to compare several potential multiple millirod treatments for a tumor with a 2 cm diameter. Finally, animal experiments using the VX2 liver cancer model in rabbits are used to validate the model conclusions.
5.2 Methods
5.2.1 Drug distribution model
Doxorubicin transport from polymer millirods was simulated with a dynamic mass balance transport model incorporating diffusion and elimination,
C DCC 2 (5.1) t where D and represent apparent diffusion and elimination coefficients, respectively.13
Drug elimination results from both perfusion and metabolism. This model was implemented in three dimensions (3-D) using the Cartesian form of the equation,
C 2C 2C 2C D C (5.2) t x2 y2 z2
Three-dimensional models of tested scenarios were discretized using the fine mesh setting of COMSOL 3.3 (Burlington, MA) to generate approximately 80,000 nodes per model. The resulting system was then solved using the finite element solver of
COMSOL to obtain drug concentrations from 0 to 8 days.
149
Figure 5-1. Multiple implant scenarios simulated using 3-D model.
Ten different multiple implant treatments for a 2.0 cm diameter tumor with a 1.8 cm diameter ablated tumor core were tested. Total doxorubicin doses are shown below each design. Two scenarios used only a single central implant measuring either 8 mm (A) or
16 mm (F) in length. Four of the tested scenarios used only peripherally placed, 8 mm implants (B-E), while the remaining four combined peripheral 8 mm implants with a single 16 mm implant in the center (G-J).
150 5.2.2 Scenarios tested
To evaluate the drug distributions from a multiple implant treatment strategy, two different tissue geometries were simulated. In the first, a 1.8 cm diameter ablated liver tissue region was placed in the center of non-ablated liver extending to a radius of 4 cm.
This scenario was performed to emulate placement of implants in an ablated rabbit liver for model validation. An ablated tumor geometry was also simulated. This geometry consisted of a 2.0 cm diameter tumor in which a central sphere measuring 1.8 cm in diameter had been treated with RF ablation. This case was designed to mimic the situation in which a tumor has been incompletely ablated, leaving a peripheral rim of untreated tumor. This geometry was used as the basis for comparing the multiple implant tumor treatments.
A battery of multiple implant scenarios as shown in Figure 5-1 was then tested on the ablated tumor geometry. Two scenarios used only a central implant (center), four scenarios used only peripheral implants (peripheral), and the remaining four scenarios used peripheral implants combined with a longer central implant (length = 16 mm). All peripheral implants had a length of 8 mm and were placed at a radius of 7 mm from the center of the ablated region. Total doxorubicin doses for these scenarios ranged from 3.4 to 23.6 mg, whereas for a typical intravenously administered therapeutic dose scaled to a rabbit would be 7.9-14.8 mg.14 All simulated doses were well below the estimated lethal dose (LD50) of 63.3 mg.15
151 5.2.3 Boundary and initial conditions
Boundary conditions for the 3-D simulations were chosen to approximate experimental measurements. At the implant surfaces, the inner boundary of the model,
r RIB :() C f t (5.3) where f(t) is the time dependent concentration. For all simulations, this time dependent concentration was obtained from experimental measurements taken in Chapter 3 for implants placed in ablated tumors. At the outer normal tissue boundary,
r ROB :0 C (5.4) where ROB is the maximum extent of normal liver tissue included in the model. The concentration at this boundary was assumed to go to zero because of the elimination process. Initially, the tissue drug concentration was zero,
tC0: 0 (5.5) because there was no doxorubicin present in the tissue before the implants were placed.
Drug flux and concentration were assumed continuous at all interior boundaries.
5.2.4 Tissue parameters
Drug transport parameters in the simulated tissues were set using previously estimated parameter values. Doxorubicin diffusion and elimination rates in nonablated and ablated liver tissue were estimated by Qian et al. in a published work.13 Values for
-11 2 -1 -4 -1 these parameters were: Dliver = 6.7 x 10 m s , liver = 9.6 x 10 s , Dablated liver = 1.1 x
-11 2 -1 -1 10 m s , and ablated liver = 0 s . Estimation of doxorubicin transit rates in tumor was
-11 2 -1 described in Chapter 4. In non-ablated tumor, Dtumor = 5.0 x 10 m s and tumor = 0.6 x
10-4 s-1 s-1. In ablated tumors, time and radially dependent values of D and γ were used as
152 described in Chapter 4. Spatial variation of diffusion was linearly scaled up to the larger ablation radius,
rD 4.2 mm : center ablated tumor D (5.6) ablated tumor centerr 4.2 mm center 4.2 mm < r < 9 mm : DDDablated tumor ablated tumor tumor 4.2 mm
This piecewise linear function for Dablated tumor was constant for the 8 day period of the study. In contrast, elimination varied as a function of time but did not vary spatially, so it did not require scaling,
t 4 days : 0 ablated tumor t 4 days day8 (5.7) t 4 days : ablated tumor 4 days
center day 8 -11 Values of the parameter constants, Dablated tumor and ablated tumor , were 8.76 ± 0.41 x 10 m2s-1 and 0.57 ± 0.04 x 10-4 s-1, respectively.
5.2.5 Evaluation of simulated drug distributions
Drug distributions were compared by calculating average drug concentrations and therapeutic coverage of doxorubicin. For these calculations, the tumor was divided into two regions, and inner core (consisting of 75% of the total volume) and an outer risk volume (outer 25% of tumor volume) as shown in Figure 2. Average drug concentrations were then calculated by integrating drug concentrations over the whole tumor volume, or alternatively, just the risk volume, and time simulated (8 days) and then dividing by the total volume and total time. Therapies were further compared by determining the fractions of the whole tumor and risk volume that were covered with drug concentrations
153
Figure 5-2. Evaluation of simulated tumors
Simulated tumors were evaluated by determining the average drug concentration in the entire tumor as well in an outer risk volume, comprised of the outer 25% of the tumor volume. This outermost 25% of the tumor (red) is likely to be at greater risk of tumor recurrence than the inner tumor volume (gray) because of lower heat exposures at the periphery.
154 greater than a therapeutic target concentration. This therapeutic target was set at 12.8
µg/g, or 2 times the known therapeutic value of doxorubicin in VX2 tumor, 6.4 µg/g.16,17
This value was chosen to ensure that all areas denoted covered received at least twice the previously estimated therapeutic concentration of DOX.
5.2.6 Manufacture of polymer implants
Doxorubicin-containing millirods were produced by compression molding of 65% poly(D,L-lactide-co-glycolide) (PLGA) microspheres, 13.5% doxorubicin (DOX), and
21.5% NaCl (w/w) using the procedure described in Chapter 2.18 The mixture was blended with a mortar and pestle, packed into a Teflon tube (1.6 mm outside diameter), and compressed with steel plungers (1.6 mm inside diameter) at 90º C for 2 hours. Final implants were cylindrical with an approximate diameter of 1.5 mm and length of 8.0 mm.
5.2.7 Animal model and treatment
One multiple implant scenario selected from the simulated scenarios was tested both in ablated liver tissue and ablated liver tumors. To test in ablated liver, the abdomens of NZW rabbits (n = 2) were opened just below the sternum. The liver was gently exposed and lifted, and a 17-gauge, 1 cm exposed tip ablation probe (Radionics,
Burlington, MA) was inserted into the middle lobe of the liver. Tissue in contact with the tip was heated to a temperature of 90ºC for 9 minutes. After ablation, polymer millirods were inserted into the ablated region in the pattern selected. If necessary, implants were sutured into place using a small piece of resorbable gelatin foam. The abdomen was then sutured closed in 3 layers.
155 Combined treatment of rabbit liver tumors with RF ablation and doxorubicin millirods was performed largely as described in Chapter 3 and also reported in a previous publication.19 VX2 carcinomas (n = 2) were implanted in the liver and were allowed to grow for 28 days until they reached an approximate diameter of 2.0 cm. After 28 days, the abdomen was reopened, and a 17-gauge ablation probe with a 2-cm exposed tip
(Radionics) was placed into the center of the tumor. The tissue was heated and maintained at a temperature of 90ºC for 9 minutes to ablate a sphere with a diameter of approximately 1.8 cm in the center of the tumor. After ablation doxorubicin-containing millirods were placed into the ablated tumor. In each of the groups, one of the rabbits was euthanized at each time point, 4 and 8 days after ablation.
5.2.8 Tumor evaluation
Tumors were removed from the surrounding liver tissue and sliced in half parallel to the front surface of the liver. Half of the tumor was fixed in formalin solution and the other half of the tumor was frozen at -20ºC. The fixed tissue was embedded in paraffin, sliced, and stained with hematoxylin and eosin (H&E) or Masson’s trichrome (MTC).
Frozen liver sections 100 µm thick were sliced from each section using a cryostat microtome (Leica CM3050S) and then scanned with a fluorescent imager (Molecular
Dynamics Fluorimager SI). In each section, the ablation background was subtracted using a background subtraction algorithm described in in Chapter 3.19 Tumor net fluorescence intensity (NFI) was converted to doxorubicin concentration using a the empirical relationship, NFI = 194·[DOX]0.67, where [DOX] is the doxorubicin concentration in µg/g.20
156 5.3 Results
5.3.1 Simulated drug distributions using multiple implants
To determine if using multiple implants provided an advantage over a single implant in treating incompletely ablated tumors, a comparison was made between using 1 central implant and 4 peripheral implants. Drug distributions determined from model output for the two scenarios are shown in Figure 5-3. On day 4, drug concentrations in the ablated tissue were high in both scenarios. However, doxorubicin content of the non- ablated tumor rim, or risk volume, was much higher in the multiple implant case. This advantage continued to day 8, when it was more evident because of decreasing drug concentrations in the single implant case. Over the 8 day period, the average drug concentration in the whole tumor was 119 µg/g using 1 implant and 290 µg/g using 4 peripheral implants. When considering only the risk volume, average concentrations over 8 days were 16.9 and 99.3 µg/g for the two scenarios. Much of this advantage was expected, as the total dose of doxorubicin had been increased by a multiple of 4.
However, the risk volume exposure increased roughly 6 fold, indicating that repositioning the implants offered more than a dose dependent increase in drug delivery to the periphery. Additionally, on day 4 100% of the risk volume was covered by the therapeutic target concentration with 4 implants, compared to only 78% of the risk volume covered using a single implant.
157 5.3.2 Quantitative comparison of multiple implant configurations
Multiple implant strategies were quantitatively compared using measures of average concentration and time in which 100% of the risk volume was above the target concentration. Graphs of average drug concentration in the whole tumor are plotted as a function of total doxorubicin dose in Figure 5-4. Strategies were divided into three categories, center, peripheral, or peripheral + center, as described in Section 5.2.2.
Increasing the number of implants, and hence the total doxorubicin dose, almost linearly increased the whole tumor’s average DOX exposure. This effect was largely independent of implant configuration. On the other hand, concentrations in the risk volume depended on the implant distribution in the tumor. For the same DOX dose, peripheral configurations added approximately 20 µg/g to the average risk volume DOX concentration over the equivalent peripheral + center configurations.
Implant configurations were also compared based on the amount of time the risk volume was 100% covered with DOX concentrations greater than the target concentration (Figure 5-5). Central only configurations never reached 100% coverage of the risk volume, nor did the peripheral configuration with only 2 millirods. All configurations containing 10 mg or more of doxorubicin achieved 100% coverage, and the length of time 100% coverage rose with increasing doxorubicin dose. Little difference was seen between peripheral and peripheral + center treatment strategies.
Configurations were also compared based on the speed with which they reached 100% coverage. Lower times are more desirable because the cells around the periphery have had less time to recover from their sublethal hyperthermia exposure and may have a
158
Figure 5-3. One versus multiple implants in a single tumor, model output.
Simulated drug concentrations from using a single implant in the center (A, C) or 4 implants spaced around the periphery (B,D) of a 2.0 cm diameter tumor with the inner 1.8 cm ablated. Concentrations are shown on day 4 (A, B) and day 8 (C, D).
159
Figure 5-4. Simulated drug concentrations in whole tumor and risk volume.
Average doxorubicin concentration over the entire 8 day simulated period for the whole tumor (A) or the outer risk volume only (B) for the three different treatment strategies: center implants, peripheral implants only, or peripheral + center implants. Results are plotted as a function of doxorubicin dose, and numerals on the graph indicate the number of peripheral implants used in the treatment.
160
Figure 5-5. Total time of and time to reach 100% coverage of risk volume.
Simulated length of time that the risk volume was 100% covered with drug concentrations above 12.8 µg/g (A) and time required for the risk volume to reach 100% drug coverage (B) plotted against total doxorubicin dose for the three treatment strategies. The center implant strategy is not shown in (B) because the risk volume never reaches 100% coverage. Numerals on the graph indicate the number of peripheral implants used in the treatment.
161
Figure 5-6. Simulated coverage from two configurations with a 13.5 mg DOX dose.
Simulated drug concentrations on day 8 of two equivalent doxorubicin doses, one with four peripheral implants (length = 8 mm) (A) and one with a central implant (length = 16 mm) and two peripheral implants (length = 8 mm) (B). Isosurfaces at 12.8 µg/g for the same treatments (C, D) show the regions covered with drug concentrations higher than the therapeutic target on day 8.
162 decreased tolerance to doxorubicin. Again, increasing the number of implants lowered the time to reach 100% coverage. At high doses of DOX, the peripheral + center strategy began to offer an advantage, primarily because it increased the speed at which drug was delivered to the north and south poles of the tumor. Otherwise, all therapies that reached
100% coverage did so within a range of 11-68 hours,
5.3.3 Comparison of two configurations with equivalent doxorubicin dose
Two configurations with equivalent total DOX doses of 13.5 mg were simulated to provide a qualitative assessment of how the two treatments differ. One of the treatments consisted of 4 peripherally placed implants 8 mm in length, while the other treatment consisted of 2 peripherally placed 8 mm implants and a central implant 16 mm in length. Simulated drug distributions from these scenarios are shown in Figure 5-6. As expected, the peripheral implants provided strong coverage of the tumor equator with less coverage at the poles of the tumor. The strength of the peripheral + center strategy was good coverage of the poles of the tumor with less drug exposure to two of the sides of the tumor.
5.3.4 In vivo treatment of ablated liver with multiple polymer millirods
Two rabbit livers were treated with RF ablation followed by treatment with 4 doxorubicin millirods in the configuration shown in Figure 5-1D. Photographs showing the liver implanted with the millirods illustrate successful placement of the implants
(Figure 5-7). Doxorubicin concentrations predicted from the model and measured from experimental data are shown in Figure 5-8. Drug concentrations in the model simulation
163 were elevated in the tissue surrounding the implants on day 4 and continued to distribute throughout the ablated region, particularly into the center of the ablated region around the ablation track, by day 8. A similar trend was observed in the experimental data, but the observed drug concentrations were markedly higher. Gaps and cracks in the experimental images arose from fracture of the brittle, coagulated ablated tissue during slicing. A summary of drug concentration information is compiled in Table 5-1. Median drug concentrations in the ablated region were almost three times higher in experimental data than predicted by the model. However, the model was accurate in predicting what fraction of the tissue would be covered with the target doxorubicin concentration, which was more than 98% at both time points. In experimental data, the drug penetration distance, defined as the distance from the ablation center where drug concentrations dropped below the therapeutic target, was 8.5 mm in the day 4 sample and 9.5 mm in the day 8 sample.
5.3.5 In vivo treatment of ablated liver tumors with multiple polymer millirods
VX2 liver tumors in rabbits were treated with ablation and the same configuration of polymer millirods used in the previous section. Although the intended pretreatment tumor size was 2 cm in diameter, tumors were closer to 2.5 cm and had a cystic core when treated. Drug concentration distributions from model simulations and experimental slices are shown in Figure 5-9. Experimental measurements were substantially more irregular in experimental tumors than in the simulated spheres, reflecting the underlying asymmetry of the liver tumors. A substantial portion of the experimental drug
164
Figure 5-7. Photographs of implantation of 4 polymer millirods.
Photograph of a rabbit liver just after ablation and implantation of 4 doxorubicin millirods (A) shows the ablation probe track (white arrowhead) and sutures over the 4 implant locations (black arrowheads). Photograph of ex vivo tissue extracted after 4 days and sliced through the center (B) again shows the ablation track (white arrowhead) and previous locations of the extracted implants (black arrowheads).
165
Figure 5-8. Model versus experimental drug concentrations in ablated liver.
Treatment of a 1.8 cm diameter region of ablated liver with 4 millirods was simulated and performed experimentally, and representative central slices are shown. Model estimated doxorubicin distributions on day 4 (A) and day 8 (C) are compared to experimentally measured drug concentrations on day 4 (B) and day 8 (D). The ablation boundary
(dashed line) is shown along with the ablation probe track (white dot) and 4 polymer millirod (white asterisks) locations in the experimental measurements. Scale bars are 5 mm.
166 Table 5-1. Doxorubicin coverage of ablated rabbit liver.
Model Experimental Day 4 Day 8 Day 4 Day 8 Area of ablated liver (mm3) 254 254 144 182 Ablated liver over therapeutic target (%) 98.3% 98.8% 98.0% 98.3% 1st quartile 117 192 166 361 [DOX] in ablated liver (µg/g) median 205 295 590 1008 3rd quartile 430 398 1311 1796
167
Figure 5-9. Model versus experimental drug concentrations in ablated tumor.
Treatment of a 2 cm tumor with RF ablation and 4 millirods was simulated and performed experimentally, and representative central slices are shown. Model estimated doxorubicin distributions on day 4 (A) and day 8 (C) are compared to experimentally measured drug concentrations on day 4 (B) and day 8 (D). The outer boundary of the ablated tumor (dashed line) and the non-ablated tumor (dotted line) are indicated. The central portion of the ablated tumor region is not visible in the experimental measurements because extensive central necrosis had liquefied that portion of the tumor.
Scale bars are 5 mm.
168 Table 5-2. Doxorubicin coverage of ablated liver tumors.
Model Experimental Day 4 Day 8 Day 4 Day 8 Area of ablated tumor (mm3) 254 254 343 418 Area of non-ablated tumor (mm3) 60 60 195 90 Non-ablated tumor over therapeutic target (%) 100% 61.3% 84.8% 64.9% 1st quartile 78 8 30 7 [DOX] in non-ablated tumor (µg/g) median 140 20 79 26 3rd quartile 232 56 173 87
169 distribution data is absent because the center of the extracted tumor was liquefied and could not be sliced, but the periphery of the ablated tumor was preserved. Qualitatively, drug distributions in the outer rim of non-ablated tumor, the risk volume, appeared similar in model and experimental distributions. A quantitative summary of DOX concentrations in the tumors is given in Table 5-2. The areas of non-ablated and ablated tumor were larger in the experimental data, corresponding to the slightly larger size of the tumors when treated. Coverage of the non-ablated tumor rim over the target concentration was similar to model predicted values, although the experimentally measured value of 84.8% did not reach the estimated 100% coverage on day 4. Median drug concentrations were also similar between model predictions and experimental measurements. On day 4, the median predicted concentration in the non-ablated rim was
140 µg/g compared to 79 µg/g, with considerable overlap between the middle 50% of the points. The model predicted average concentration on day 8, 20 µg/g, corresponded well to the experimentally measured average concentration of 26 µg/g. Overall, the experimental values for drug coverage of the outer rim were close to the model-predicted values, and both experimental and model data exhibited the same trend of drug coverage peaking on day 4 and decreasing slightly by day 8.
5.4 Discussion
In this chapter, a finite element method (FEM) was used to simulate the use of multiple polymer millirods to treat an RF ablated tumor. The FEM model was a useful method for distinguishing between different prototype tumor treatments. Simulated drug distributions could be compared based on more information than would be readily
170 available from animal experiments, such as the average drug concentration over 8 days and the percentage of areas that were covered with drug concentrations above a predetermined level. Additionally, this chapter introduces the concept of a risk volume, or a region of a tumor that is more prone to recurrence and should be preferentially targeted with drug. In the case of RF ablation, this risk volume is the tumor periphery, which may be exposed to sublethal doses of heat during ablation.
5.4.1 Value of drug transport modeling for millirod therapies
A finite element model offers some advantages over solving the drug transport model analytically. First, FEM can be used to solve systems of arbitrary geometric complexity without consideration of symmetry or regularity. This property made FEM ideal for this chapter, in which asymmetric placements of multiple implants in a tumor were tested. Second, a FEM model can accept a wide range of inputs in terms of boundary conditions and tissue properties, and these values are not limited to analytical functions. This was particularly useful given the time and spatially dependent values of diffusion and elimination parameters determined in Chapter 4. Third, the FEM model could be easily extended to simulate realistic tumor scenarios using imaging data, a potential future use of this tool.
Model simulation of drug distributions provided several unique insights about using multiple implants to treat an ablated tumor 2.0 cm in diameter. In general, increasing the number of implants raised the average drug exposure of the tumors almost linearly, a finding which was relatively independent of the arrangement of implants within the ablated tumor. The increase in average exposure was not surprising, as using
171 more implants increased the total doxorubicin dose. The lack of an effect from repositioning the implants on the total tumor exposure resulted from the properties of ablated tumor: relatively high diffusion and almost no elimination. Because of these properties, drug from the implants redistributed easily through the ablated tumor, where it acted like a reservoir for drug distribution into the surrounding non-ablated tumor and normal liver. However, if drug concentration in the peripheral tumor was valued over exposure to the whole tumor, implant placement became more important. Using some combination of peripheral implants allowed the entire tumor to be covered with drug concentrations above the desired concentration of 12.8 µg/g; this achievement was not equaled using a single central implant measuring either 8 or 16 mm in length.
Model simulation also indicated that using peripheral implant placement would be superior to the use of peripheral implants along with a central implant. Peripheral implant placement boosted average drug doses to the risk volume by 20 µg/g compared to peripheral + central strategies with the same doxorubicin dose. In the scenarios using a central implant, much of the drug dose from the center implant was delivered to the core of the ablated region tumor, where it may not be needed because of high thermal doses during ablation. In the situations shown in Figure 5-6, the weakness of the peripheral strategy was the poles of the tumor, while the weakness of the peripheral + central strategy was the tumor equator. In the context of RF ablation, drug coverage of the tumor poles may be less important because these areas are closer to the ablation probe, where ablation heating should be more effective. For this reason, peripheral only placement of implants has an additional advantage. Equivalent drug exposures could be achieved with the peripheral + central implant strategy, but required increasing the total doxorubicin
172 dose, and potentially the cost to the patient in terms of side effects. For this reason, subsequent validation experiments were performed using peripheral placement of 4 millirods, which the model predicted would completely cover the risk volume with concentrations above 12.8 µg/g for more than 3 days.
5.4.2 In vivo validation of drug transport model
Animal experiments placing 4 millirods into ablated rabbit liver and liver tumors were used for validation of the drug transport model. Millirod implantation was performed successfully in all of the animal subjects, and no serious side effects of the treatment were noted. In ablated liver, experimentally measured drug concentrations extended almost 1 cm away from the ablation probe track. Experimentally measured concentrations varied considerably from the model prediction, with the model substantially underestimating drug accumulation in the center of the ablated region. This discrepancy could reflect several potential problems with the model in ablated liver. For this simulation, ablated tissue parameters were taken from values for ablated rat liver, which may not accurately reflect the values in ablated rabbit liver. Additionally, the implant boundary condition was taken from a single implant placed in a small ablated tumor (Chapter 3). Although this boundary condition is the best available experimental data for this implant formulation, this function may inadequately represent boundary drug concentrations for this simulation in which multiple implants were in close proximity in the ablated region. Despite limitations in accurately predicting the magnitude of drug concentrations, the model accurately predicted the overall trend in average drug
173 concentrations. Moreover, 98% coverage of the ablated region with therapeutic target concentrations as predicted by the model was experimentally confirmed.
Model predictions in ablated tumors were further validated by placing millirods into ablated VX2 liver tumors in rabbits. Tumors were larger than anticipated and necrotic in the center, limiting the data that could be collected from the center of the treated tumors. Exact implant locations could not be verified, as a distinct implant path was not visible in the semi-solid core. However, the periphery of the ablated tumors, particularly the non-ablated rim, was largely preserved and could be assessed for doxorubicin content. The peripheral rim contained doxorubicin concentrations well above therapeutic levels, and there was a strong correlation between model predictions and experimental measurements both in average concentration and therapeutic coverage.
Total tumor coverage on day 4, which was predicted by the model, was not achieved experimentally, yet more than 85% of the tumor rim was covered. Inability to cover the remaining 15% could reflect the larger size of the tumors as treated or experimental error in measuring drug concentrations. Additionally, because the core of the tumor was cystic when treated, drug may have behaved differently than would be expected in a completely solid ablated tumor. This difference between the simulated model and experimental conditions could have resulted in differences in drug concentrations at the tumor periphery. Nevertheless, the tumor areas, both measuring > 5 cm2, had peripheries largely covered with therapeutic concentrations of drug. These tumors were largest regions treated by polymer millirods to date, a major step toward the use of these implants to treat larger tumors.
174 5.4.3 Limitations of the study
While using a drug transport model to develop a multiple implant strategy led to promising simulated and experimental results, this chapter has several limitations which should be noted. The major limitation of this chapter is the small number of animal subjects, which restricts the extent of the conclusions that can be drawn. In the future, studies with larger numbers of animal subjects in the experimental groups could confirm these results and extend them into a variety of different treatment situations. However, the animal results in this chapter provide a useful demonstration of modeling different treatment scenarios and experimentally validating the results in a tumor model. A second limitation is the use of previously estimated parameters and boundary conditions which may not be the best possible measurements. This factor is particularly important in the simulations of implants in ablated liver tissue because the properties of ablated rabbit liver be different from the parameters from a previous study in ablated rat liver.
Additionally, the boundary conditions developed from data in Chapter 3 may vary considerably from the situation simulated. Placing several implants in close proximity in the ablated region may lead to considerably different implant release rates, and there is no experimental data to better approximate the boundary. This limitation could be accommodated by performing additional experiments in ablated rabbit liver to determine better tissue parameters and boundary conditions. Finally, experimentally treating cystic tumors with semisolid cores may not accurately reflect what would happen in a solid ablated tumor. It is difficult to place the millirods in these tumors, and the liquid core may increase distribution of the drug by acting as a drug reservoir with little elimination.
This caveat reflects a challenge to using the VX2 tumor model, which is largely solid at
175 sizes below 1.5 cm in diameter but rapidly becomes necrotic as it increases in size. A way to address this problem is either to closely monitor the growing tumors with ultrasound to determine the time of treatment, ensuring that the tumors are not necrotic when treated, or to switch to an alternate tumor model which grows more slowly and does not form a necrotic core.
5.5 Conclusion
Despite the limitations of the study, model simulation allowed for prototyping of several different multiple implant tumor treatments and demonstrated the value of peripheral implant placement. This model shows considerable potential for future use in testing different implant strategies. A small set of animal experiments demonstrated the value of the model predictions and the feasibility of the treatment strategy. The combination of modeling and experimental results from this chapter represents a major stride toward treatment of larger tumors with RF ablation and polymer millirods. The use of drug transport modeling and multiple implants is discussed further in the next chapter, which describes their role in the future development of polymer millirods as a treatment for human tumors.
5.6 References
1. Ueno S, Tanabe G, Sako K, Hiwaki T, Hokotate H, Fukukura Y, Baba Y, Imamura Y, Aikou T 2001. Discrimination value of the new western prognostic system (CLIP score) for hepatocellular carcinoma in 662 Japanese patients. Cancer of the Liver Italian Program. Hepatology (Baltimore, Md 34(3):529-534.
2. de Baere T, Rehim MA, Teriitheau C, Deschamps F, Lapeyre M, Dromain C, Boige V, Ducreux M, Elias D 2006. Usefulness of guiding needles for radiofrequency ablative treatment of liver tumors. Cardiovascular and interventional radiology 29(4):650-654. 176 3. Bilchik AJ, Wood TF, Allegra D, Tsioulias GJ, Chung M, Rose DM, Ramming KP, Morton DL 2000. Cryosurgical ablation and radiofrequency ablation for unresectable hepatic malignant neoplasms: a proposed algorithm. Arch Surg 135(6):657-662; discussion 662-654.
4. Curley SA, Izzo F, Delrio P, Ellis LM, Granchi J, Vallone P, Fiore F, Pignata S, Daniele B, Cremona F 1999. Radiofrequency ablation of unresectable primary and metastatic hepatic malignancies: results in 123 patients. Ann Surg 230(1):1-8.
5. Guerin C, Olivi A, Weingart JD, Lawson HC, Brem H 2004. Recent advances in brain tumor therapy: local intracerebral drug delivery by polymers. Invest New Drugs 22(1):27-37.
6. Menei P, Jadaud E, Faisant N, Boisdron-Celle M, Michalak S, Fournier D, Delhaye M, Benoit JP 2004. Stereotaxic implantation of 5-fluorouracil-releasing microspheres in malignant glioma. Cancer 100(2):405-410.
7. Menei P, Capelle L, Guyotat J, Fuentes S, Assaker R, Bataille B, Francois P, Dorwling-Carter D, Paquis P, Bauchet L, Parker F, Sabatier J, Faisant N, Benoit JP 2005. Local and sustained delivery of 5-fluorouracil from biodegradable microspheres for the radiosensitization of malignant glioma: a randomized phase II trial. Neurosurgery 56(2):242-248; discussion 242-248.
8. Grimm P, Sylvester J 2004. Advances in Brachytherapy. Rev Urol 6(S4):S37-48.
9. Kaplan ID, Meskell P, Oldenburg NE, Saltzman B, Kearney GP, Holupka EJ 2006. Real-time computed tomography dosimetry during ultrasound-guided brachytherapy for prostate cancer. Brachytherapy 5(3):147-151.
10. Yamada Y, Bhatia S, Zaider M, Cohen G, Donat M, Eastham J, Rabbani F, Schupak K, Lee J, Mueller B, Zelefsky MJ 2006. Favorable clinical outcomes of three- dimensional computer-optimized high-dose-rate prostate brachytherapy in the management of localized prostate cancer. Brachytherapy 5(3):157-164.
11. Goldberg SN 2001. Radiofrequency tumor ablation: principles and techniques. Eur J Ultrasound 13(2):129-147.
12. Yu HC, Cheng JS, Lai KH, Lin CP, Lo GH, Lin CK, Hsu PI, Chan HH, Lo CC, Tsai WL, Chen WC 2005. Factors for early tumor recurrence of single small hepatocellular carcinoma after percutaneous radiofrequency ablation therapy. World J Gastroenterol 11(10):1439-1444.
13. Qian F, Stowe N, Liu EH, Saidel GM, Gao J 2003. Quantification of in vivo doxorubicin transport from PLGA millirods in thermoablated rat livers. J Control Release 91(1-2):157-166.
14. Wallace KB 2003. Doxorubicin-induced cardiac mitochondrionopathy. Pharmacol Toxicol 93(3):105-115.
177 15. Bertazzoli C, Chieli T, Grandi M, Ricevuti G 1970. Adriamycin: toxicity data. Experientia 26(4):389-390.
16. Ridge JA, Collin C, Bading JR, Hancock C, Conti PS, Daly JM, Raaf JH 1988. Increased adriamycin levels in hepatic implants of rabbit Vx-2 carcinoma from regional infusion. Cancer Res 48(16):4584-4587.
17. Swistel AJ, Bading JR, Raaf JH 1984. Intraarterial versus intravenous adriamycin in the rabbit Vx-2 tumor system. Cancer 53(6):1397-1404.
18. Qian F, Szymanski A, Gao J 2001. Fabrication and characterization of controlled release poly(D,L-lactide-co-glycolide) millirods. J Biomed Mater Res 55(4):512-522.
19. Weinberg BD, Blanco E, Lempka SF, Anderson JM, Exner AA, Gao J 2007. Combined radiofrequency ablation and doxorubicin-eluting polymer implants for liver cancer treatment. J Biomed Mater Res A 81(1):205-213.
20. Weinberg BD, Ai H, Blanco E, Anderson JM, Gao J 2007. Antitumor efficacy and local distribution of doxorubicin via intratumoral delivery from polymer millirods. J Biomed Mater Res A 81(1):161-170.
178 Chapter 6 Future prospects of polymer implants for the treatment of human tumors
6.1 Overall findings of this thesis
In this thesis, an attempt to develop a new class of minimally invasive tumor therapy has been described. This therapy was based on placing biodegradable polymer millirods directly into solid tumors to release drugs into the adjacent malignant tissue.
The primary obstacle to the success of this approach was the limited depth of drug penetration from the implant boundary, a result which has been noted in other attempts to use implants to treat tumors.1,2 Combining the millirods with radiofrequency (RF) ablation supplemented the implant approach and nearly doubled the depth of drug distribution away from the implants, confirming results that had been previously established in ablated liver.3 However, the first half of the thesis showed that the combined treatment was still insufficient to completely eradicate small liver carcinomas in rabbits. In these experiments, residual tumors were found within 1 cm of the implants, just outside the ablation coagulated zone. Problems resulting from the limited drug penetration in rabbits would likely be exaggerated in typically larger human liver tumors, thus requiring further modification of the approach.
The second half of this thesis described an integrated mathematical modeling and experimental approach to establish a multiple implant strategy for overcoming these limitations. Model-based parameter estimation of doxorubicin transport rates in liver tumors provided new insights on local drug transit in tumors. Particularly, the model was able to quantify the effects of ablation on drug transport rates. The flexibility of the finite
179 element method in solving the transport model allowed, for the first time, consideration of functions that varied over time and space. The estimated parameter values demonstrated the mechanism by which ablation improved drug distribution: it tipped the balance between diffusion and elimination in favor of increased drug spread. Although ablating the tumors both increased diffusion and decreased elimination, the change in elimination was more drastic and likely had the more dominant effect.
Based on the estimated parameters, modeling created opportunities for developing new treatment strategies using polymer millirods. Simulation provided a new means of evaluating potential treatments without performing a large number of cumbersome and costly animal experiments. Additionally, the concept of emphasizing drug distribution in one region of a tumor was introduced. This approach was used to test the concept of using multiple implants to treat a larger liver tumor. Simulated drug distribution demonstrated that multiple implants placed around the periphery of a tumor could provide improved drug delivery over a single implant. Preliminary animal experiments validated that implantation of multiple polymer millirods was feasible and could provide therapeutic drug concentrations throughout a region of 2 cm in diameter over an 8 day period. This finding represented a substantial improvement over areas covered by previous approaches with a single implant and could be nearing the treated volume necessary to be truly relevant for treatment of human tumors.
6.2 Limitations of the VX2 animal model
When evaluating the results of this thesis, the relevance and limitations of the tumor model must be considered. The goal of this thesis was to develop a minimally-
180 invasive, combined treatment for hepatocellular carcinoma (HCC). The VX2 carcinoma in rabbits described in this work is the most widely used model for testing experimental
HCC treatments. Although commonly used as a model of HCC, the VX2 cell line is not hepatic in origin and was originally derived from virus induced papillomas in rabbit skin.
For this reason, VX2 tumors are expected to differ from primary liver tumors in several ways. First, the VX2 may have substantially different density and vascularity than HCC.
Both of these properties are expected to have effects on diffusion and elimination of drugs within the tumors. Second, cells of hepatic origin are known to have high levels of metabolic enzymes and detoxifying molecules, such as glutathione, and VX2 cells are unlikely to express these enzymes. For this reason, doxorubicin elimination in VX2 tumors may be lower than the corresponding elimination in primary HCC tumors.
Additionally, doxorubicin may be more toxic to the VX2 cells, which may be less protected by this metabolic activity. Third, the VX2 tumors are very poorly differentiated and may be more aggressive and invasive than typical HCC, which may have an effect on the overall morphology of the tumors. Each of these factors must be kept in mind when interpreting the results, which may limit their direct applicability to human tumors.
Despite these factors, the VX2 model is currently the best, most cost effective tumor model available for testing this combined treatment. Its frequent use in literature makes it well known to other investigators, and a dearth of other suitable options makes it a common choice in studies of minimally invasive tumor treatments. Many alternative tumor models exist in mice and rats, but these animals have small livers that make for very difficult testing of interventional treatments in situ. The only other liver tumor
181 model of comparable size is an autochthonous model of HCC in woodchucks. These woodchucks are infected with woodchuck hepatitis virus at a young age and develop spontaneous liver tumors similar in morphology to human HCC induced by hepatitis.
Because of the time required to induce the tumors, these woodchucks are expensive and difficult to obtain. For this reason, the VX2 rabbit liver model provides a balance of practicality and relevance to human applications. However, the considerations described here are important in understanding the broad validity of the results, particularly to human cancers. Further discussion of the origin and limitations of the VX2 model is provided in Appendix A.
6.3 Insight based on findings of this work.
Several general themes were developed throughout this thesis and provide insight for the future implementation of locoregional tumor treatments. While in this work these ideas were developed in the context of an intratumoral implant, they are applicable to a wide range of minimally invasive therapies. Specifically, lessons learned from this work include the benefit of using combination treatments, the value of modeling as an evaluation and planning tool, and the potential to overcome limited drug penetration.
6.3.1 Value of combination treatments
This work clearly demonstrated the value of combined treatment in cancer therapy. Tumor treatments always face the risk of tumor recurring from a small pool of tumor cells left untreated. Combining treatments provides a reliable means for eliminating a larger fraction of cells and often improves the effects of the individual
182 treatments. Reports in the literature have also consistently proven the value of combination approaches to tumor treatment. For example, RF ablation has been shown to increase the accumulation of liposomal doxorubicin in tumors,4 and arterial embolization before ablation has been used to increase the extent of destruction from ablation.5 In a variety of circumstances, combining treatments has benefited both therapies, as was the case in this thesis. There were at least two compelling reasons for including RF ablation with polymer millirod. First, the ablation eradicated the bulk of the tumor, considerably reducing the volume of tumor cells needing treatment from the drug.
Drug released from the implant was only responsible for killing cells that were not destroyed by the initial ablation. Second, ablation facilitated drug distribution to further distances from the implant by changing the drug transport properties of the tissue. Future minimally invasive therapies will also likely benefit from these combination effects.
6.3.2 Use of modeling to design a drug delivery system
A second theme developed in this work is the value of modeling as a tool for treatment development. A model provides a low cost means for rapidly prototyping changes in the implant strategy. In Chapter 5, ten different treatment options for a 2 cm tumor were simulated and compared. Any experimental method of testing these implant strategies would have involved more than 40 tumor-bearing rabbits, and using the model minimized this use of animal subjects. While limited to testing the use of multiple implants in this thesis, model simulation could also be used to assess other implant changes. For instance, polymer millirods with different release profiles have already been developed,6,7 and using the model it would be possible to determine the potential
183 benefits of using these implants in tumors before experimentally testing them. Then, animal tests can be limited to only those scenarios which require more extensive study. It is also possible to anticipate potential changes to other parameters in the treatment, such as modulating blood flow to the tumor or limiting the inflammatory response after ablation. Modeling allows consideration of a wider range of scenarios by offering a way to test all the conceivable changes before unnecessarily investing time and effort in them.
Future work can certainly benefit by incorporating drug transport modeling and parameter estimation.
6.3.3 Overcoming limited penetration distance
A third theme developed in this thesis is that there are practical ways to overcome the limited drug penetration distance when using the implants. The method tested in this thesis was perhaps the most straightforward: using multiple implants placed throughout a larger tumor region. Using multiple implants increased the total doxorubicin dose to the tumor and allowed that dose to be distributed in a way that targeted a specific region of the tumor. Not unexpectedly, peripheral placement of implant improved coverage of the outer tumor rim without markedly affecting the center of the ablated region. In the experimental trials, 4 implants provided extensive doxorubicin coverage of a larger ablated region and ablated tumors measuring more than 2 cm in diameter. These results provide direct evidence that these implants may be able to provide enough drug coverage to treat human-scale liver tumors. Of course, the multiple implant strategy is not the only available method to increase the penetration distance of drug from the implant. One feasible alternative method is utilizing a prodrug strategy to enhance the drug transport
184 properties. Published reports have shown that drugs can be modified by conjugation with polyethylene glycol (PEG) to reduce elimination values, leading to their increased spread from implants.8 Another alternative is to modify the physiological properties of the surrounding tissue to improve drug spread. Reducing blood flow to the tumor region would almost certainly improve tumor drug spread by reducing elimination through transport of drug into nearby blood vessels. Previous reports have used drugs that induce vasoconstriction, such as arsenic trioxide, halothane, or epinephrine, to reduce blood flow to tumors before ablation.9 Extension of this approach could be used to improve drug delivery as well. Another means of reducing tumor blood flow is transarterial chemoembolization (TACE), which could be applied prior to millirod implantation to reduce blood flow in the region surrounding the tumor.10 Through the use of one or a combination of these methods to improve drug distribution distances, the ability to treat larger tumors can become more feasible.
6.4 Future directions of millirod research
In light of the progress described in this thesis, several avenues for future research using polymer millirod implants exist. Each of these research directions is based on previous findings using polymer millirods, the results of this thesis, and directions of current published literature. Although many possibilities for future research could be pursued, a few development paths seem to be the most likely, including: performing experimental validation, refining implant design, developing the drug transport model, and incorporating developments in ablation technology.
185 6.4.1 Further experimental validation
One of the largest potential areas where work is needed for future millirod development is performing additional experimental validation studies in animal subjects.
One immediate need is a long-term study of tumor response to establish the therapeutic efficacy of the treatment strategy. Such a study should meet two criteria; it should examine a time period sufficient to assess the completeness of the tumor response, and it should include larger experimental groups of animal subjects. Tumor studies performed in this thesis were ended either 4 or 8 days after the treatment to maximize the amount of information available from drug distributions. However, these early time points prevented assessment of the long-term efficacy of the implants. A future study should incorporate animal groups at longer times, such as 4 or 8 weeks, so a determination can be made as to whether a tumor has been completely treated and is not likely to recur.
Incorporation of ultrasound or other imaging monitoring of the treatment site should allow for such a long study to be done in a way that minimizes suffering to the animal subjects. Any future study should also incorporate more animal subjects so that statistically significant conclusions about the treatment outcome could be drawn. The emphasis of the studies in this thesis was on drug distribution, so only a small number of animal subjects was used in each case. While the small studies permitted assessment of drug distributions, they limited the conclusions that could be made about tumor treatment efficacy. A larger study could use 10-20 tumors per time point to assess the difference between RF ablation and polymer millirods and a more conventional treatment, such as systemically administered doxorubicin. Successfully demonstrating that polymer millirods provide a treatment advantage over other available treatments would greatly
186 advance the case for using millirods to treat human tumors. Because of the high cost and difficulty of treating VX2 tumors in rabbit livers, performing this study in mouse or rat tumor model and placing the tumors in a subcutaneous location could reduce the effort required to perform a sizeable study.
Another valuable animal study that could be performed is further validation of the drug transport model. The small group of animal subjects reported in Chapter 5 provided some initial idea of the value of using the model. However, the experimental groups were not a statistically relevant size. Moreover, the treated tumors were cystic and larger than expected at the time of treatment. Another study could include a greater number of animals and use ultrasound monitoring of tumors to ensure that the pretreatment size is appropriate. Such a study would allow for much greater certainty about the validity of the model conclusions, and could even allow for refinement of model parameters and boundary conditions. Ultimately, this study would be useful in refining the drug model and using it to evaluate other treatment scenarios.
6.4.2 Refinement of implant design
Another direction of future work which could help the progression of polymer millirod therapies is the incorporation of design changes to the implants themselves. One design change is to use implants with different drug release rates. As described extensively in Chapter 1, several types of implants have been created that were not tested in this thesis, including implants with sustained-release and dual-release profiles.6,7 In studies in ablated liver, dual-release implants considerably improved the speed with which the ablated region was covered with drug,11 and this could ultimately have a
187 beneficial effect on tumor treatment with the millirods. It is not immediately clear if this effect would translate into additional tumor treatment efficacy; in this thesis, tissue properties such as doxorubicin diffusion and elimination rates appeared to have greater effects on drug distribution than implant release rates. Other implant modifications could attempt to improve local drug distribution by modifying the tissue surrounding the implant. One such modification is the reduction of inflammation associated with ablation,12 which has been previously accomplished by incorporating dexamethasone into the implants.13 Reducing inflammation after ablation should have two beneficial effects: slowing fibrous capsule formation and minimizing new blood vessel formation. It is well known that the immune system is important to destruction of tumor cells after local therapies,14 so broadly suppressing the inflammatory response with dexamethasone after ablation may reduce the effectiveness of the treatment by limiting the body’s ability to kill residual cancer cells. If this proves problematic, an agent with more specific effects, such as a vascular-disrupting agent,15 could be incorporated into the implants as an alternative.
6.4.3 Maturation of modeling
In conjunction with additional animal studies, refining and developing new uses for the drug transport model could be quite beneficial for polymer millirod development.
To start, incorporating data from additional animal studies could be used to improve the existing model. Tissue properties could be determined for more than just the initial 8 day period to allow for modeling of longer periods of time, and experimental measurements of drug concentrations could provide more accurate boundary conditions. New
188 experiments could be used to further validate that the model predictions are correct, building upon the animal validation already reported in Chapter 5. Furthermore, with enough animal data it would be possible to correlate cumulative drug exposure with cell death. This advancement would allowing the model to not only estimate average drug exposure over time but also to predict the likelihood that tumor cells would be killed by that exposure. Also, the model could be used to predict benefits of using the implant modifications described in the section above, accelerating technological advances in implant design.
One implementation of the model that could be developed in future work is a modeling tool for imaging-based treatment planning. The principle of such a tool would be to use imaging methods, such as MRI or CT, to determine tumor properties and geometry before treatment. Ablation of one or more areas of the tumor could be planned, perhaps even using a tool to predict which regions would be successfully treated by ablation as well as which regions were likely to recur. Then, the predictive model as described above could be used to determine what would be the best possible placement of implants to maximize the probability that the entire tumor would be successfully treated.
The added value of this predictive tool is that it would be individualized for each patient based on the tumor’s original geometry and subsequent ablation. If millirods are ultimately used for treating human tumors, this software could easily be translated into a clinical planning tool, much like those used to plan doses of radioactivity in brachytherapy.16
189 6.4.4 Incorporation of novel ablation technology
A final direction of treatment optimization is the incorporation of new developments in tumor ablation into the combined therapy. Over the past several years, new techniques for ablation have become more available, such as microwave coagulation therapy (MCT)17 and cryoablation.18 Each of these therapies has potential advantages over radiofrequency ablation that could be investigated. MCT, for example, has showed some benefit in ablating larger regions than radiofrequency ablation.19 Cryoablation, on the other hand, has the benefit of being able to treat large regions and to monitor tumor destruction in real time using computed tomography (CT) or ultrasound.20 For each of these methods, the tumor treatment benefit versus RF ablation is controversial,21,22 but they may eventually prove to have some benefit. In addition, it is possible that different ablation techniques could have a different effect on drug transport. In this thesis, it has already been shown that RF ablation facilitates drug distribution by increasing drug diffusion and lowering elimination in tumors. The effects of these other ablation techniques on drug transit are unknown because the methods of tissue destruction are slightly different. It is possible, for instance, that the destruction of tissue through freezing has a greater impact on drug diffusion through the ablated tissue. For this reason, these other ablative techniques should be investigated to determine if they have some characteristics which further benefit drug delivery to tumors. Such tests could eventually lead to greater applicability and increased utility for polymer millirod treatments.
190 6.5 Potential value of millirods as a human tumor treatment
The ultimate goal of this work is to develop a treatment that could be used to treat human tumors. Given this objective, it is important to consider how the results described in this thesis and the prospect of future advances fit in to the development of clinical liver tumor treatments. Initial results using the implant treated only small regions around the implant which would have limited clinical use, but combination with ablation and the use of multiple implants increased the size of the treated area such that it may be reasonable to consider using these implants in human scenarios. The future work described above could supplement the current results and improve the treatment further, leading to a treatment that is clinically applicable. If the millirods were to be used in a clinical scenario, it is likely they would be used in one of two situations: as part of a combined treatment or as salvage therapy.
6.5.1 Use of millirods as part of a combined treatment
The most direct clinical extension of the work described in this thesis would be the use of polymer millirods as part of a combined treatment with surgery or ablation.
The gold standard of liver cancer treatment is surgery,23 but even after resection locoregional recurrence occurs in more than 64% of patients.24 In this case, several polymer millirods could be inserted around the resection boundary to kill any microscopic foci of cancer cells remaining after resection. For the larger fraction of patients that are not good candidates for resection, RF ablation is a good alternative treatment but it has also been plagued by frequent recurrence.25 With ablation, the main tumor mass could be ablated one or more times until all apparent regions of the tumor
191 had been successfully treated. Then, polymer millirods could be placed throughout the tumor to minimize the risk of recurrence. Using polymer millirods in conjunction with other ablative techniques, such as laser, microwave, or ultrasound, could also be considered, and animal studies with these techniques could determine if they could benefit from concomitant drug administration as well. Using polymer millirods as part of a combined treatment has several advantages. First, these patients are receiving a procedure already and millirod implantation does not subject the patients to the added risk of having another procedure. Second, the fairly high rates of tumor recurrence after these treatments could benefit from even a modest improvement in tumor destruction.
Currently, combination treatment is likely the best avenue for the use of polymer millirods in humans.
6.5.2 Use of millirods as a part of salvage therapy
Many patients with primary liver tumors or colorectal metastases to the liver are not considered good candidates for any therapy because of the advanced state of their disease. Hepatocellular carcinoma is an aggressive cancer, and in most cases severe disease or extrahepatic involvement indicates that the focus of care should be shifted to supportive and palliative measures.26 Additionally, many cases of colorectal metastases to the liver are too advanced to be successfully treated.27 Systemic chemotherapy is often the only option available in these cases, yet the response rate to systemic chemotherapy is less than 35% at best.28 In each of these cases, polymer millirods might be used as a life- prolonging measure to slow the growth of the main tumor masses and reduce patient suffering. The advantage of using polymer millirods is that they could be applied
192 percutaneously or laparoscopically, likely with a minimal hospital stay and risk to the patient. Furthermore, total doses of drug applied with the millirods are small compared to typically systemic doses, which would likely limit the extent of side effects. Although the implants would not provide a cure for this type of disease, it is possible that they could prolong survival or reduce pain associated with these advanced cases.
For patients with less advanced disease but who are still not good candidates for surgery, polymer millirods might be used as a bridge to resection or transplant. The general principle behind this technique is to administer treatment prior to resection or transplantation to reduce the size of tumors, making them more amenable to surgery and increasing the number of patients eligible for resection. In a previous study, transarterial chemoembolization (TACE) downstaging of tumors prior to resection was shown to significantly increase patient survival, although not conferring the same advantage to patients subsequently undergoing transplantation.29 A more recent study demonstrated that yttrium 90 microspheres could also be used to downstage tumors, increasing the number of patients who were candidates for liver transplantation.30 Polymer millirods could perhaps be administered in the same way, used prior to surgery to downstage tumors and permit subsequent surgical treatment of the disease. One benefit of using millirods in this way is that the implants would not be responsible for complete eradication of the tumor but only for providing some tumor regression, which they have shown their ability to do in this work. On the whole, salvage therapy is a potentially promising opportunity for human use of polymer millirod therapies.
193 6.5.3 Limitations to progress
Despite promising modeling and experimental results as well as a large population of patients which would be good candidates to receive the treatment, polymer millirods face several obstacles which prevent aggressive implementation of these treatment strategies in humans. The first, and possibly largest, impediment to human use of millirods is the lack of a large study showing the conclusive value of the implants over other treatments. In order for the implants to truly advance, a large study must demonstrate that these implants are better than current available treatments and have limited toxicity and side effects. Without this conclusive evidence, polymer millirods are unlikely to be used in the clinical trials which would be necessary to transition them into widespread clinical use. Second, none of the current implants have been produced in accordance with current good laboratory practice (cGLP) or current good manufacturing practice (cGMP) standards. If the implants are to be used in large preclinical animal studies or clinical trials, the manufacturing technique must be modified in such a way that implants can be produced according with these guidelines and sterilized. While this change can be successfully made, it will certainly increase the cost of performing these studies. Both these limitations are primarily engineering problems which might be best addressed by a company more suited to performing these kinds of tasks. A company with access to better manufacturing facilities could address the manufacturing issue, and a strong financial incentive would propel the animal studies necessary to drive these implants into development. If vigorously pursued, these factors could be addressed over a relatively short period of time and could open the gateway for using polymer millirods in clinical trials.
194 6.6 Overall conclusions
This thesis has demonstrated the potential value of using polymer millirods for tumor treatment by illustrating the treatment of liver tumors in an animal model. Use of polymer millirods alone provided volume control of small tumors over a limited region, and combination with ablation and the use of multiple implants set the stage for treatment of larger lesions. Furthermore, the development of a drug transport model provided a valuable tool that can be used for more rapidly evaluating and improving the implant strategy. There is a large clinical need for these devices, which could improve the success rates of other treatments and alleviate suffering in patients who are not eligible for any current treatments. However, there is currently a gap between the established knowledge and the requirements necessary to transition to clinical trials using these devices. For this reason, polymer millirods are at a critical juncture to determine if future studies can close this gap, eventually leading to the implementation of these devices as a clinically available treatment for human tumors.
6.7 References
1. Strasser JF, Fung LK, Eller S, Grossman SA, Saltzman WM 1995. Distribution of 1,3-bis(2-chloroethyl)-1-nitrosourea and tracers in the rabbit brain after interstitial delivery by biodegradable polymer implants. J Pharmacol Exp Ther 275(3):1647-1655.
2. Wang CC, Li J, Teo CS, Lee T 1999. The delivery of BCNU to brain tumors. J Control Release 61(1-2):21-41.
3. Qian F, Stowe N, Liu EH, Saidel GM, Gao J 2003. Quantification of in vivo doxorubicin transport from PLGA millirods in thermoablated rat livers. J Control Release 91(1-2):157-166.
4. Ahmed M, Monsky WE, Girnun G, Lukyanov A, D'Ippolito G, Kruskal JB, Stuart KE, Torchilin VP, Goldberg SN 2003. Radiofrequency thermal ablation sharply increases intratumoral liposomal doxorubicin accumulation and tumor coagulation. Cancer Res 63(19):6327-6333.
195 5. Yamakado K, Nakatsuka A, Kobayashi S, Akeboshi M, Takaki H, Kariya Z, Kinbara H, Arima K, Yanagawa M, Hori Y, Kato H, Sugimura Y, Takeda K 2006. Radiofrequency ablation combined with renal arterial embolization for the treatment of unresectable renal cell carcinoma larger than 3.5 cm: initial experience. Cardiovascular and interventional radiology 29(3):389-394.
6. Qian F, Nasongkla N, Gao J 2002. Membrane-encased polymer millirods for sustained release of 5-fluorouracil. J Biomed Mater Res 61(2):203-211.
7. Qian F, Saidel GM, Sutton DM, Exner A, Gao J 2002. Combined modeling and experimental approach for the development of dual-release polymer millirods. J Control Release 83(3):427-435.
8. Fleming AB, Haverstick K, Saltzman WM 2004. In vitro cytotoxicity and in vivo distribution after direct delivery of PEG-camptothecin conjugates to the rat brain. Bioconjugate chemistry 15(6):1364-1375.
9. Horkan C, Ahmed M, Liu Z, Gazelle GS, Solazzo SA, Kruskal JB, Goldberg SN 2004. Radiofrequency ablation: Effect of pharmacologic modulation of hepatic and renal blood flow on coagulation diameter in a VX2 tumor model. J Vasc Interv Radiol 15(3):269-274.
10. Qian J, Feng GS, Vogl T 2003. Combined interventional therapies of hepatocellular carcinoma. World J Gastroenterol 9(9):1885-1891.
11. Qian F, Stowe N, Saidel GM, Gao J 2004. Comparison of doxorubicin concentration profiles in radiofrequency-ablated rat livers from sustained- and dual- release PLGA millirods. Pharm Res 21(3):394-399.
12. Blanco E, Qian F, Weinberg B, Stowe N, Anderson JM, Gao J 2004. Effect of fibrous capsule formation on doxorubicin distribution in radiofrequency ablated rat livers. J Biomed Mater Res A 69(3):398-406.
13. Blanco E, Weinberg BD, Stowe NT, Anderson JM, Gao J 2006. Local release of dexamethasone from polymer millirods effectively prevents fibrosis after radiofrequency ablation. J Biomed Mater Res A 76(1):174-182.
14. Goldberg EP, Hadba AR, Almond BA, Marotta JS 2002. Intratumoral cancer chemotherapy and immunotherapy: opportunities for nonsystemic preoperative drug delivery. J Pharm Pharmacol 54(2):159-180.
15. Tozer GM, Kanthou C, Baguley BC 2005. Disrupting tumour blood vessels. Nature reviews 5(6):423-435.
16. Kaplan ID, Meskell P, Oldenburg NE, Saltzman B, Kearney GP, Holupka EJ 2006. Real-time computed tomography dosimetry during ultrasound-guided brachytherapy for prostate cancer. Brachytherapy 5(3):147-151.
196 17. Ohmoto K, Tsuduki M, Shibata N, Takesue M, Kunieda T, Yamamoto S 1999. Percutaneous microwave coagulation therapy for hepatocellular carcinoma located on the surface of the liver. AJR Am J Roentgenol 173(5):1231-1233.
18. Han KR, Belldegrun AS 2004. Third-generation cryosurgery for primary and recurrent prostate cancer. BJU international 93(1):14-18.
19. Xu HX, Lu MD. 2006. Comparison between radiofrequency ablation and percutaneous microwave coagulation therapy for small hepatocellular carcinomas - a reply. ed.: The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China. p 801-802.
20. Atwell TD, Farrell MA, Callstrom MR, Charboneau JW, Leibovich BC, Patterson DE, Chow GK, Blute ML 2007. Percutaneous Cryoablation of 40 Solid Renal Tumors with US Guidance and CT Monitoring: Initial Experience. Radiology.
21. Martin RC 2006. Hepatic tumor ablation: cryo versus radiofrequency, which is better? The American surgeon 72(5):391-392.
22. Ohmoto K, Yamamoto S 2006. Comparison between radiofrequency ablation and percutaneous microwave coagulation therapy for small hepatocellular carcinomas. Clinical radiology 61(9):800-801; author reply 801-802.
23. Little SA, Fong Y 2001. Hepatocellular carcinoma: current surgical management. Semin Oncol 28(5):474-486.
24. Yang Y, Nagano H, Ota H, Morimoto O, Nakamura M, Wada H, Noda T, Damdinsuren B, Marubashi S, Miyamoto A, Takeda Y, Dono K, Umeshita K, Nakamori S, Wakasa K, Sakon M, Monden M 2007. Patterns and clinicopathologic features of extrahepatic recurrence of hepatocellular carcinoma after curative resection. Surgery 141(2):196-202.
25. Barnett CC, Jr., Curley SA 2001. Ablative techniques for hepatocellular carcinoma. Semin Oncol 28(5):487-496.
26. Clark HP, Carson WF, Kavanagh PV, Ho CP, Shen P, Zagoria RJ 2005. Staging and current treatment of hepatocellular carcinoma. Radiographics 25 Suppl 1:S3-23.
27. Bentrem DJ, Dematteo RP, Blumgart LH 2005. Surgical therapy for metastatic disease to the liver. Annu Rev Med 56:139-156.
28. Leung TW, Johnson PJ 2001. Systemic therapy for hepatocellular carcinoma. Semin Oncol 28(5):514-520.
29. Majno PE, Adam R, Bismuth H, Castaing D, Ariche A, Krissat J, Perrin H, Azoulay D 1997. Influence of preoperative transarterial lipiodol chemoembolization on resection and transplantation for hepatocellular carcinoma in patients with cirrhosis. Ann Surg 226(6):688-701; discussion 701-683.
197 30. Kulik LM, Atassi B, van Holsbeeck L, Souman T, Lewandowski RJ, Mulcahy MF, Hunter RD, Nemcek AA, Jr., Abecassis MM, Haines KG, 3rd, Salem R 2006. Yttrium-90 microspheres (TheraSphere) treatment of unresectable hepatocellular carcinoma: downstaging to resection, RFA and bridge to transplantation. Journal of surgical oncology 94(7):572-586.
198 Appendix A VX2 tumor model
A.1 Introduction
The tumor model used throughout this work was the VX2 liver carcinoma in rabbits. The model is an aggressive, allograft carcinoma that can be implanted in a variety of anatomical sites. In the liver, the tumors are widely used as a model of human hepatocellular carcinoma (HCC).1 This appendix provides details on the experimental use and observed properties of the VX2 model that are not frequently described in the literature.
The VX2 model has several properties that make it useful for the study of tumor treatments. First, the animal host, the New Zealand white (NZW) rabbit, is large enough that it can reasonably maintain a large tumor burden. Tumors as large as 2 cm in diameter can be grown in rabbit tissue, allowing testing of clinically relevant versions of equipment or treatment strategies that could be used in humans. Other animals, such as mice and rats, are unsuitable for this work because of their small size. Larger animals, such as pigs or dogs, might also be used for this type of work but lack reliable tumor models for testing treatments in malignant tissues. Thus, treatments in these animals are often limited to testing in normal tissues. Second, the ability of the tumors to grow in several locations allows for testing of treatments against a variety of background tissues.
For example, similar tumors can be tested in liver and kidney to determine what effects, if any, the surrounding tissue parenchyma has on the treatment. Additionally, these tumors grow in immunocompetent animals, where the immune contribution to tumor treatment is likely to be more similar to that in human tumor treatment. Third, the tumor
199 grows rapidly after implantation, allowing for timely studies with relatively short waiting periods. This property facilitates prompt studies and minimizes the expenses involved in performing animal experiments. Finally, the aggressive nature of the model means that results from VX2 tumor treatment can be more easily extrapolated into situations with aggressive human tumors.
A.1.1 Origin of tissue type
VX2 tumor cells were isolated from a virally induced epithelial carcinoma and have been extensively propagated over the past 50 years. The discovery of this tumor model was facilitated by reports from hunters in northwestern Iowa, who reported that cottontail rabbits there developed warts that were discovered to be virus-induced papillomas in 1933.2,3 The first report that these papillomas could develop into invasive carcinomas was published in 1935 by Rous and Beard.4 It was later reported that the tumor could be passed into additional rabbits through surgical transplantation of tumor cells, which began to grow more aggressively after 14 successive transplantations.5 By
1952, the tumors had been propagated through at least 72 generations, at which point they no longer induced immunity to the original papilloma virus.6 Because cells have been propagated through many generations since these first reports, the VX2 tumors have become considerably less differentiated and more aggressive. Currently, standard VX2 tissue is available from the National Cancer Institute (NCI) Tumor Repository.
200 A.1.2 Previous use in other research
The VX2 tumor model in rabbits is extensively used in a variety of research, particularly in the testing of novel tumor treatments. To date, there are more than 700 publications concerning the model in the Pubmed database, and the number continues to increase. The studies have been done in a variety of anatomical sites, including muscle,7 bone,8 liver,9 and kidney.10,11 Because of the size of the tumor model, VX2 carcinomas in rabbits are ideally suited for the study of minimally invasive tumor treatment procedures. Almost every form of ablation (e.g. radiofrequency,10 laser,12 microwave,13 and ethanol or acetic acid injection1) has been tested in the VX2 model, as have other minimally invasive tumor treatments such as transarterial chemoembolization.14 Imaging studies which require a tumor model, such as developing new contrast agents15 or investigating new imaging techniques,16 is another area in which VX2 tumors have been heavily used. Additionally, the VX2 tumor has served as a model for the study of cancer biology, for instance, in the study of the lymphatic spread of head and neck cancers.17
For these uses, tumors were implanted in a variety of locations using slightly different techniques. However, in general, the VX2 model has proven to be a useful tumor model in a wide range of situations and circumstances.
A.2 Procedures
This section provides an overview of procedures used throughout this work as well as references providing more information about the procedures. Detailed experimental protocols are found at the end of this appendix in Section A.6.
201 A.2.1 Cell propagation
VX2 cells are propagated via injection or implantation into a rabbit either intramuscularly or subcutaneously. While previous publications have reported in vitro propagation of the tumor cells,18 this technique is not commonly reported in current VX2 literature because of the limited ability of the cells to proliferate in culture. Most commonly, authors propagate the tumor through a live, donor rabbit.9,19 To grow the tumors in the donor rabbit, a small piece of tumor or a cell suspension is implanted into the recipient rabbit, either in the subcutaneous space or into muscle. Subcutaneous injection offers the advantage that tumor growth is easy to monitor by palpation; however, intramuscular placement of the tumor into hind leg is quite common in the literature.20 For subcutaneous implantation, a small piece of tumor is implanted in a pocket created directly under the skin. For intramuscular implantation, the tumor tissue is implanted in a small muscle puncture made by a needle. Alternatively, cells can be minced into a cell suspension and injected directly into either site without an incision.
Techniques for making the cell suspension include mechanically chopping the tumor piece with scissors and a scalpel19 or blending the tissue piece with a homogenizer. The tumor subsequently grows into a size suitable for harvesting (1-2 cm in diameter) over a period of 4-6 weeks.
202 A.2.2 Cell harvesting
After the tumors reach 1-2 cm in diameter, they can be harvested either for immediate implantation into another rabbit or frozen for future use. In both cases, tumor tissue should be collected using sterile technique to minimize the risk of bacterial contamination of the tumor tissue. The rabbits are anesthetized prior to tumor resection, and the tissue surrounding the implant (either subcutaneous connective tissue or muscle, depending on the implantation site) is removed using scissors and a scalpel. The rabbits can be euthanized by sodium pentobarbital injection either immediately before or just after the tumor was resected; euthanasia prior to tumor resection does not appear to adversely affect the viability of the extracted tumor cells. After resection, any non-tumor tissue should be removed along with any regions of the tumor that were necrotic or liquefied. The remaining tumor pieces are then chopped into small pieces (2 x 2 x 2 mm cubes) and stored in cold media until further use.
A.2.3 Cell freezing
For long term storage, pieces of VX2 tumor cells can be frozen in liquid nitrogen.
Because most of the literature describes the use of live tissue donors, published methods for freezing VX2 tumor pieces are limited. To freeze the VX2 tumor, several small pieces are placed in a combination of media supplemented with fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO). In this work, VX2 pieces were frozen in a solution of
90% FBS and 10% DMSO, but other solutions incorporating media may be suitable as well. Dulbecco’s modified Eagle medium (DMEM) has been used for washing and temporarily storing tumor pieces and may be suitable for freezing, but was not used in
203 this work. Once in vials, the cells should then be frozen at a rate of approximately -
1ºC/min to a temperature of -80ºC. These vials should be transferred to liquid nitrogen for long term storage. The length of time for which these tumors can be stored in liquid nitrogen was not tested, but is expected to be on the order of years. When thawing these cells, the vials are brought to room temperature as quickly as possible. Viability of thawed cells was tested using a Trypan blue dye exclusion assay. In this assay, 40 µL of
0.4% Trypan blue solution was added to 400 µL of cells that had been minced with scissors and a scalpel or using a tissue homogenizer, as described above. After 5 minutes, solutions were visualized with a microscope (Figure A-1). Viable cell counts for each image are shown in Table A-1. Viability of the frozen cells was just higher than
40% for both techniques, and homogenization did not appear to adversely affect cell viability. However, it should be noted that cell viability is expected to be different for each batch of cells.
A.2.4 Liver tumor implantation
To grow the VX2 liver tumors, a piece of tumor tissue was surgically implanted in the liver. Some investigators have studied the possibility of injecting tumor cell suspensions into the liver, but frequent peritoneal and lung metastases have limited the effectiveness of this approach.19 For the tumors used in these studies, pieces of tumor tissue were implanted in the rabbit livers using a technique most closely described in the literature by Geschwind et al.20 The abdomen was opened just distal to the sternum, and the liver lifted out for easier implantation. The capsule of the liver was punctured with an
204
Figure A-1. VX2 cell viability prior to implantation.
Previously frozen and thawed cells separated by manual chopping with a scalpel blade
(A) or after mixing with a tissue homogenizer (B). Cells were stained with Trypan blue dye for 5 minutes to indicate cell viability. Blue staining cells did not exclude the dye and were considered non-viable. Scale bars are 100 µm.
205
Table A-1. Qualitative viability of VX2 cells using two suspension techniques.
Cell counts Viable Non-viable Viability (%) Minced cells 114 143 44% Homogenized cells 115 156 42%
Viable and non-viable cells from representative 10x fields were counted immediately after thawing and mechanical separation using scissors and a scalpel (minced cells) or using a tissue homogenizer (homogenized cells).
206 angiocatheter, and a small piece of tumor was placed in the puncture site. In Chapter 2, a small piece of abdominal fat was then sutured over the liver puncture to minimize bleeding. However, the piece of adipose tissue complicated histological interpretation of extracted tumors. Therefore, in subsequent chapters, this piece of adipose tissue was replaced with a small piece of absorbable gelatin sponge (Surgifoam). The abdomen was then sutured in three layers for wound closure. The tumor was allowed to grow for the desired amount of time prior to treatment.
A.2.5 Liver tumor treatment
Liver tumor treatment is described in detail in each chapter, but the general procedure is repeated here. The abdomen was opened just distal to the xiphoid as described above, and the tumor located. Any adhesions between the liver or tumor and the abdominal wall were then gently separated using blunt dissection. The tumor- containing portion of the liver was then gently lifted for treatment. In cases when ablation was applied, a 17-gauge RF ablation probe (Radionics) was inserted into the tumor and the desired extent of ablation applied. For polymer millirod implantation, a small hole was punctured in the desired implantation site and the implant was placed into the puncture. In chapter 2, implants were then sutured into place with a piece of fat and a single suture over the top to minimize the risk of the implant shifting. In chapter 3, the piece of fat was replaced with a piece of Surgifoam, while in chapter 5 the implants were not sutured into place at all because of fear of damaging the liver through the placement of 4 sutures. The abdomen was then again closed in three layers as described above.
207 A.3 VX2 tumor properties
A.3.1 Gross appearance
The gross appearance of VX2 liver tumors is summarized by photographs in
Figure A-2. The tumor had a white to yellow appearance, which was considerably lighter than the dark brown surrounding liver tissue. Tumors were typically visible from the surface of the liver prior to slicing, allowing for relatively easy location of the tumors for treatment. Small tumors less than 1.0 cm in diameter, such as that shown in Figure
A-2A, were typically completely surrounded by liver tissue on all sides. Furthermore, this size tumor was relatively solid throughout the entire tumor volume and contained no pockets of necrotic fluid. As tumors continued to grow for 20-28 days after implantation
(Figure A-2C and E), they became progressively larger and began to protrude from the surface of the liver, largely because the tumor was thicker than the liver itself (typically
1.0 cm thick at its maximum). These tumors consisted of a dense, white outer region surrounding a cystic and fluid-filled necrotic core. The fluid in the center of the tumor was typically dark red to purple and consisted of edema, blood, and necrotic tumor cells.
Central necrosis in the VX2 tumor occurred because the rate of tumor growth was faster than new vasculature to supply the tumor can be formed. For this reason, the ideal time for tumor treatment was roughly 12-20 days after implantation, when the tumor size should be less than 1.5 cm in diameter.
208 A.3.2 Computed tomography
To gain further insight about the development of the VX2 tumors in the liver, computed tomography scans were performed on two untreated tumors at two time points after tumor implantation. Contrast free images were obtained using a Philips MX8000
CT scanner using the following settings: 120 kVp, 250 mAs, and voxel size 0.57 x 0.57 x
5 mm. Images from these scans are seen in Figure A-3. The liver tumors were found in the front of the liver and appeared hypointense compared to the surrounding liver parenchyma. Tumors imaged on day 11 were relatively small, but by day 27 the tumors had expanded substantially and occupied the majority of the liver thickness. Although the tumors were largely necrotic by this time, there were no visible differences in the two regions on the image. Contrast-enhanced CT would likely be able to further differentiate the viable and necrotic regions. By measuring the volume of the tumors in the CT images, the relative growth rate of these two tumors was established and is shown in
Figure A-3. The tumors increased in volume almost exponentially with time, underscoring the importance of carefully selecting treatment times to ensure that the tumors are an appropriate size.
A.3.3 Histological appearance
The histological appearance of the VX2 tumor was indicative of a rapidly growing, poorly differentiated carcinoma. Representative H&E stained sections are shown in Figure A-5. The tumor featured two regions corresponding to the two areas seen in gross pathology: a necrotic core and a dense viable rim. The core of the tumor consisted of interspersed areas of viable tumor and necrotic debris. The viable tumor was
209
Figure A-2. Gross appearance of VX2 tumors in the rabbit liver.
Photographs of gross morphology showing VX2 tumor development 16 (A, B), 20 (C,
D), and 28 days (E, F) after tissue implantation. Note the transition between a solid tumor with little necrosis (B) to a cystic lesion (D, F) after approximately 2 weeks. The day 28 tumor appears small after slicing (F) because the tumor collapsed after drainage of the cystic core. Scale bars are 1 cm.
210
Figure A-3. Computed tomography scans of VX2 tumors in liver.
Computed tomography scans of two different rabbit tumors 11 days after implantation
(A, C) and those same tumors 28 days after implantation (B, D). The tumors appear as a hypointense region on the font face of the liver (red dashed circles).
211
Figure A-4. Growth rate of VX2 tumors in rabbit liver.
Measurement of volumes of tumors using CT shows the trend of growth in VX2 tumor volume. Error bars shown the range of the measurements (n = 2).
212 Figure A-5. Histology of untreated
VX2 tumors in rabbit liver.
H&E stained histology images from a typical VX2 tumor within the core of the tumor (A) as well as at the boundary between tumor and normal liver (B). A higher magnification image (C) shows infiltration of malignant cells into the surrounding normal tissue. Scale bars are 200 µm.
213 heavily populated with tumor cells characterized by low differentiation, high nucleus/cytoplasm ratio, densely staining cytoplasm, and evidence of rapid cell division, such as mitotic figures. These viable cells were clustered around areas where blood vessels were adequate to provide oxygen and nutrients to the rapidly growing tumor cells.
Adjacent to the viable tumor regions were areas of necrosis, identified by light staining regions with lack of overall structure, few intact cell membranes, the presence of edema, and some blood cells. In the core of the tumor, these necrotic regions made up more than half of the tumor area. At the tumor periphery, the tumor cell morphology was the same as the viable cells in the core, but the overall balance of cells was shifted in favor of viable tumor. Only small and intermittent regions of necrosis were present. Just past the boundary of the main tumor, nets of tumor cells were seen extending into normal tissue, confirming the infiltrative and aggressive nature of the tumor.
A.4 Concerns and limitations
Despite many advantages of using these tumors, the VX2 model had several limitations. First, implantation success was sometimes limited, i.e. implanted pieces do not always successfully grow into tumors. The take rate when implanting tumor pieces has been estimated in some cases to be as low as 59%.21 This limitation was countered by using a live donor and measuring cell viability before implantation to ensure that a sufficient number of viable cells is present. Furthermore, implanting a slightly larger tumor piece helped ensure enough viable cells are present to seed a viable tumor. The second limitation was the variable growth rate of the tumor, which caused a wide range in
214
Figure A-6. Ultrasound image of large VX2 tumor in rabbit liver.
The image shows an ultrasound of a rabbit tumor taken 26 days after tumor implantation.
The diameter of the tumor is indicated by the dotted line, which is 25.5 mm in diameter.
In this tumor, a hypoechoic core (*) was surrounded by a hyperechoic, dense tumor rim
(**). This pattern was indicative of a large, cystic tumor with a necrotic center.
215 the pretreatment size of tumors. Again, measuring cell viability prior to implantation and adjusting the size of the implanted piece could be used to adjust for this. Perhaps more practically, pretreatment tumor monitoring, such as ultrasound, can be used to periodically check the size of the tumor to determine the appropriate time to perform the treatment. Third, the VX2 tumors also formed cystic, necrotic cores (Figure A-6). If a tumor is highly necrotic when treated, the effects of the treatment could be vastly different than if used to treat a completely solid tumor. To reduce problems from treating cystic tumors, studies can be planned so as to treat tumors when they were less than 2 cm in diameter. For each of these limitations, careful study design can help overcome the potential problems.
A.5 Conclusion
The VX2 model in rabbits has a number of advantages that makes it suitable for use in these tumor treatment studies. Its widespread use, flexibility in anatomical location, rapid growth rate, large size of the animal host, and the ability to grow in immunocompetent animals makes a desirable model for testing polymer millirods. The tumor’s predictable characteristics, such as gross pathology, histology, and imaging appearance, can be used for comparison with treated tumors. Through careful study design, many of the limitations of the VX2 model can be addressed and their effects minimized.
216 A.6 Protocols
A.6.1 Tumor cell injection
1. Remove a cryotube containing frozen tumor pieces from liquid nitrogen storage,
taking care to wear goggles because the rapid temperature change can cause
explosion of the tube contents.
2. Place the tubes in a 37ºC bath until the ice has completely thawed.
3. Carefully remove the tumor pieces from the cryotube with a sterile pipet and
place into a sterile Petri dish.
4. Wash these tumor pieces 2-3 times with DMEM (supplemented with 5% FBS and
L-glutamine), approximately 10 mL per wash.
5. Repeat washing 2-3 times with 10 mL HBSS.
6. Place the tumor pieces in a test tube with 2 mL cold HBSS.
7. Insert the tissue homogenzer approximately ½-1” into the solution.
8. Turn on the homogenizer to the lowest available speed for 5-10 seconds. Attempt
to minimize the amount of time homogenizing, as the cell viability will go down
with longer or faster homogenization.
9. Load 1 mL syringes with 0.25 mL of the solution using an 18 or 21 gauge needle.
10. Keep cold until used.
11. Administer 1 mL of acetylpromazine as pre-anesthesia (3 mg/kg).
12. Anesthetize with isoflurane gas to desired depth in chamber. Reasonable
anesthesia may be 4% isoflurane with an oxygen flow rate of 1 L/min.
217 13. Remove animal from chamber and place in anesthesia cone. Tape the cone
around nose.
14. Shave the regions where injections are planned.
15. Locate desired injection site, either skin location for subcutaneous injection or
muscle belly for intramuscular injection.
16. Insert needle to desired depth and inject cell suspension.
17. To minimize the chance and effects of tumor leakage, leave the needle in place
for 10-15 s after injection, and remove slowly to reduce reflux down needle tract.
A.6.2 Tumor cell harvesting
1. Once the tumor is detected, the animal can be euthanized to harvest the tumor
pieces. This is a sterile procedure and should be performed with fully aseptic
techniques.
2. Anesthetize the rabbit and shave the area surrounding the tumor. A large area
should be shaved so that a large incision can be made for tumor removal.
3. Lay the rabbit on the surgical table so that the muscle containing the tumor faces
up.
4. Scrub the area with betadine (2x), betadine - soap (2x), and isopropanol (2x).
5. Drape the area with 4 sterile towels.
6. If necessary, the rabbit may be euthanized at this time. The preferred method is
by intravenous or intracardiac sodium pentobarbital (Fatal Plus) overdose.
7. Begin the procedure by making a large incision with a scalpel across the equator
of the tumor. Go through the skin in this manner.
218 8. Make the incision X-shaped by making a similar incision perpendicular to the
original incision.
9. Repeat this process through the overlying layers of muscle or skin, alternating
directions and removing extra tissue as necessary. Try to keep the area clean
using 4x4 gauze and cotton swabs as needed.
10. When the tumor perimeter is reached, puncture it with care using the scalpel
blade. Be careful when performing this procedure, as it is common for the
necrotic core of the tumor to spray or ooze necrotic fluid. Use gauze to try to
minimize bleeding from the tumor site and to clean up the core.
11. Gradually cut the surrounding tissue away from the tumor perimeter, attempting
to remove the entire tumor mass from the underlying tissue.
12. After removing the entire tumor, remove as much necrotic debris and blood as
possible. Use the scalpel and/or scissors to cut away as much of the surrounding
muscle as possible. Remember, it is much easier to remove muscle tissue at this
stage than if you wait until placing the tumor in media.
13. The resulting tumor tissue should be pinkish white, tough, and slightly harder than
the surrounding muscle.
14. Place the tumor in cold HBSS for storage until it can be processed for long term
storage.
219 A.6.3 Long term tumor storage or tumor use.
1. Prepare the appropriate number of cryotubes by filling them with approximately
1.8 mL of 90% FBS with 10% DMSO. Chill those tubes throughout the rest of
the procedures.
2. Take the tumor pieces as removed in the tumor removal and place them in a
sterile Petri dish. Fill 75% of the bottom of the dish with HBSS.
3. Using a combination of scissors, forceps, and scalpels, remove any remaining
muscle and non-tumor tissue.
4. Move to a new dish and fill again with fresh HBSS.
5. Cut into intermediate size pieces (0.5 cm x 0.5 cm), removing any suspect non
tumor tissue. If you even think that it might not be tumor, then get rid of it.
6. Move these pieces to a new dish with HBSS.
7. Cut away any remaining connective tissue (will appear whiter than remaining
tissue) or suspect tissue. Cut tumor into uniform pieces approximately 3 mm x 3
mm. At this point, tumor pieces may be used for cell suspension or direct
implantation if fresh tissue pieces are desired.
8. Place 2-4 tumor pieces into each chilled cryotube.
9. Place all cryotubes at -80ºC for a day. It will be beneficial to use a device which
provides an intermediate cooling rate of -1ºC/min. After 1-2 days, transfer the
tubes into liquid nitrogen storage. If liquid nitrogen storage is not available, cells
can be maintained at -80ºC.
220 A.6.4 Liver tumor implantation
1. Anesthetize the rabbit and shave the majority of the abdomen from about 2 cm
above the sternum to the middle of the abdomen. Shave a small area on the back
to make contact with the electrocautery ground.
2. Lay the rabbit on the surgical table on its back so the abdomen is facing up. Make
sure the animal is not twisted and is appropriately oriented. Tape the legs to the
side of the surgical table to prevent minor movements during the surgery.
3. Scrub the area with betadine (2x), betadine - soap (2x), and isopropanol (2x).
4. Without touching the sterile field, inject 0.5 mL marcaine along the surgical
incision site.
5. Drape the area with 4 sterile towels.
6. Make a midline incision with a scalpel just distal to the xiphoid process. The
appropriate length of this incision is likely 4-5 cm. Stop any serious bleeding
with hemostats and/or electrocautery.
7. Continue to open the skin and subcutaneous layers with the scalpel.
8. When the muscle is reached, begin opening the muscle with scissors. Take care
to approach slowly, as some rabbits have a thin muscle layer, and puncturing the
liver, stomach, or other abdominal contents is a distinct risk. Again, make sure to
stop as much bleeding as possible.
9. When the peritoneal membrane is reached, open it carefully with scissors, careful
not to cut any of the abdominal contents.
10. After opening the abdomen, expose the front surface of the middle lobe by lifting
the lobe out of the abdomen with two clean swabs. Check to make sure that this
221 lobe is not the right lobe (which has the gall bladder immediately behind it) or the
left lobe (which is closest to the stomach).
11. Remove a small piece of tumor from the DMEM vial using a swab and wash it in
the vial of cold HBSS. Cut off a small piece of the tumor of a size desired for the
implantation.
12. Puncture a small hole approximately 5 mm deep in the liver surface using the
outer cannula of a 24 gauge IV catheter (i.e. using only the plastic part).
13. Using small forceps, grasp the piece of tumor and place it into the puncture site,
ensuring that it is completely concealed by the liver surface.
14. Place a small piece of absorbable gelatin foam (Surgifoam, 4 mm x 4 mm x 2
mm) on top of the puncture site.
15. Suture the Surgifoam in place with a single biodegradable suture (Monocryl 6-0).
16. Check the liver for bleeding, and repair any torn areas with interrupted sutures
(Monocryl 6-0).
17. Lower the liver back into the abdomen and add 5-10 mL of saline to the
abdominal cavity.
18. Close the muscle layer (making sure to include the peritoneal membrane) with
interrupted biodegradable sutures (Vicryl, 3-0).
19. Close the subcutaneous layer with a continuous biodegradable suture (Vicryl 3-0)
suture.
20. Close the skin with interrupted nylon sutures (Ethilon 4-0).
21. Prepare the rabbit wound by placing some gentamicin ointment on the site,
covering it with a 4 x 4 gauze, and putting on the surgical sleeve.
222 22. Give the rabbit buprenex, penicillin, and 20 mL saline SQ.
23. Allow the rabbit to recover in a warm incubator and monitor every 30 minutes
until sternal.
A.6.5 Liver tumor treatment (with or without RF ablation)
1. Perform the first ten steps of as described in the liver tumor implantation section.
If ablation is to be performed, a larger area on the back should be shaved so that
the entire grounding pad can be fit on the rabbit’s back.
2. After opening the abdomen, locate the original tumor location and check for any
adhesions between the tumor site and other liver lobes, the abdominal wall,
intestinal fat, or the stomach. If there are adhesions, try to remove them gently
using a cotton swab, cutting only as a last resort, as any bleeding will have to be
repaired.
3. Expose the tumor by lifting the lobe out of the abdomen with two clean swabs.
4. Carefully puncture the center tumor with an 18 Ga needle.
5. Ablation: If ablation is being performed, insert the ablation probe in the puncture
created by the needle and begin the ablation, performing for the specified time at
the predetermined temperature.
6. Insert any polymer implants using small forceps. Make sure that the entire
implant is in the liver, while still ensuring that it does not punch through the liver.
7. Place a small piece of absorbable gelatin foam (Surgifoam, 4 mm x 4 mm x 2
mm)on top of the puncture site.
223 8. Suture the Surgifoam in place with a single single biodegradable suture
(Monocryl 6-0).
9. Check the opposite side of the liver to make sure there is no bleeding and that the
ablation probe or implant have not punched through the liver.
10. Close the abdomen as described in the liver tumor implantation.
A.7 References
1. Shah SS, Jacobs DL, Krasinkas AM, Furth EE, Itkin M, Clark TW 2004. Percutaneous ablation of VX2 carcinoma-induced liver tumors with use of ethanol versus acetic acid: pilot study in a rabbit model. J Vasc Interv Radiol 15(1 Pt 1):63-67.
2. Shope RE 1932. A transmissible tumor-like condition in rabbits. J Exp Med 56(6):793-802.
3. Shope RE, Hurst EW 1933. Infectious papillomatosis of rabbits: with a note on the histopathology. J Exp Med 58(5):607-624.
4. Rous P, Beard JW 1935. The progression to carcinoma of virus-induced rabbit papillomas (shope). J Exp Med 62(4):523-548.
5. Kidd JG, Rous P 1940. A transplantable rabbit carcinoma originating in a virus- induced papilloma and containing the virus in masked or altered form. J Exp Med 71(6):813-838.
6. Rous P, Kidd JG, Smith WE 1952. Experiments on the cause of the rabbit carcinomas derived from virus-induced papillomas: II. Loss by the vx2 carcinoma of the power to immunize hosts against the papilloma virus. J Exp Med 96(2):159-174.
7. Hazle JD, Stafford RJ, Price RE 2002. Magnetic resonance imaging-guided focused ultrasound thermal therapy in experimental animal models: correlation of ablation volumes with pathology in rabbit muscle and VX2 tumors. J Magn Reson Imaging 15(2):185-194.
8. Tsuda N, Tsuji T, Kato N, Fukuda Y, Ando K, Ishikura R, Nakao N 2005. Potential of superparamagnetic iron oxide in the differential diagnosis of metastasis and inflammation in bone marrow: experimental study. Investigative radiology 40(10):676- 681.
9. Miao Y, Ni Y, Mulier S, Yu J, De Wever I, Penninckx F, Baert AL, Marchal G 2000. Treatment of VX2 liver tumor in rabbits with "wet" electrode mediated radio- frequency ablation. Eur Radiol 10(1):188-194. 224 10. Boehm T, Malich A, Goldberg SN, Hauff P, Reinhardt M, Reichenbach JR, Muller W, Fleck M, Seifert B, Kaiser WA 2002. Radio-frequency ablation of VX2 rabbit tumors: assessment of completeness of treatment by using contrast-enhanced harmonic power Doppler US. Radiology 225(3):815-821.
11. Horkan C, Ahmed M, Liu Z, Gazelle GS, Solazzo SA, Kruskal JB, Goldberg SN 2004. Radiofrequency ablation: Effect of pharmacologic modulation of hepatic and renal blood flow on coagulation diameter in a VX2 tumor model. J Vasc Interv Radiol 15(3):269-274.
12. Purdie TG, Sherar MD, Lee TY 2003. The use of CT perfusion to monitor the effect of hypocapnia during laser thermal therapy in a rabbit model. Int J Hyperthermia 19(4):461-479.
13. Demura K, Morikawa S, Murakami K, Sato K, Shiomi H, Naka S, Kurumi Y, Inubushi T, Tani T 2006. An easy-to-use microwave hyperthermia system combined with spatially resolved MR temperature maps: phantom and animal studies. The Journal of surgical research 135(1):179-186.
14. Yoon CJ, Chung JW, Park JH, Yoon YH, Lee JW, Jeong SY, Chung H 2003. Transcatheter arterial chemoembolization with paclitaxel-lipiodol solution in rabbit VX2 liver tumor. Radiology 229(1):126-131.
15. Choi SH, Moon WK, Hong JH, Son KR, Cho N, Kwon BJ, Lee JJ, Chung JK, Min HS, Park SH 2007. Lymph node metastasis: ultrasmall superparamagnetic iron oxide-enhanced MR imaging versus PET/CT in a rabbit model. Radiology 242(1):137- 143.
16. Thorstensen O, Isberg B, Jorulf H, Svahn U, Westman L, Venizelos N, Nennesmo I 2004. Detection of small implanted tumors growing during repeated magnetic resonance imaging of the rabbit liver: application of an interpretation model. Acta Radiol 45(5):547-555.
17. Dunne AA, Mandic R, Ramaswamy A, Plehn S, Schulz S, Lippert BM, Moll R, Werner JA 2002. Lymphogenic metastatic spread of auricular VX2 carcinoma in New Zealand white rabbits. Anticancer Res 22(6A):3273-3279.
18. Osato T, Ito Y 1967. In vitro cultivation and immunofluorescent studies of transplantable carcinomas Vx2 and Vx7. Persistence of a Shope virus-related antigenic substance in the cells of both tumors. J Exp Med 126(5):881-886.
19. Lin WY, Chen J, Lin Y, Han K 2002. Implantation of VX2 carcinoma into the liver of rabbits: a comparison of three direct-injection methods. The Journal of veterinary medical science / the Japanese Society of Veterinary Science 64(7):649-652.
20. Geschwind JF, Ko YH, Torbenson MS, Magee C, Pedersen PL 2002. Novel therapy for liver cancer: direct intraarterial injection of a potent inhibitor of ATP production. Cancer Res 62(14):3909-3913.
225 21. van Es RJ, Dullens HF, van der Bilt A, Koole R, Slootweg PJ 2000. Evaluation of the VX2 rabbit auricle carcinoma as a model for head and neck cancer in humans. J Craniomaxillofac Surg 28(5):300-307.
226 Appendix B Tumor treatment animations
Figure B-1. Tumor recurrence after RF ablation.
Ablation of liver tumors often successfully treats the bulk of the tumor region, but the risk of tumor recurrence is high. Tumors can recur locally at the periphery of the ablated region or at a site in the liver relatively far from the original tumor. In the worst case scenario, tumors can metastasize to other organs, significantly worsening the prognosis for the patient.
227
Figure B-2. Millirod only treatment of liver tumors.
In Chapter 2, liver tumors in rabbits were treated by placing polymer millirods into the center of the tumor. The desired outcome is sustained delivery of drug to the tumor, ultimately leading to death of the surrounding tumor cells extending all the way to the tumor periphery.
228
Figure B-3. Combined treatment with RF ablation and polymer millirods.
To improve drug distribution from the implants, in Chapter 3 tumors were treated with
RF ablation before implanting the millirods. The goal of this strategy was to destroy the majority of the tumor with thermal ablation and kill any remaining cells with drug delivered to the tumors through polymer implants placed in the ablated region.
229 Appendix C Annotated publication list
C.1 Journal articles
Lazebnik, R.S., B.D. Weinberg, M.S. Breen, J.S. Lewin, and D.L. Wilson. Three- dimensional model of lesion geometry for evaluation of MR-guided thermal ablation therapy. Acad Radiol 2002; 9: 1128-38.
RATIONALE AND OBJECTIVES: High-radiofrequency energy is used clinically to ablate pathologic tissue with interventional magnetic resonance (MR) imaging. For many tissues, resulting lesions have a characteristic appearance on contrast- enhanced T1- and T2-weighted MR images, with two boundaries enclosing an inner hypointense region and an outer hyperintense margin. Geometric modeling of three- dimensional thermal lesions in animal experiments and patient treatments would improve analyses and visualization. MATERIALS AND METHODS: The authors created a model with two quadric surfaces and 12 parameters to describe both lesion surfaces. Parameters were estimated with iterative optimization to minimize the sum of the squared shortest distances from segmented points to the model surface. The authors validated the estimation process with digital lesion phantoms that simulated varying levels of segmentation error and missing surface information. They also applied their method to in vivo images of lesions in a rabbit model. RESULTS: For simulated phantom lesions, the lesion geometry was accurate despite manual segmentation error and incomplete surface data. Even when 50% of the surface was missing, the median error was less than 0.5 mm. For all in vivo lesions, the median distance from the model surface to data was no more than 0.58 mm for both inner and outer surfaces, less than a voxel width (0.7 mm). The interquartile range was 0.89 mm or less for all data. CONCLUSION: The authors' model provides a good approximation of actual lesion geometry and is highly resistant to missing segmentation information. It should prove useful for three-dimensional lesion visualization, volume estimation, automated segmentation, and volume registration.
230 Sawant, A., H. Zeman, S. Samant, G. Lovhoiden, B. Weinberg, and F. DiBianca. Theoretical analysis and experimental evaluation of a Csl(TI) based electronic portal imaging system. Med Phys 2002; 29: 1042-53.
This article discusses the design and analysis of a portal imaging system based on a thick transparent scintillator. A theoretical analysis using Monte Carlo simulation was performed to calculate the x-ray quantum detection efficiency (QDE), signal to noise ratio (SNR) and the zero frequency detective quantum efficiency [DQE(0)] of the system. A prototype electronic portal imaging device (EPID) was built, using a 12.7 mm thick, 20.32 cm diameter, Csl(Tl) scintillator, coupled to a liquid nitrogen cooled CCD TV camera. The system geometry of the prototype EPID was optimized to achieve high spatial resolution. The experimental evaluation of the prototype EPID involved the determination of contrast resolution, depth of focus, light scatter and mirror glare. Images of humanoid and contrast detail phantoms were acquired using the prototype EPID and were compared with those obtained using conventional and high contrast portal film and a commercial EPID. A theoretical analysis was also carried out for a proposed full field of view system using a large area, thinned CCD camera and a 12.7 mm thick CsI(TI) crystal. Results indicate that this proposed design could achieve DQE(0) levels up to 11%, due to its order of magnitude higher QDE compared to phosphor screen-metal plate based EPID designs, as well as significantly higher light collection compared to conventional TV camera based systems.
231 Lazebnik, R.S., B.D. Weinberg, M.S. Breen, J.S. Lewin, and D.L. Wilson. Sub-acute changes in lesion conspicuity and geometry following MR-guided radiofrequency ablation. J Magn Reson Imaging 2003; 18: 353-9.
PURPOSE: To evaluate MR signal and lesion zone volume evolution through the sub-acute phase following image-guided radiofrequency (RF) thermal ablation. MATERIALS AND METHODS: For many tissues, including muscle and liver, thermal lesions that result from RF heating have a characteristic two-boundary appearance featuring an inner core (zone I) surrounded by a hyper-intense margin (zone II) and normal tissue (zone III), found in both T(2) and contrast enhanced (CE) T(1)-weighted MR images, both immediately post-ablation and four days later. First, we compared corresponding points between manually segmented zone boundaries apparent in T(2)- and CE T(1)-weighted images. Second, we examined the contrast-to-noise ratio (CNR) between all zone combinations. Third, we quantified the volume of zone I, zone II, and the entire lesion using a three-dimensional lesion geometry model fitted to segmented images. RESULTS: On a slice-by-slice basis, no statistically significant differences were found between zone boundaries apparent in T(2) and CE T(1)-weighted images. The contrast to noise ratio (CNR) of zone I vs. zone II, zone I vs. background muscle, and zone II vs. background muscle was always equal or greater for T(2)-weighted images than for CE T(1)-weighted images. In addition, by day four, zone II significantly increased in intensity compared to background muscle. The median Zone I volume increase was 44.2% (42.6%) using T(2) weighted images and 55.5% (68.7% interquartile range) using CE T(1)- weighted images. This expansion likely corresponds to an enlargement of the ablated, coagulative necrosis, region. The median Zone II volume increase was 15.0% (42.6%) using T(2)- weighted images 1.5% (38.8%) using CE T(1)- weighted images. CONCLUSIONS: 1) There are no significant differences between the apparent zone boundaries in T(2)- and CE T(1)-weighted images; 2) CNR is equal or greater for T(2)-weighted images as compared to CE T(1)-images; and 3) both the inner and outer lesion zone volumes typically increase several days post-ablation.
232 Szymanski-Exner, A., A. Gallacher, N.T. Stowe, B. Weinberg, J.R. Haaga, and J. Gao. Local carboplatin delivery and tissue distribution in livers after radiofrequency ablation. J Biomed Mater Res A 2003; 67: 510-6.
This study investigated the local drug pharmacokinetics of intralesional drug delivery after radiofrequency ablation of the liver. We hypothesized that the tissue architecture damaged by the ablation process facilitates the drug penetration in the liver and potentially enlarges the therapeutic margin in the local treatment of cancer. The delivery rate and tissue distribution of carboplatin, an anticancer agent, released from poly(D,L-lactide-co-glycolide) implants into rat livers after radiofrequency ablation were quantified by atomic absorption spectroscopy. Results showed that carboplatin clearance through blood perfusion was significantly slower in the ablated livers, leading to a more extensive tissue retention and distribution of the drug. The concentration of Pt at the implant-tissue interface ranged from 234 to 1440 microg Pt/(g liver) in the ablated livers over 144 h versus 56 to 177 microg Pt/(g liver) in the normal tissue. The maximum penetration distance at which Pt level reached above 6 microg/g (calculated based on a reported IC90 value for carboplatin) was 8-10 mm and 4-6 mm in ablated and normal liver, respectively. Histological analysis of the necrotic lesions showed widespread destruction of tissue structure and vasculature, supporting the initial hypothesis. This study demonstrated that intralesional drug delivery could provide a sustained, elevated concentration of anticancer drug at the ablation boundary that has the potential to eliminate residual cancer cells surviving radiofrequency ablation.
233 Blanco, E., F. Qian, B. Weinberg, N. Stowe, J.M. Anderson, and J. Gao. Effect of fibrous capsule formation on doxorubicin distribution in radiofrequency ablated rat livers. J Biomed Mater Res A 2004; 69: 398-406.
In this study, we report the histological findings of a combined therapy using radiofrequency ablation and intratumoral drug delivery in rat livers, with special attention to wound-healing processes and their effects on drug transport in post-ablated tissue. Doxorubicin-loaded millirods were implanted in rat livers that had undergone medial lobe ablation. Millirods and liver samples were retrieved upon animal sacrifice at time points ranging from 1 h to 8 days. Results demonstrate a clearly defined area of coagulative necrosis within the ablation boundary. The wound-healing response, complete with the appearance of inflammatory cells, neovascularization, and the formation of a fibrous capsule, was also observed. At the 8-day time point, fluorescence imaging analysis showed a higher concentration of doxorubicin localized within the ablation region, with its distribution hampered primarily by fibrous capsule formation at the boundary. Given the variant nature of ablated liver, a mathematical model devised previously by our laboratory describes the data well up to 4 days, but loses reliability at 8 days. These results provide useful mechanistic insights into the wound-healing response after radiofrequency ablation and polymer millirod implantation, as well as the impact this natural corollary has on drug distribution. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 69A: 398-406, 2004
234 Exner, A., B.D. Weinberg, N.T. Stowe, A. Gallacher, D.L. Wilson, J.R. Haaga, and J. Gao. Noninvasive monitoring of local carboplatin chemotherapy using x-ray computed tomography. Acad Radiol 2004; 11: 1326-1336.
Rationale and Objectives. Computed tomography (CT) was utilized to noninvasively monitor local drug release from polymer implants in rat livers before and following radiofrequency ablation. Methods. Polymer matrixes containing carboplatin (a platinum-containing chemotherapeutic agent) were implanted into the livers of male rats either immediately after radiofrequency ablation (n=15) or without prior treatment (n=15). The animals were divided into 5 sub-groups (n=3 per group) and subjected to a terminal CT scan at 6, 24, 48, 96 or 144 hrs. Carboplatin concentration in tissue and within the implant matrix was correlated with CT intensity, and standard curves were produced for each environment. This correlation was used to evaluate the differences in drug transport properties between normal and ablated rat livers. A quantitative image analysis method was developed and employed to evaluate the release rate and tissue distribution of carboplatin in normal and ablated liver tissue. The CT data was validated by previously reported atomic absorption spectroscopy measurement of implant and tissue drug levels. Results. Correlation of carboplatin concentration and Hounsfield Units results in a linear relationship with correlation coefficients (slopes) of 15 and 4 HU/(mg/ml), for carboplatin in tissue and polymer, respectively. Noninvasive monitoring of local pharmacokinetics in normal and ablated tissues indicates that ablation prior to local carboplatin delivery increases the retention of carboplatin within the polymer matrix and drastically increases the drug retention in the ablated tissue volume (over 3-fold difference) resulting in a higher average dose to the surrounding tissue. Drug penetration is more extensive in the ablated tissue, with the drug concentration peaking at 48 hrs. At 1.6 mm from the implant boundary, carboplatin concentration is significantly higher in ablated tissue at 48, 96 and 144 hrs (p<0.05). The drug concentration in ablated tissue reaches 4.7, 3.3, and 3.6 mg/ml at 48, 96 and 144 hrs, respectively. In comparison, the concentration in normal liver at 1.6 mm reaches 0.7, 0.5, 0.9 mg/ml at 48, 96 and 144 hrs, respectively. The drug reaches 3.1 mm in ablated liver compared to 2.3 mm in normal liver at 48 hrs. After 144 hrs, the drug is still detected at 3.1 mm in ablated liver but drops to 0 mm in normal liver. The differences are significant (p<0.05) at 48 and 144 hours. Correlation with chemical analysis suggests that CT data accurately predicts the kinetics in this system and this is true in both ablated and normal livers. Conclusion. This work demonstrates that X-ray CT imaging is a useful and promising technique for in vivo monitoring of the release kinetics of locally delivered radiopaque agents.
235 Nasongkla, N., X. Shuai, H. Ai, B.D. Weinberg, J. Pink, D.A. Boothman, and J. Gao. cRGD-Functionalized Polymer Micelles for Targeted Doxorubicin Delivery. Angew Chem Int Ed Engl 2004; 43: 6323-6327.
No Abstract.
236 Ai, H., C. Flask, B. Weinberg, X.-T. Shuai, M.D. Pagel, D. Farrell, J. Duerk, and J. Gao. Magnetite-Loaded Polymeric Micelles as Ultrasensitive Magnetic-Resonance Probes. Adv Mater 2005; 17: 1949-1952.
No abstract.
237 Lazebnik, R.S., B.D. Weinberg, M.S. Breen, J.S. Lewin, and D.L. Wilson. Semiautomatic Parametric Model-Based 3D Lesion Segmentation for Evaluation of MR- Guided Radiofrequency Ablation Therapy. Acad Radiol 2005; 12: 1491-1501.
Rationale and Objectives Interventional magnetic resonance imaging (iMRI) allows real-time guidance and optimization of radiofrequency ablation of pathologic tissue. For many tissues, resulting lesions have a characteristic two-boundary appearance featuring an inner region and an outer hyper-intense margin in both T2 and contrast- enhanced (CE) T1 -weighted MR images. We created a geometric model-based semiautomatic method to aid in real-time lesion segmentation, cross-sectional/three- dimensional visualization, and intra/posttreatment evaluation. Materials and Methods Our method relies on a 12-parameter, 3-dimensional, globally deformable model with quadric surfaces that describe both lesion boundaries. We present an energy minimization approach to quickly and semiautomatically fit the model to a gray-scale MR image volume. We applied the method to in vivo lesions (n = 10) in a rabbit thigh model, using T2 and CE T1 -weighted MR images, and compared the results with manually segmented boundaries. Results For all lesions, the median error was 1.21 mm for the inner region and 1.00 mm for the outer hyper-intense region, values that favorably compare to a voxel width of 0.7 mm and distances between the borders manually segmented by the two operators. Conclusion Our method provides a precise, semiautomatic approximation of lesion shape for ellipsoidal lesions. Further, the method has clinical applications in lesion visualization, volume estimation, and treatment evaluation.
238 Weinberg, B.D., S.J. Schomisch, M. Rahmatalla, W.H. Finlay, A. Chaturvedi, G.R. Wojtkiewicz, and Z. Lee. Mapping of PET-measured aerosol deposition: a comparison study. J Aerosol Sci 2005; 36: 1157-1176.
Three-dimensional positron emission tomography (PET) can be used to assess the spatial distribution of inhaled aerosols, and with lung models of airway parameters this data can be converted into aerosol deposition information for each airway generation. Two alternative methods for extracting this generational data, the analytical and the arithmetic reconstruction technique (ART), are investigated in conjunction with two different airway branching models, monopodial and dichotomous, to determine pulmonary deposition in a canine model. All solutions revealed two regions with high deposition: the first 10 generations past the trachea (1-10) and the deepest lung generations (18-23). Post-imaging autoradiographic images were in agreement with the deposition pattern in the larger airway generations (1-10). In comparing the different techniques, the dichotomous lung model yielded large variance in mapped activity but similar concentrations because of variation in generation volumes, while ART showed a greater ability to discern small differences in deposition patterns between subjects.
239 Blanco, E., B.D. Weinberg, N.T. Stowe, J.M. Anderson, and J. Gao. Local release of dexamethasone from polymer millirods effectively prevents fibrosis after radiofrequency ablation. J Biomed Mater Res A 2006; 76: 174-82.
Recent studies show that after radiofrequency (RF) ablation, fibrosis occurs at the ablation boundary, hindering anticancer drug transport from a locally implanted polymer depot to the ablation margin, where tumors recur. The purpose of this study is to investigate strategies that can effectively deliver dexamethasone (DEX), an anti- inflammatory agent, to prevent fibrosis. Polymer millirods consisting of poly(D,L- lactide-co-glycolide) (PLGA) were loaded with either DEX complexed with hydroxypropyl beta-cyclodextrin (HPbeta-CD), or an NaCl and DEX mixture. In vitro release studies show that DEX complexed with HPbeta-CD released 95% of the drug after 4 days, compared to 14% from millirods containing NaCl and DEX. Rat livers underwent RF ablation and received either DEX-HPbeta-CD-loaded millirods, PLGA millirods with an intraperitoneal (i.p.) DEX injection, or control PLGA millirods alone. After 8 days in vivo, heightened inflammation and the appearance of a well-defined fibrous capsule can be observed in both the control experiments and those receiving a DEX injection (0.29 +/- 0.08 and 0.26 +/- 0.07 mm in thickness, respectively), with minimal inflammation and fibrosis present in livers receiving DEX millirods (0.04 +/- 0.01 mm). Results from this study show that local release of DEX prevents fibrosis more effectively than a systemic i.p. injection.
240 Krupka, T.M., B.D. Weinberg, N.P. Ziats, J.R. Haaga, and A.A. Exner. Injectable Polymer Depot Combined With Radiofrequency Ablation for Treatment of Experimental Carcinoma in Rat. Invest Radiol 2006; 41: 890-897.
OBJECTIVE:: The purpose of this study was to investigate whether an intralesional chemotherapy depot with or without a chemosensitizer could improve the efficacy of radiofrequency (RF) ablation in treatment of experimental carcinoma in rats. MATERIALS AND METHODS:: Eighteen BD-IX rats were inoculated with bilateral subcutaneous tumors via injection of DHD/K12TRb rat colorectal carcinoma cells in suspension. Four weeks after inoculation, one tumor in each rat was treated with RF ablation at 80 degrees C for 2 minutes and the other with RF ablation followed by intralesional chemotherapy with carboplatin. The drug was administered via 2 different in situ-forming poly(D,L-lactide-coglycolide) (PLGA) depot formulations either with or without a chemosensitizer. Treatment efficacy was assessed by comparing the change in tumor diameter compared with control, percent of coagulation necrosis and a rating of treatment completeness. RESULTS:: Tumors treated with ablation and carboplatin + sensitizer (n = 9) showed a diameter decrease of 49.4 +/- 24.5% at the end point relative to ablation control, while those treated with ablation and carboplatin only (n = 8) showed a 7.1 +/- 12.6% decrease. Use of sensitizer also showed increased tissue necrosis (81.9 +/- 9.7% compared with 68.7 +/- 26.7% for ablation only) and double the number of complete treatments (6/9 or 66.7%) compared with ablation control (3/9 or 33.3%). CONCLUSIONS:: From these results, we conclude that intralesional administration of a carboplatin and sensitizer-loaded polymer depot after RF ablation has the potential to improve the outcome of ablation by increasing effectiveness of local adjuvant chemotherapy in preventing progression of tumor unaffected by the ablation treatment.
241 Weinberg, B.D., H. Ai, E. Blanco, J.M. Anderson, and J. Gao. Antitumor efficacy and local distribution of doxorubicin via intratumoral delivery from polymer millirods. J Biomed Mater Res A 2007; 81: 161-170.
The purpose of this study was to evaluate the antitumor efficacy and local drug distribution from doxorubicin-containing poly(D,L-lactide-co-glycolide) (PLGA) implants for intratumoral treatment of liver cancer in a rabbit model. Cylindrical polymer millirods (length 8 mm, diameter 1.5 mm) were produced using 65% PLGA, 21.5% NaCl, and 13.5% doxorubicin. These implants were placed in the center of VX2 liver tumors (n = 16, 8 mm in diameter) in rabbits. Tumors were removed 4 and 8 days after millirod implantation, and antitumor efficacy was assessed using tumor size measurements, tumor histology, and fluorescent measurement of drug distribution. The treated tumors were smaller than the untreated controls on both day 4 (0.17 +/- 0.06 vs. 0.31 +/- 0.08 cm(2), p = 0.048) and day 8 (0.14 +/- 0.04 vs. 1.8 +/- 0.8 cm(2), p = 0.025). Drug distribution profiles demonstrated high doxorubicin concentrations (>1000 mug/g) at the tumor core at both time points and drug penetration distances of 2.8 and 1.3 mm on day 4 and 8, respectively. Histological examination confirmed necrosis throughout the tumor tissue. Biodegradable polymer millirods successfully treated the primary tumor mass by providing high doxorubicin concentrations to the tumor tissue over an eight day period.
242 Weinberg, B.D., E. Blanco, S.F. Lempka, J.M. Anderson, A.A. Exner, and J. Gao. Combined radiofrequency ablation and doxorubicin-eluting polymer implants for liver cancer treatment. J Biomed Mater Res A 2007; 81: 205-13.
Previously, biodegradable polymer implants (polymer millirods) to release chemotherapeutic agents directly into tumors have been developed. The purpose of this study is to evaluate local drug distribution from these implants in liver tumors treated with radiofrequency (RF) ablation and determine if the implants provide a therapeutic improvement over RF ablation alone. Cylindrical implants were fabricated using 65% poly(D,L-lactide-co-glycolide) (PLGA), 21.5% NaCl, and 13.5% doxorubicin. Control or drug-containing millirods were implanted inside VX2 liver tumors (11 mm diameter) in rabbits after RF ablation. Therapeutic efficacy was assessed 4 and 8 days after treatment using tumor size, histology, and fluorescence measurement of drug distribution. Tumors in both test groups recurred at the boundary of the ablated region. Therapeutic doxorubicin concentrations were found in more than 80% of the ablated area, but concentrations declined rapidly at the boundary between normal and ablated tissue. This region was characterized by a developing fibrous capsule with resolving inflammation, which restricted drug transport out of the ablated zone. The intratumoral doxorubicin implants delivered high concentrations of drug within the ablated region but only limited amounts outside the ablation zone. Future studies will focus on overcoming the fibrotic transport barrier and enhancing drug delivery to the periphery of the ablation region to prevent tumor progression.
243 Krupka, T.M., B.D. Weinberg, H. Wu, N.P. Ziats, and A.A. Exner. Effect of Intratumoral Injection of Carboplatin Combined with Pluronic P85 or L61 on Experimental Colorectal Carcinoma in Rats. Exp Biol Med 2007; In press.
Pluronic, a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymer, has been shown to enhance the cytotoxic activity of anticancer drugs in various cell lines. In the current study, we examined the effect of Pluronic P85 (P85) and Pluronic L61 (L61) on intratumoral chemotherapy of an experimental adenocarcinoma in rats. A total of 120 subcutaneous tumors (4 per rat) were inoculated in 30 BDIX rats and were treated weekly for 4 weeks with intratumoral injection of the anticancer drug carboplatin (CPt) alone or with either P85 or L61. Tumors were monitored weekly and were excised at the endpoint for histological evaluation. The effect of Pluronic on levels of intracellular ATP was explored as a possible mechanism of sensitization. Results showed that tumors treated with low dose CPt (1 mg/ml) and P85 or L61 exhibited significantly reduced growth after 28 days (112.7 ± 34.4%, n=15; 131.3 ± 55.6%, n=8) compared to tumors treated with free drug (339.4 ± 75.0%, n=16). Control tumors treated with either P85 or L61 alone or saline showed growth of 1079.4 ± 143.6% (n=16), 729.4 ± 202.2% (n=7) and 1119.2 ± 6.1% (n=16), respectively. Treatment with high dose CPt (7.5 mg/ml) led to a 79.3 ± 4.2% reduction in tumor volume, and no differences were noted with addition of P85 or L61. In vitro ATP measurements showed that P85 significantly reduced levels of intracellular ATP to 44.7 ± 1.5% of control, while L61 depleted ATP levels completely under the same conditions. Results from these studies confirm that Pluronic P85 and L61 act as potent sensitizers to carboplatin chemotherapy of the experimental colorectal carcinoma leading to a significant reduction of tumor growth in vivo when compared to carboplatin alone. ATP depletion is a possible mechanism for these observed differences.
244 Weinberg, B.D., E. Blanco, and J. Gao. Polymer Implants for Intratumoral Drug Delivery and Cancer Therapy. J Pharm Sci 2007; In press.
To address the need for minimally invasive treatment of unresectable tumors, intratumoral polymer implants have been developed to release a variety of chemotherapeutic agents for the locoregional therapy of cancer. These implants, also termed “polymer millirods,” were designed to provide optimal drug release kinetics to improve drug delivery efficiency and antitumor efficacy when treating unresectable tumors. Modeling of drug transport properties in different tissue environments has provided theoretical insights on rational implant design, and several imaging techniques have been established to monitor the local drug concentrations surrounding these implants both ex vivo and in vivo. Preliminary antitumor efficacy and drug distribution studies in a rabbit liver tumor model have shown that these implants can restrict tumor growth in small animal tumors (diameter < 1cm). In the future, new approaches, such as three-dimensional (3D) drug distribution modeling and the use of multiple drug-releasing implants, will be used to extend the efficacy of these implants in treating larger tumors more similar to intractable human tumors.
245 Weinberg, B.D., T.M. Krupka, J.R. Haaga, and A.A. Exner. A combination of sensitizing pretreatment and radiofrequency ablation evaluated in a rat carcinoma model. Radiology 2007; Accepted.
Purpose: To determine if the block copolymer Pluronic P85 (P85) sensitizes cancer cells to hyperthermia, and to examine if intratumorally or intravenously (IV) administered copolymer improves the therapeutic outcome of radiofrequency (RF) ablation tumor treatment. Materials and Methods: The effects of P85 and mild hyperthermia in vitro were tested on DHD/K12/TRb rat colorectal carcinoma cells using mitochondrial enzyme activity and cell proliferation assays. BD-IX rats were inoculated with bilateral, subcutaneous tumors which were treated with intratumoral P85 and ablation (n=13), IV P85 and ablation (n=15), or ablation alone (n=14). Treatment efficacy was assessed using weekly tumor size measurements and histology. Acute effects of P85 on size of the ablation-induced coagulation necrosis were measured in 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) stained slices in additional tumors (n=18). Results: In vitro, 7% P85 reduced overall viability by 22±5% (p=0.0002) at 43ºC compared to 37ºC, and this finding was confirmed by a cell proliferation assay. In vivo, ablated tumors increased 16±28% in volume after 14 days while tumors ablated after local and systemic P85 pretreatment decreased 55±14% (p=0.03) and 59±14% (p=0.02), respectively. TTC-measured coagulation was reduced by 44% relative to control (p=0.03) after intratumoral P85 injection but was unchanged for systemic P85. Conclusion: Tumor pretreatment with Pluronic P85 improved the outcome of RF ablation in an experimental carcinoma model by decreasing the tumor volume and residual tumor detected after two weeks. IV P85 may be superior to intratumoral P85 because it provides similar benefits and does not have an acute effect on the ablation- induced coagulation necrosis.
246 C.2 Book chapter
Weinberg, B.D., F. Qian, and J. Gao, "Development and Characterization of Dual- Release Poly(D,L-lactide-co-glycolide) Millirods for Tumor Treatment." In: Polymeric Drug Delivery II: Polymeric Matrices and Drug Particle Engineering, S. Svenson, Editor: Oxford University Press. 2006; 169-185.
In recent years, minimally invasive treatments of solid tumors, such as image- guided radiofrequency (RF) ablation, have emerged as a powerful alternative therapy to surgery for patients with unresectable tumors. One major limitation of RF ablation is frequent recurrence of tumors due to incomplete destruction of cancerous cells at the tumor boundary. Biodegradable polymer millirods composed of poly(D,L-lactide-co- glycolide) (PLGA) and impregnated with anti-cancer agents have been designed to be implanted in tumors after RF ablation to deliver drugs to the surrounding tissue and kill the remaining tumor cells. By tailoring device design, it is possible to create dual-release polymer implants that incorporate both a burst release to rapidly raise the surrounding tissue drug concentrations as well as a sustained release to maintain those drug concentrations for an extended period of time. Combination of RF ablation with local drug therapy will provide a minimally invasive paradigm for the effective treatment of solid tumors.
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