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insoluble copper(II) hydroxide precipitate. Furthermore, analysis of the topotecan loaded liposomes indicated that only the active lactone form of the drug was ...
COPPER-TOPOTECAN COMPLEXATION: DEVELOPMENT OF A NOVEL LIPOSOMAL FROMULATION OF TOPOTECAN by

A M A N D E E P S. T A G G A R B.Sc. (Chemistry, Honours), University of British Columbia, 2003

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE

in

T H E FACULTY OF GRADUATE STUDIES (PATHOLOGY AND LABORATORY MEDICINE)

The University of British Columbia April, 2006

© Amandeep S. Taggar, 2006

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Previously it has been reported that transition metal complexation reactions can be used as a method to encapsulate the anticancer drug doxorubicin into liposomes. It has also been reported that a therapeutically promising formulation of topotecan could be prepared using methods that relied on encapsulated manganese(II) ions (Mn ) and a divalent cation/proton ionophore A23187. In this formulation, however, it was not clear whether topotecan encapsulation was achieved as a result of an established pH gradient or as a consequence of transition metal cation complexation with topotecan. The studies described here assess the role of transition metal ions in the encapsulation of topotecan into liposome prepared from l,2-distearoyl-^n-glycero-3-phosphocholine (DSPC) and cholesterol (CH) (55:45, mole ratio). Liposomes with Mn , copper(II) (Cu ), zinc(II) 2+

2+

(Zn ) or cobalt(II) (Co ) ion gradients (metal inside) were prepared. Subsequently, 2+

2+

topotecan was added to the outside of these liposomes (final drug to lipid ratio (mol:mol) of 0.2) and drug encapsulation was measured as a function of time and temperature. Consistent with previous results, topotecan could be encapsulated into Mn -containing 2+

liposomes only in the presence of A23187. This result suggested that a transmembrane pH gradient was necessary for topotecan loading. No drug loading was achieved with liposomes containing Co or Zn . However, Cu -containing liposomes, in the presence 2+

2+

2+

or absence of an imposed pH gradient, efficiently encapsulated topotecan. It has been 2"F

reported that Cu

can form a complex with camptothecin and for this reason the

complexation reaction between topotecan and Cu

was characterized in solution as a

function of pH. These studies demonstrated that topotecan inhibited formation of an

insoluble copper(II) hydroxide precipitate. Furthermore, analysis of the topotecan loaded liposomes indicated that only the active lactone form of the drug was encapsulated and that the inactive, carboxylate form, could not be encapsulated. Therefore, transition metal ion complexation reactions define a viable methodology to prepare liposomal topotecan formulation.

TABLE OF CONTENTS

ABSTRACT

ii

TABLE OF CONTENTS

iv

LIST OF TABLES

vi

LIST OF FIGURES

vii

ABBREVIATIONS

viii

ACKNOWLEDGEMENTS DEDICATIONS

x xi

CHAPTER 1 - INTRODUCTION

1

1.1 Project Overview

1

1.2 Transition metals in medicine 1.2.1 Transition metal pharmaceuticals 1.2.2 Influence of transition metals on therapeutic outcome

3 4 6

1.3 Liposomes 1.3.1 Liposomes as drug carriers 1.3.2 Components of Liposomes 1.3.2.1 Phospholipids 1.3.2.2 Cholesterol 1.3.2.3 Non-conventional excipients used in bilayers 1.3.3 Liposome Classification 1.3.4 Formation of liposomes: thermodynamic considerations 1.3.5 Gel-to-liquid crystalline phase transition 1.3.6 Drug Encapsulation and retention 1.3.6.1 Encapsulation methods 1.3.6.1.1 Passive encapsulation 1.3.6.1.2 Active Encapsulation (remote loading) 1.3.6.1.2.1 Ionophores

7 7 11 11 15 16 17 19 21 23 24 26 26 27

1.4 Complexation reactions with active anticancer agents: their use in liposome formulations

28

1.5 Camptothecins 1.5.1 Camp to thecin Chemistry 1.5.2 Camptothecins in liposomes

31 32 34

iv

1.5.3 Camptothecin-metal interactions 1.6 Hypotheses and research objectives

CHAPTER 2 - MATERIALS AND METHODS 2.1 Materials 2.2 Liposome Preparation 2.3 Ion gradient preparation and topotecan loading 2.4 Topotecan assays 2.5 pH gradient and trapped volume determination 2.6 pH titrations of topotecan and/or copper containing solution 2.7 Cryogenic transmission electron microscopy (Cryo-TEM)

CHAPTER 3 - RESULTS CHAPTER 4 - DISCUSSION CHAPTER 5 - FUTURE DIRECTIONS REFERENCES

LIST O F TABLES

Table 1.1

List of FDA approved liposomal formulations of some therapeutic agents

8

Table 1.2

Melting points of some saturated alkanes and their 1-unsaturated derivatives

14

Table 3.1

Conversion of topotecanfromring closed to ring open form at 60°C in pH 7.4 buffer

58

vi

LIST OF FIGURES

Figure 1.1

Chemical structures of drugs that can complex with metal ions

5

Figure 1.2

Liposome target site accumulation - EPR effect.

10

Figure 1.3

Structure of a phospholipid molecule and some examples of head groups, acyl chains, and other excipients found in lipid bilayers

13

Figure 1.4

Basic structures of phospholipids

20

Figure 1.5

Gel-to-liquid crystalline phase transition

22

Figure 1.6

Drug encapsulation methods

25

Figure 1.7

Structures of ionophore

29

Figure 1.8

Topotecan structure and E-ring hydrolysis

33

Figure 3.1

Topotecan encapsulation into DSPC/CH (55:45) liposomes with preentrapped MnS0 at 40°C or 60°C, (A) in the presence of ionophore A23187, (B) in the absence of ionophore

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Topotecan encapsulation into DSPC/CH (55:45) liposomes with preentrapped MnS0 , C 0 S O 4 , CuS0 and ZnS0 at 60°C

47

Determination of transmembrane pH gradient before and after addition of drug/ionophore to DSPC/CH (55:45) liposomes formulated with unbuffered MnS0

49

Topotecan encapsulation into DSPC/CH (55:45) liposomes with preentrapped MnS0 , CoS0 , CuS0 and ZnS0 at 60°C in the presence of ionophore Nigericin

51

Topotecan encapsulation into DSPC/CH (55:45) liposomes with preentrapped CuS0 at 40°C or 60°C or 20°C, (A) in the presence of ionophore A23187, (B) in the absence of ionophore, (C) encapsulation in liposomes formulated with buffered pH 7.5 CuS0 solution. Inset: representative Cryo-TEM images of liposomes loaded with topotecan under various conditions

53

NaOH induced precipitation of copper in the presence or absence of topotecan

56

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Figure 3.2

4

Figure 3.3

4

4

4

Figure 3.4

4

Figure 3.5

4

4

4

4

4

Figure 3.6

vii

Figure 3.7

Influence of lactone ring hydrolysis on topotecan loading into DSPC/Ch liposomes prepared in unbuffered CuS0 solution

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Possible coordination sites in topotecan

67

4

Figure 4.1

vm

ABBREVIATIONS

ao

area of phospholipid head group

CH

Cholesterol

Co

cobalt(II) ions

2+

CPP

critical packing parameter

CPT Cryo-TEM

camptothecin cryogenic-transmission electron microscopy

Cu

copper(II) ions

2+

DNA

deoxyribonucleic acid

dsDNA DOTAP

double strand DNA 1,2-dioleoyl-3 -trimethylammonium-propane

DPSC

l,2-distearoyl-src-glycero-3-phosphocholine

EDTA

ethylene diamine tetraacetic acid

EPR

enhanced permeability and retention

ESR FATMLV

electron spin resonance freeze and thawed multilamellar vesicles

FDA

Food and Drug Administration

HBS HEPES

HEPES buffered saline N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)

HPLC

high performance liquid chromatography

IR

infrared resonance

iv

intravenous

lc

critical chain length

LUV

large unilamellar vesicles

MLV

multilamellar vesicles

Mn

manganese(II) ions

2+

MPS

mononuclear phagocytic system

NMR

nuclear magnetic resonance

NSAID

non-steroidal anti-inflammatory drugs

PC

phosphocholine

PE

phosphoethanolamine

PEG

polyethanol glycol

PI

phosphoinositol

PS

phosphoserine

SEC

size exclusion column

SHE

sucrose HEPES EDTA

viii

SUV

small unilamellar vesicles

TEA

triethylamine

T Topo-I

transition temperature

UV

ultra-violet

V

volume of a phospholipid molecule

V

volume trapped within liposomes (ul/umol)

m

Vis Zn

2+

3

H-CHE

topoisomerase-I enzyme

visible light zinc(II) ions H-cholesterylhexadecyl ether

3

ACKNOWLEDGEMENTS

This thesis in no part is an individual effort. Rather it is love and support of many people through out my life that made it possible for me to achieve this. First of all I would like to thank Marcel Bally for taking me on as a graduate student and believing in me, although there were instances even I thought I wouldn't finish this project. Thank you Marcel, for guidance, helpful discussions, resources and not letting me work on 5-FU stuff. I would also like to thank other members of Bally lab especially Euan Ramsay for answering my countless pestering questions, Anitha Thomas for all the helpful suggestions, Malathi Anantha for teaching me the art of A A , H P L C and number of other machines and gadgets around the lab, Catherine Tucker for that wonderful smile and positive attitude to lift me up whenever I encountered a problem, Janet and Brian for just being there. I would like to send special thanks to Spenser Kong for fixing "all the problems" and whose presence was direly missed in the lab during my second year. To all the members of the Advanced therapeutics group - thanks for the wonderful discussions, lunches, parties (and for not trashing my place during those parties) and lots of other fun times. Special thanks to Fuloi restaurant for shanghai noodles, Wrap-zone and G G W . . .kept me going for wee hours into the night I am also very grateful to Dr. Chris Orvig for many discussions on metals, metal chemistry and explaining me some key elements crucial to this work. I would like to extend my warmest thanks to my family. Mom, dad thank you very much for everything you have done for me. Your love and support has made it possible for me to achieve everything in my life. Deep & Jatinder thank you for always supporting me and believing in me and don't worry, I am getting somewhere and won't always be a student. Lastly, I am very grateful to Paveen for listening to all my concerns, cheering me up with her brilliant smile, editing my work, helping me practice my talks and for everything else in my life. Thank you.

x

DEDICATIONS

This thesis is dedicated to my mother and father Kewal and Kuldev Taggar Thank you for your patience and support And also to the loving memory of my uncle Darshan Singh Taggar Who passed away due to cancer in 2005

Chapter 1 INTRODUCTION

1.1

Project overview

Cancer is a disease characterized by heterogeneous populations of cells that have the ability to generate their own growth signals, thereby reducing dependence on growth inhibitory signals; signals released both internally and externally [1]. Surgery and/or radiation therapy, where applicable, are the primary modes of treatment for locally developed tumours. Although technological advances in surgical equipment, surgical techniques and radiation methodology have resulted in an improvement in prognosis of various forms of cancers, their application is limited to local and readily accessible tumours. These interventions are ineffective for patients where tumours are present in hard to reach areas and are not appropriate for use in treatment of tumours that have metastasised. The introduction of chemotherapy about half a century ago has resulted in the development of effective therapeutic interventions for metastatic disease and is now commonly used in conjunction with surgery and radiation. Unfortunately, the use of chemotherapy drugs has its problems. Primarily these include toxicity to the normal healthy tissue, such as the dividing cells of the gut and bone marrow. Moreover the use of chemotherapy drugs is also associated with development of cellular resistance and survival mechanism [2, 3]. Thus treatment may initially result in disease remission but when the disease recurs it is often more aggressive and chemotherapy resistant. Clearly, an important long-term goal is to improve the outcome of cancer patients undergoing chemotherapy and this goal has been directed towards development of new drugs as well

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as improving the activity of existing drugs. The latter includes methods, such as chemical modification of drugs through medicinal chemistry [4, 5], the use of alternative dosing schedules, such as infusions [6, 7] and metronomic dosing [8, 9] as well as the use of drug delivery systems. There are many different drug delivery technologies comprising a wide range of materials that include naturally occurring and synthetic lipids and polymers. There are benefits and drawbacks to each specific type of drug delivery technology. However, it can be argued that for intravenous applications lipid based drug delivery systems are the most advanced [10]. It has been well established that chemically superior pharmaceuticals can also be generated through formation of coordination complexes of selected agents with transition metals [11]. A number of reasons have been given to justify improved activity of metaldrug compounds. These include, generation of free radicals by transition metals [12], metal-based catalysis of radical generation by pharmaceutical agents [12-14] and protection of pharmaceuticals from metabolic mechanisms in the blood compartment. It has recently been shown that metal-drug complexation can also be used to encapsulate selected drugs into liposomes [15-17]. In this approach, for example, the ability of doxorubicin to form complexes with transition metals was utilized to drive encapsulation of the drug into pre-formed liposomes. It will, therefore, be important to evaluate the use of transition metal complexation to develop liposomal formulations for other drug classes capable of forming coordination complexes. This thesis is focused on characterizing copper-topotecan (a camptothecin derivative) complexation and establishing, for the first time, that this complexation reaction is suitable for preparing a liposomal topotecan formulation.

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1.2

Transition metals in medicine

Empirical evidence suggests that utility of metal-based therapeutics has existed for centuries. Perhaps this is best exemplified by the use of copper jewellery to treat arthritic related pains. Recent gains in theoretical understanding of metal chemistry have resulted in growth of transition metal based pharmaceuticals. Transition metals possess the ability to form coordination complexes with a wide variety of natural as well as synthetic bio-organic molecules [18-20]. This property of transition metals along with the discovery of metalloproteins and transition metal catalyzed reactions in vivo has lead to the formulation of new scientific disciplines, such as bio-inorganic chemistry [18], and medicinal inorganic chemistry [20]. With a better understanding of the nature of interactions between transition metals and their biological ligands, many newer applications of transition metals have emerged. For example, the ability of transition metals to form complexes with protein residues has been utilized to treat many infectious diseases; these include, for example, use of transition metal complexes to suppress the activity of disease causing viruses [21, 22]. Furthermore, coordination complexes of biomacromolecules and radio isotopes of transition metals, such as gadolinium (Gd), technetium (Tc) and copper (Cu) have been utilized for imaging purposes [20, 23, 24]. Nonetheless, the role of transition metals has not been limited to form complexes with proteins or being used as imaging agents; coordination complexes of many pharmaceutical agents have also been produced and shown to be more effective than the parent drugs [11, 25, 26].

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1.2.1 Transition metal pharmaceuticals

Over the past number of years transition metals have been utilized in combination with pharmaceutical agents to mediate therapeutic effects against number of illnesses through disruption of DNA. For example, the impact of DNA damaging agents can be enhanced by metal-based catalysis offreeradical production by these agents [12-14, 27, 28] or coordination complexes can be manufactured that can intercalate and damage DNA [29-31]. Examples of such coordination complexes include platinum(II) based drugs such as cisplatin and carboplatin, which are integral to the standard-of-care for malignancies of the lung, breast, testes and ovaries [29, 31, 32]. Other studies suggest that complexes of pre-existing drugs containing chemical groups capable of forming coordination bonds are also possible, and this complexation improves their therapeutic activity. For example, the metal-drug compounds of acidic anti-inflammatory arthritic drugs have been shown to be more effective than the parent acids [11, 33]. These studies also describe numerous other transition metal complexes that are incorporated in therapies against rheumatoid arthritis [33, 34]. Furthermore, metal complexes of preexisting anticancer drugs have also been shown to possess a unique potential to target cancer cells [24, 31, 35-37]. This observation prompted investigations into transition metal complexes formed with chemotherapy agents, such as anthracyclines [38, 39], nitrogen mustards [40], anti-inflammatory drugs [41] and camptothecins [14, 42, 43]. Figure 1.1 illustrates the chemical structures of the drugs that can form complexes with transition metal ions.

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OH O

OH

° o

H3C-0

CH COOH 2

anthracycline - Doxorubicin

anti-inflammatory drug Indomethacin

Cl

/

CH2 CH2 -

H C—N 3

/ \

CHo-CHo \ Cl

OH

Nitrogen Mustard

Camptothecin

O

Figure 1.1 Examples of chemical structures of some drugs that can complex with transition metal ions.

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1.2.2 Influence of transition metals on therapeutic outcome

Therapeutic agents are complexed with transition metals with the belief that the resultant compound will be more effective than the parent drug. It is believed that transition metals can enhance the efficacy of drugs via i) generation offreeradicals [12], ii) catalysis offreeradical production reactions involving therapeutic agents [14, 26, 38, 39], iii) complexation that can alter the drug's in vivo metabolism and interaction with target enzymes [26, 40, 41, 44], or iv) complexation reactions that can be used to package drugs in delivery systems, such as liposomes and polymers [15-17, 45]. Doxorubicin, an anthracycline antibiotic, is the best example where liposomal encapsulation via complexation reaction enhanced a drug's anti-tumour effect [15-17]. The authors suggested that transition metal based doxorubicin had a lower plasma elimination rate. Moreover, results showed that in this formulation a higher drug-to-lipid ratio was maintained for a longer period of time compared to other formulations. Similarly, efficacy of a large number of other pharmaceutical agents that are capable of forming coordination complexes with transition metal ions can also be improved via this methodology. This thesis is focused on use of this technology to prepare a liposomal formulation of a camptothecin analogue, topotecan. We focus on topotecan, in part, because of existing literature that indicated that camptothecin can complex with metals, such as copper (Cu) [14, 43].

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1.3

Liposomes

In the 1960's, it was recognized that artificial vesicles made from phospholipids can be used to study cell membrane properties such as membrane-membrane interaction and diffusion of molecules across the phospholipid bilayers [46-49]. Soon after the potential of these vesicles (later referred as liposomes) to carry therapeutic agents was recognized [48, 50]. Since then it has been established that liposomal mediated changes in the pharmacokinetics and bio-distribution of chemotherapeutic agents can result in improved efficacy [51-54]. Moreover, there is also some evidence that in select cases liposomal encapsulation can reverse the cellular drug resistance [55, 56]. Therefore, researchers have strived to refine liposome technology and over the past forty years many improvements have been made in this field; examples include development of longcirculating sterically-stabilized (Stealth®) liposomes [57-59], thermosensitive liposomes [60], and targeted liposomes [61-64]. With the advancement in liposomal technology many liposomal-drug formulations have made their way into the clinic (Table 1.1).

1.3.1 Liposomes as drug carriers

It is clear from past evidence that liposomes can be employed as drug carriers to improve the therapeutic index of the encapsulated agents and this improvement is a consequence of liposomal mediated changes in drug circulation lifetimes and tissue biodistribution characteristics. These properties are regulated via two processes: 1) liposomal accumulation in the target tissue, and 2) drug retention and release rate from

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Table 1.1 List of FDA approved Liposomal formulations of some therapeutic agents [65]

Therapeutic agent

Disease treated

Amphotericin- B

Systemic Fungal Infections

Cytarabine Daunorubicin

Doxorubicin

Verteporfin

Lymphomatous Meningitis AIDS-related Kaposi's Sarcoma Ovarian and Breast Cancer AIDS-related Kaposi's Sarcoma Metastatic Breast Cancer Age-related macular degeneration

Liposomal Product name

Company Name

FDA approval year

Ambelcet®

Enzon

1995/1996

Ambiosome®

Gilead Sciences

1997

Amphotec®

Alza Corp.

1997

DepoCyt®

SkyePharma/Enzon

1999

DaunoSome®

Gilead Sciences

1996

Caelyx®*

Schering-Plough

1999

Doxil®*

Alza Corp.

1995

Myocet®

Elan Corp.

2000

Visudyne®

QLT/Novartis Opthalmics

2000

*Caelyx® and Doxil® are the same products but approved under different names in different countries

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liposomes, within blood compartment and/or at the site of accumulation. Previous studies [66-69] indicate that certain tissues, such as liver, spleen, sites of solid tumours as well as regions of infection/inflammation have associated blood vessels that exhibit small gaps (fenestrations, typically 380-780nm) between adjacent endothelial cells lining the vascular endothelium. These studies, and others have also shown that macromolecules [67, 70] and liposomes [54, 71] are able to diffuse out of the blood compartment through these gaps and accumulate in the extravascular space. This process has been defined by what is called the enhanced permeability and retention (EPR) effect [67, 70] (Figure 1.2) whereby the concentration of the therapeutic agent increases at sites where there is leaky vasculature and normal tissue with associated normal blood vessel structure is exposed to lower concentrations of the drug. This alters the therapeutic outcome of the drug. For example, the liposomal encapsulation of doxorubicin improved efficacy by increasing its tumour localization [53] and decreasing the drug associated cardiac toxicity [72, 73]. Along with bio-distribution attributes, the ability of liposomes to retain the associated drug for a sufficient amount of time and release it with an appropriate rate also significantly affects efficacy. Therefore, therapeutically optimized liposomes would be able to efficiently evade normal clearance mechanisms of the body, such as mononuclear phagocytic system (MPS) and opsonin-binding, have a long circulation life time, such that maximum accumulation will occur at the target site, have optimal drug retention/release rates to exert maximum therapeutic effects. Hence, the behaviour of liposomes in vivo is the most important factor governing whether a particular liposomal formulation will be active. The three properties that affect the in vivo behaviour of liposomes are lipid composition, vesicle size, and dose. In the next three sections I will

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Figure 1.2 Liposome target site accumulation - Enhanced permeability and retention (EPR) effect. A - liposomes circulate in the blood compartment for extended periods of time where they may encounter different cell types; B - Liposomes escape blood compartment into extravaszular tumour tissue through gaps between the endothelial cells; C - liposomes release their packaged contents at the site to tumour cells; D - macrophages uptake liposomes via endocytosis, which release their contents after cellular digestion, liposomes also attach to membranes of tumour cells due to surface bound ligands that may increase the specificity. Liposomes are also retained in the site following extravasation because lymphatic drainage is not efficient in sites of tumour growth. Figure adaptedfromPHD thesis of Gigi NC Chiu.

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briefly discuss these properties but for detailed review readers are referred to [54].

1.3.2 Components of liposomes

Since they were first discovered in the 1960s to the present, liposomes have evolved in many different ways. This evolution has direct impact on the clinical utility of liposomes and is related to the improvements in liposome compositions. The early liposomes were primarily derived from natural lipids such as egg-PC [74], whereas today many synthetic and/or semi-synthetic phospholipids are being used to prepare liposomes. Figure 1.3 illustrates the main components currently used to prepare liposomes. Primarily, they are: phospholipids, cholesterol, and PEG polymers. Additionally, other components (not shown in the Figure 1.3), such as lyso phospholipids, proteins and peptides are also used in some formulations. Finally, the properties of these components are sometimes also influenced by the associated therapeutic agent.

1.3.2.1 Phospholipids Phospholipids are amphipathic molecules that form the bulk of the lipid bilayers. A phospholipid can be divided into three parts: an organic R-group containing the phosphate head group, hydrocarbon acyl chains and a glycol molecule linking the two parts (see Figure 1.3). The phospholipid molecules orient themselves in such a way that head groups are pointed toward the aqueous environment and acyl chains are directed away from the aqueous environment (readers are referred to [75-77] for extensive review of chemical principles behind this orientation). Briefly, the head groups interact with

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aqueous environment through electrostatic and dipolar interactions and through steric forces with each other, which results in this portion of the molecule orienting towards the aqueous environment. The organic R-group attached to phosphate moiety gives the head group its unique characteristics (Figure 1.3 lists some common R-groups). For example, the mono-positively charged R-groups, such as choline and ethanolamine give the head group a zwitterionic character, whereas serine (carboxylate containing) or inositol (hydroxyl containing) produce negatively charged head groups. Positively charged molecules such as trimethyammonium when attached to the glycol molecule without phosphate linker produce positively charged head groups. The charge on the head group plays an important role during its interaction with other bilayers, aqueous salts and blood components such as opsonins and lipoproteins [78]. Furthermore, it can be anticipated that head group composition will influence the interactions between transition metals and liposomes used in this thesis. For example, it has been reported that metals such as copper and silver can bind to choline head groups [79-83]. The hydrophobic acyl chains, on other hand, form the interior of bilayers and interact with each other through van-der-Waals forces [75, 84]. These acyl chains have variable chain lengths (number of carbons) and variable degrees of saturation (number of double bonds). The chain length and degrees of saturation have direct impact on the melting points of alkyl chains (Table 1.2), hence they influence the fluidity of the bilayer. The general trend is that melting temperature increases with increasing chain length and decreases with the introduction of double bonds.

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Head group

Fatty Acyl Chains

Glycol

Common Acyl Chains

Common Head groups Zwitterionic Choline (PC)

CH H C--CH,

N*

3

1

CH Ethanolamine (PE)

H C-—CH 3

Positive

CH

CH, 3

N*H

2

Melting point

Saturated

3

3

Laurie (12:0)

CH (CH )i COOH

43-44°C

Myristic (14:0)

CH (CH ) COOH

52-54 °C

Palmitic (16:0)

CH (CH ) COOH

59-63°C

Stearic (18:0)

CH (CH ) COOH

69-72 °C

3

3


N

3

2

12

14

16

Unsaturated CH

+

CH

2

0

3

Palmitoleic(16:1 )

CH (CH ) CH=CH(CH ) COOH

Oleic(18:1 )

CH (CH ) CH=CH(CH ) COOH

3

2

5

2

7

3 A9

(DOTAP)

2

3

A9

Trimethylammonium

2

3

2

7

2

7

3

Negative H Serine (PS)

I HC 3

N

+

CH

3

COOH HO Inositol (PI)

OH OH

H,C HO

Cholesterol

OH

OH

Polyethylene Glycol Example:

PEG-2000 (mw

= 2000)

Figure 1.3 Structure of a phospholipid molecule and some examples of head groups, acyl chains and other excipients found in lipid bilayers.

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Table 1.2 Melting points of some saturated alkanes and their 1-unsaturated derivatives

Melting Points

Alkane chain number of double bonds =

0

1*

D o d e c a n e (Ci H 6)

-10°C

-35°C

Tridecane (Ci H 8)

-5°C

-23°C

T e t r a d e c a n e (C H )

5°C

-12°C

Pentadecane

10°C

-4°C

H e x a d e c a n e (C H )

18°C

4°C

H e p t a d e c a n e (C H )

21°C

10°C

O c t a d e c a n e (Ci H )

30°C

18°C

2

3

2

2

14

30

(C15H32)

16

34

17

8

36

38

*the double bond is present between the first and second carbons of the acyl chain to form the following unsaturated hydrocarbons: 1-dodecene, 1-tridecene, 1-tetradecene, 1pentadecene, 1-hexadecene, 1-heptadecene and 1-octadecene.

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Therefore, the type of acyl chain dictates the packing order or state (gel or liquid crystalline, see section 1.3.5) of the bilayer at body temperature. The liposomes used in this thesis were prepared with DSPC as the primary phospholipids component. This is a zwitterionic lipid that contains two 18:0 acyl chains. The ability of this lipid to retain therapeutic agents is well established.

1.3.2.2 Cholesterol Cholesterol is a steroid derivative found in various eukaryotic cell membranes and is essential in regulating their physical and chemical properties [85-87]. Incorporation of cholesterol into bilayers decreases the membrane order of phospholipids in gel state while increasing order in the liquid crystalline state [88]. The structure of cholesterol (see Figure 1.3) contains four fused hydrocarbon rings with a seven member side chain and one hydroxyl group, making it amphipathic in nature. In lipid membranes cholesterol is capable of interdigitating between adjacent phospholipids, with hydroxyl group orienting toward phospholipid head groups and the rest of the molecule lying in the acyl chain region [85, 89]. This type of incorporation results in critical alterations in lipid membrane's physical properties, which are summarised as follows: first, cholesterol decreases the enthalpy of gel to liquid-crystalline phase transition of phospholipids and totally eliminates it above 33 mol% and certain phospholipid (e.g. DSPC) bilayers become liquid crystalline at body temperature [86, 90]; second, above the phase transition temperature, cholesterol inhibits the molecular motion of hydrocarbon chains, thus providing stability to the bilayers [90-92]; third, cholesterol prevents the interaction between phosphate head groups on adjacent phospholipids [91], thus lowering the stearic

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interactions between them; fourth, cholesterol decreases permeability of large ions and solutes [91, 93, 94]. From a liposome design and development perspective, incorporation of cholesterol has an additional benefit of increasing the liposome stability in the blood compartment [95]. This effect is primarily due to reduced lipid exchange with lipoproteins [96, 97]. Moreover, cholesterol, when present in >30 mol%, reduces the serum opsonin and protein binding to phospholipid membranes [93, 96-99] thus enabling lipid vesicles to evade the clearance mechanisms and increasing their circulation life spans. Liposomes containing only phospholipids and cholesterol are known as conventional liposomes. The formulation used in this thesis contained 45 mol% cholesterol, which was incorporated to aid liposome preparation as well as to enhance liposome stability as indicated above.

1.3.2.3 Non-conventional excipients used in bilayers Excipients other than phospholipids and cholesterol have also been incorporated into bilayers to produce liposomes with desired in vivo characteristics. For example, PEG conjugated lipids have been incorporated in the bilayers to increase their in vivo life span [58]. PEG molecules provide a coating around the bilayer reducing lipid-lipid interactions thus preventing them from aggregating, and also reducing their interactions with serum proteins [74, 100]. Therefore, PEG-coated liposomes are able to evade clearance mechanisms and have enhanced circulation life times. Lysolipids or one-legged lipids are also sometimes incorporated into liposomes to enhance their permeability at/above the phase transition temperature [101]. This

16

enhanced permeability is believed to occur via lipid segregation and lysolipid dissociation [102]. Therefore, these lipids are utilized in trigger-release thermosensitive liposomes, that deliver maximum drug to the target site, when the temperature reaches the phase transition temperature of bulk phospholipids. Other lipids that destabilize bilayers due to a change in the environmental pH are also sometimes used to make trigger-release liposomes [103]. Although not used in this thesis, these other lipid components can be used in an effort to improve the in vivo activity of the formulation methodology described here. Therefore, based on the lipid composition, liposomes can be grouped into three general categories: conventional liposomes (containing phospholipids and cholesterol), steric-stabilized liposomes (containing PEG conjugated phospholipids), and triggerrelease liposomes (containing temperature & pH sensitive lipids [60, 103]). However, within each group liposomes can be further classified into subgroups based on their size and number of bilayers.

1.3.3 Liposome Classification

Two important properties that are used to classify liposomes are: size and number of bilayers (readers are referred to [104] for an excellent review of these properties). Briefly, when lipid films are hydrated with aqueous solutions, spherical lipid vesicles with multiple bilayers are formed. The bilayers are separated by aqueous spaces and these vesicles are referred to as multilamellar vesicles (MLVs) [46]. MLVs typically range from 1-100 um and generally have low trapped volume (total internal aqueous volume

17

in ul/umol of lipid). Earlier studies in liposome research employed MLVs for drug delivery. These studies demonstrated that MLVs generally have very low drug encapsulation efficiency [105, 106], and are rapidly removedfromblood after i.v. administration [107, 108]. Although, the entrapped volume and consequently entrapment efficiency can be increased byfreeze-thawingthe MLVs to form FATMLVs [106] they are still cleared very rapidly, due to their large size [78]. Thus, MLVs are of limited value for applications that rely on i.v. administration. However, lamellarity and size can be altered by techniques such as extrusion and sonication [106, 109-111], both of which produce vesicles with single lamellae. Small unilamellar vesicles (SUVs, 20 - 40 nm) can be prepared via sonication [49]. SUVs have single lipid bilayer enclosing an aqueous core. However, due to their small size these liposomes are highly strained and thermodynamically unstable and tend to aggregate [78, 107]. Furthermore, they have low ratios of trapped volume, low encapsulation efficiencies and the possibility of asymmetric distributions of lipids between inner and outer monolayer [109]. Therefore, they have limited use as drug carriers. When MLVs are forced through polycarbonate filters of defined pore size under moderate pressure, large unilamellar vesicles (LUVs) are formed [111]. LUVs are similar to SUVs, but larger in size. These vesicles are homogeneously distributed and range from 50 - 200 nm in diameter. LUVs with trapped volume of 1 - 2 ul/umol are commoly employed in drug delivery systems because of their stability in the blood compartment [78], higher encapsulation efficiencies and their ability to extravasate and accumulate at the disease site [54, 71]. The studies described in this thesis use liposomes prepared by

18

extrusion methods designed to generate unilamellar vesicles, that exhibited size range from 80- 120 nm.

1.3.4 Formation of liposomes: thermodynamic considerations

Upon addition to aqueous solvents phospholipids exhibit polymorphic phase behaviour and spontaneously assemble into structures that are determined by their molecular shape (Figure 1.4). The structure that phospholipids adopt depends upon their associated free energy and is dictated by the critical packing parameter (CPP) [75, 112]. CPP is calculated according to equation 1.1 and is based on the geometrical representation of the phospholipid (Figure 1.4). CPP =

where

1.1

V

v = volume of the phospholipid ^ = area of the head group l = critical chain length c

If the CPP 1 they form inverted bilayer, such as Hn phase [75, 112]. However, if CPP is between A and 1, then bilayers are l

formed. Therefore, phospholipids that form bilayers generally have cylindrical geometry. Nonetheless, molecules of other shapes, such as cone-shaped lysolipids (Figure 1.4) or flat ring structured molecules such as cholesterol (Figure 1.2) can also be incorporated into

19

(A)

Hn phase

Figure 1.4 Basic shapes of phospholipids. Upon encountering an aqueous environment the shape the phospholipids adopt is based on their graphical representation and resultant critical packing parameter (CPP = v/(aol )). (A) Lipids with large headgroup area and small hydrocarbon area have cone-like structure and assemble into micelles. (B) Lipids cylindrical in shape with nearly head group and hydrocarbon areas assemble into bilayers. (C) Alternatively, lipids with small headgroup areas adopt an inverted lipid phases such as inverted hexagonal (Hn) phase. Figure adoptedfrom[75] and [112]. c

20

bilayers. These components are important for the stability and fluidity of the bilayers and help regulate many physical and chemical properties of biological and artificial membranes. DSPC, the primary lipid used for development of liposomes in this thesis, adopts a "cylinder" shape and readily assembles into bilayer structures.

1.3.5 Gel - to - Liquid crystalline phase transition

All phospholipids exhibit a well defined gel-to-liquid crystalline phase transition [90, 113,114], where phospholipid molecules of the bilayer transformfroma more ordered state to a less ordered state (Figure 1.5). In the gel state the acyl chains are rigid, tightly packed, and well ordered; in the liquid crystalline state they are less ordered, loosely packed and more fluid. Above the transition temperature (T ), the liquidm

crystalline phase is favoured, hence the phase transformation occurs. This transition depends on head-groups and acyl chains of phospholipids, aqueous environment (pH, presence of ions etc.) and presence of macromolecules in the bilayer. For an in depth review of these parameters, readers are referred to [84, 115-121]. Briefly, the degree of hydration of head groups and phase transition are inversely related, i.e. more hydrated head groups have lower T . On the other hand the pH and ionic strength of the aqueous m

solution surrounding the bilayer have direct impact on the T , (i.e. increasing the pH m

and/or salt concentration increases T ). The presence of alkaline or transition metal m

cations also increases T . Acyl chains also have a profound effect on the membrane m

phase transition as an increase in the alkane chain length increases the T , due to an m

increase in chain melting temperature (Figure 1.3 and Table 1.2).

21

Below phase transition temperature; lipid chains are ordered

Above phase transition temperature; lipid chains are disordered and entropically favoured

Figure 1.5 Gel-to-liquid crystalline phase transition. Generally, the permeability of the bilayer increases as the chains go from well ordered gel state to a less ordered liquidcrystalline state.

22

On the other hand, the presence of double bonds decreases the T , due to a decrease in m

chain melting temperature (see Table 1.2). The presence of macromolecules, such as proteins and cholesterol also influences T . For example, incorporation of cholesterol in m

a phospholipid bilayer disrupts the ordering of lipid chains thus increasing its fluidity. Previous studies [91] have shown that cholesterol-lipid contacts are more favourable than lipid-lipid contacts. Therefore from 0-33 mol% of cholesterol, T gradually broadens m

and it is virtually eliminated above 33 mol% [87-89, 122]. The phase transition is an important characteristic of lipid bilayers as it has direct implication on the permeability characteristics. Therefore in order to utilize liposomes as effective delivery tools, it is crucial to understand the bilayer permeability characteristics of ions, drugs and biological molecules in vitro (for drug encapsulation) and in vivo (for drug retention/release). DSPC, the primary lipid used in this thesis goes through phase transition at 55 °C, however, incorporation of 45 mol% CH eliminated this transition.

1.3.6 Drug Encapsulation And Retention

The retention of the encapsulated agent within the liposomes is crucial to engender an improvement its efficacy. As discussed in sections 1.3.2.1, 1.3.2.2 and 1.3.2.5 the type of phospholipids, incorporation of cholesterol and other excipients and the gel-to-liquid crystalline state transition play an important role in drug retention. In addition to them the amount of drug that is encapsulated within liposomes i.e. the drug-to-lipid ratio also play an important role in drug retention attributes. Recent work by Johnston et al. [123] shows that an increase in the drug-to-lipid ratio decreases the drug release rate from the

23

liposomes, hence improved retention of the drug. One way to achieve higher drug to lipid ratio is by optimizing the encapsulation process discussed in next section.

1.3.6.1 Encapsulation methods

The underlying assumption in employing liposomes as delivery systems is that pharmaceuticals can be readily and efficiently encapsulated into liposomes. Relatively few drugs are fully soluble in aqueous solutions, and most exhibit a substantial lipophilic character and may behave as weak acids or bases. In choosing a method to encapsulate the candidate drug, one must consider its chemical and physical properties, efficiency of encapsulation, stability and retention inside liposomes, and final drug-to-lipid ratios [124, 125]. Figure 1.6 illustrates two methods commonly employed to encapsulate drugs into liposomes. In general, the candidate drug can be entrapped either during liposome formation (passive encapsulation) or by loading into pre-formed liposomes (remote loading/active encapsulation). Passive loading is based on empirical evidence that hydrophilic substances will partition into the aqueous compartment. On the other hand active loading is based on observations made by Albert Roos [126], that, "preferential permeation and distribution of weak acids across the cell membranes is due to a difference in the internal and external pH." Currently, the active encapsulation can be achieved via artificially generating transmembrane gradients of ions, such as K [127, +

128] or protons [128, 129], or of transition metals [15-17].

24

a) Passive loading

Figure 1.6 Drug encapsulation methods. A - passive loading, drug is added during rehydration and un-encapsulated drug is separated by Size Exclusion Column (SEC); B active loading using pH gradient generated by either citrate buffer, (NFLj^SCv or an ionophore. Generally, hydrophilic drugs get trapped in the aqueous core of liposomes and lipophilic drugs partition into the bilayers.

25

1.3.6.1.1 Passive Encapsulation

Figure 1.6a illustrates the passive encapsulation method in which the candidate drug is added to the dried lipid film prior to the rehydration step of the liposome preparation. Depending on the physical and chemical properties of the drug, it can either incorporate into the lipid membrane (lipophilic drugs, with high methanol-water partition coefficient) or get trapped in the aqueous spacings between bilayers (hydrophilic drugs with low partition coefficient). If the drug is highly soluble in water high encapsulation efficiency can be achieved [130].

1.3.6.1.2 Active Encapsulation (remote loading)

In contrast to passive encapsulation, many drugs can be loaded into pre-formed liposomes in response to a pre-established transmembrane gradient of ions such as K , or +

H . The transmembrane gradients can be established by either: (i) translocation of K , +

+

which generates a difference in transmembrane theta (i|/) potential [111, 127], and facilitates drug permeation, or (ii) formulating liposomes with buffers exhibit substantial difference in pH, such as citrate (pH 4.0) on the inside of liposomal core and HBS (pH 7.5) on the outside, or (iii) by formulating liposomes containing ammonium sulphate ((NFL^hSCV) solution, whereby one molecule of NH3 moves out and leaves behind one proton to create a pH gradient, or alternatively, (iv) by incorporating ionophores such as A23187, which transport one divalent cation to the outside of liposomes in exchange for

26

two protons, thus creating a pH gradient. In general, the pH gradient loading technique is applicable to drugs that contain ionizable amine groups [131, 132]. This has resulted in numerous liposomal formulations currently in various stages of investigation process [132]. Briefly, prior to encapsulation the drug is uncharged and permeates across the lipid bilayer. Upon reaching the inside and encountering low pH environment the drug is protonated, hence charged. Since charged species are less permeable [133], the encapsulated drug is effectively trapped inside the liposome aqueous core. Over the past few years many drugs have been loaded into liposomes using wellestablished pH gradient loading techniques. Though citrate and (NH4)2S04 containing pH gradient techniques are used more often, formulations exhibiting pH gradient in presence of ionophore A23187 are also gaining interest.

1.3.6.1.2.1 Ionophores

Ionophores are substances that facilitate movement of ions into and/or through organic phases such as lipid membranes [134-136]. The majority of ionophores contain variety of oxygen atoms in heterocyclic rings or linear configurations, hydroxyls and/or ketonic carbonyls as well as carboxyls that focus upon a sphere which can bind to the appropriately fitting cations via ion-dipole interactions. Figure 1.7 illustrates two ionophores mentioned in this thesis and illustrates binding of a cation by an ionophore. In select liposomal formulations, ionophores such as A23187 (Figure 1.7) have been used to generate a transmembrane pH gradient. Briefly, the liposomes are prepared 2+

with a solution that contains divalent metal cations, such as manganese (Mn ) ions at

27

low pH. The ionophore A23187 is added to the bilayers of pre-formed liposomes and it translocates two protons in exchange for one divalent cation, thus maintaining the pH gradient. Investigators reporting this pH loading technique suggest that the drug uptake is primarily due to the established pH gradient, however, the presence of the transition metal ion gradient in these formulations cannot be dismissed. For example, studies [1517] show that doxorubicin can be loaded into Mn -containing liposomes even in the 2+

absence of ionophore and these investigators propose that this encapsulation is due to interaction between doxorubicin molecules and M n

2+

ions. This observation and the

plethora of evidence indicating the ability of transition metals to bind organic molecules, biological substances, nucleic acids and chemotherapy drugs warrants further investigation into drug loading techniques relying on metal-drug interactions. Previous evidence shows that transition metal ions are able to form complexes with anthracyclines [38, 39, 137], nitrogen mustards [40], non-steroidal anti inflammatory drugs [41, 138] and camptothecins [14, 43]. Therefore, it is reasonable to investigate the applicability of transition metal complexation as a possible mechanism to encapsulate drugs into liposomes.

1.4

Complexation Reactions with active anticancer agents: their use in liposome formulaions

Previously, the complexation reactions between transition metals and active anticancer agents were used to improve the therapeutic value of selected agents

28

Figure 1.7 Structures of ionophores. (A) Nigericin - a monovalent cation ionophore that translocates a M+ ion in exchange for a proton; (B) A23187 - a divalent cation ionophore that translocates a M++ ion in exchange for two protons; (C) illustration of ionophore oxygen atoms focusing on and binding a cation.

29

[38-40, 137]. Recently, it has been reported that transition metal complexation reactions can also be used to drive the accumulation of anticancer drugs like doxorubicin into 2~F

liposomes [15-17]. These studies used encapsulated manganese (Mn ) ions. When doxorubicin was added to the outside of Mn -containing liposomes the drug permeated 2+

across the liposomal lipid bilayer and subsequently formed a complex with Mn ; a reaction that involved deprotonation of the hydroxy-anthraquinone moiety and an associated formation of a 6-membered chelate compound [16]. These encapsulation methods relying on the use of transition metal complexation reactions to drive the accumulation of drugs into preformed liposomes were novel. In the case of doxorubicinMn complexation, the loading reaction was shown to occur in the absence of a 2+

transmembrane pH gradient [16, 17]. This observation was surprising for several reasons. First, others have described doxorubicin loading methods that were dependent on the use of liposomes with a pH gradient [132, 139, 140] and, further, these studies strongly suggested that the stability of the resulting formulation was dependent on maintaining that pH gradient following loading [139, 141]. However, the loading reaction that relied on Mn

complexation to encapsulate

doxorubicin was not dependent on the pH gradient. Furthermore drug release rates, albeit faster than those observed for liposomal formulations with a pH gradient, were slow [16]. It was postulated that the rate of drug release in these formulations was dependent on the dissociation rate of the metal-drug complex. Further, it is believed that this strategy would be applicable to other drugs that possess coordination sites capable of complexing transition metals. The typical coordinating sites within drug molecules include alcohols, carboxylic acids, thiols, amines (primary, secondary and tertiary), Schiff bases, ketones,

30

esters and amides. In particular, drugs already known to have chemical groups capable of complexing transition metals are anthracyclines in addition to doxorubicin [38, 39], bleomycin [142, 143], antibacterial antibiotics such as amikacin [13], nonsteroidal antiinflammatory drugs (NSAIDs) [41, 138] as well as camptothecins [14, 43]. See Figure 1.1 for the structures of these drugs.

1.5

Camptothecins

The camptothecins represent an emerging class of effective anticancer drugs that are known to form coordination complexes with transition metal ions [14, 43]. The camptothecins, originally extractedfroma Chinese plant called camptotheca acuminata, inhibit the nuclear enzyme topoisomerase I (Topo-I). Topo-I mediates temporary DNA strand breaks that are necessary for DNA synthesis [144, 145]. Briefly, during replication, topo-I forms a cleavable complex with dsDNA and mediates temporary single strand breaks which relieve the torsional strain of the DNA molecule. Upon arrival of the replication fork this cleavable complex dissociates, and single strand break points are re-ligated during this dissociation step. Camptothecins stabilize the DNA-Topo-I cleavable complexes, an action that ultimately leads to apoptosis [146-148]. The first analogue of the camptothecins, though believed to be highly potent against cancer, was of limited use due to its poor water solubility [146]. An alternative method to improve efficacy of camptothecin was proposed, in which a coordination complex of the drug and copper ions (Cu ) was formed and irradiated with UV-light to 2+

induce DNA damage [43]. Nonetheless, efforts were continued to produce water-soluble

31

analogues of camptothecin. Currently, two water-soluble analogues, irinotecan and topotecan are approved by the US Food and Drug Administraion (FDA). Regardless of the incorporation of camptothecins into chemotherapy regimens, the outcome has not substantially improved. This is in part due to the fact that the biological activity of all camptothecins is dependent on the integrity of E-ring «-hydroxy-5-lactone (Figure 1.8).

1.5.1 Camptothecin Chemistry

The E-ring a-hydroxy-r5-lactone moiety of camptothecins is believed to be integral for their biological activity, therefore it is preserved throughout all analogues. However, the lactone ring (closed form) undergoes pH dependent reversible hydrolysis to an inactive carboxylate form (ring open form) (Figure 1.8) [149]. Under physiologic conditions (pH 7.2) the equilibrium shifts to therightand the carboxylate form of the drug is favoured [149]. Serum proteins, such as albumin have high affinity for the carboxylate form of the drug [150, 151], thus shifting the equilibrium more to the right. Therefore, the levels of biologically active lactone form are reduced in the presence of serum proteins. It is suggested that this conversion has great impact on the pharmacokinetics, bio-distribution and clearance of camptothecins [152]. Moreover, the openringform is considered inactive, due to reduced membrane permeability and decreased affinity for the active site of topo-I [144, 145].

32

Figure 1.8 Topotecan structure and E-ring hydrolysis. All camptothecins undergo pH induced reversible hydrolysis of the E-ringfromlactone to carboxylate (carboxy) form. The carboxylate form is believed to be biologically inactive.

33

1.5.2 Camptothecins in liposomes

Methods to achieve stabilization of the camptothecins in the active form have been actively researched and one approach that has garnered interest involves use of liposome encapsulation. Liposomal formulations of various camptothecin analogues have been produced and tested for efficacy [130, 153-157]. The liposomal mediated alterations in pharmacokinetics, clearance rate, biodistribution, bioavailability and stabilization of the lactone moiety will engender an improvement in therapeutic outcome in camptothecin therapy. For example, Burke et al. demonstrated, that topotecan (a water soluble camptothecin derivative) can be encapsulated into liposomes prepared in a low pH (pH 5) topotecan containing buffer [130] and the liposome associated drug was stabilized in the lactone form due to the low internal pH of the liposome. Abraham et al. and later Tardi et al. described the development of a therapeutically effective liposomal formulation of topotecan prepared using an internally trapped transition metal (Mn ) ions [158, 159]. 2+

The method used, however, appeared to be dependent on use of a transmembrane pH gradient and there was no evidence suggesting that the encapsulated metal ions interacted with topotecan [158]. In brief, addition of a transmembrane ionophore (A23187) that translocates two protons to the interior of liposomes in exchange for a divalent cation (e.g. Mn ) was essential for topotecan accumulation. This method was, therefore, 2+

analogous to other previously described techniques that relied on use of a pH gradient (acidic interior) to drive drug uptake. Liu et al. demonstrated, for example, that topotecan can be encapsulated into liposomes at ratios of 0.185 wt/wt (drug-to-lipid ratio) using encapsulated (NH^SC^ as a means to generate a pH gradient across the lipid bilayer

34

(acidic inside) and drive topotecan accumulation[160]. As expected on the basis of the pioneering workfromThomas Burke's laboratory, these formulations, with their acidic interiors, helped to maintain the encapsulated camptothecin derivative in its lactone form. This thesis is focused on the development of a liposomal formulation of topotecan, which relies on a coordination complexation reaction to drive encapsulation.

1.5.3 Camptothecin-metal interactions

Coordination complexes of camptothecins (copper-camptothecin, Cu-CPT (1:1)) were first described by Kuwahara et al. [42, 43]. The coordination complexes were pursued in part to overcome the hydrophobicity of the parent compound, however, it was found that in the presence of UV-radiation the Cu-CPT was able to cause DNA damage via free radical generation. These findings were later confirmed by Brezova et al. [14] through ESR techniques, who also suggested coordination of CPT via hydroxyl on the Ering (see topotecan structure Figure 1.6). An interaction at this level would be interesting as it could potentially stabilize the biologically active lactone moiety. The evidence of camptothecins being able to form a coordination complex with copper in combination with the fact that liposomal formulations of camptothecins can be developed form the basis for this thesis.

35

1.6

Hypotheses and research objectives

Previously it has been shown that topotecan can be efficiently encapsulated by using the transition metal ion (Mn ) and the ionophore A23187 to generate a pH 2+

gradient. The drug was maintained in the lactone form as exhibited by increased efficacy [158, 159]. However, the question pertaining to the direct role of transition metal ions in the encapsulation process was left unanswered. Evidence shows that camptothecins have the ability to form complexes with select transition metal ions. The studies presented here were designed to ask the question whether transition metal ion complexation could be used to prepare, in a pH gradient independent fashion, a liposomal formulation of topotecan that is maintained in the lactone form. The specific objectives for the research contained in this thesis are as follows: i)

To demonstrate that encapsulated Cu ions could drive the accumulation of 2+

topotecan. ii)

To establish that this loading reaction was not dependent on a transmembrane pH gradient and the resulting formulation maintained the drug in the lactone form.

iii)

To establish the presence of an interaction between C u

2+

and topotecan using

solution chemistry.

36

Chapter 2 MATERIALS AND METHODS

2.1

Materials

l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) was purchased from Avanti Polar Lipids (Alabaster, AL). Topotecan hydrochloride for injection (Smithkline Beecham Pharmaceuticals, ON, Canada) was purchased from the pharmacy of the British Columbia Cancer Agency (Vancouver, BC, Canada). It is also known as Hycamtin® for injection and is supplied as a sterile lyophilized, buffered, light yellow to greenish powder available in single-dose vials. Each vial contains topotecan hydrochloride equivalent to 4 mg of topotecan asfreebase. The reconstituted solution ranges in color from yellow to yellow-green. Inactive ingredients in the vial are mannitol, 48 mg, and tartaric acid, 20 mg. Hydrochloric acid and sodium hydroxide may be used to adjust the pH. The solution pH for the reconstituted drug ranges from 2.5 to 3.5. [ H]cholesterylhexadecyl ether ( H-CHE) and Pico-Fluor 15 scintillation cocktail was 3

purchasedfromPerkinElmer Life Sciences Products (Woodbridge, ON, Canada). [ C]14

methylamine hydrochloride was purchasedfromGE Healthcare (Piscatatway, NJ). Cholesterol (CH), ionophores A23187 & Nigericin, Sephadex G-50 size exclusion gel and other chemicals (reagent grade) were purchasedfromSigma-Aldrich (Oakville, ON, Canada).

37

2.2

Liposome Preparation

DSPC and CH were dissolved in chloroform at a 55:45 molar ratio. H-CHE was 3

added to achieve a specific activity of 5uCi/100 pmol of lipid. The bulk chloroform was removed with a gentle stream of nitrogen and the residual chloroform was removed by placing the sample under high vacuum for approximately 3 hours. The dried lipid films were rehydrated with 300 rnM solutions of: i) CuSC^, ii) MnS04, iii) C0SO4, iv) ZnSCU. The pH of each of the rehydration solutions was adjusted to 3.5 with 1M HC1 or to 7.5 by using HEPES and triethyl amine (TEA). The resulting multilamellar vesicles (MLVs; final lipid concentration of lOOpmol/ml) were subjected to five freeze-thaw cycles (freezing in liquid nitrogen and thawing at 65 °C) followed by extrusion 10 times through two stacked polycarbonatefilters(0.1 and/or 0.08pm) at 65 °C using water a jacketed extruder (Northern Lipids, Inc., Vancouver, Canada). The mean diameter of the resultant large unilamellar vesicles (LUVs) was 100-120 nm as determined by quasi-elastic light scattering techniques using NICOMP sub-micron particle sizer model 370/270, operating at X= 632.8 nm and/or a Brookhaven ZetaPals particle sizer operating at X= 660.0 nm. Liposomal lipid concentration was measured through use of the radiolabled marker [ H]3

CHE which was detected by liquid scintillation counting methods (Packard 1900TR Liquid Scintillation Analyzer). Samples prepared for cryo-EM analysis in the laboratory of Dr. Katerina Edwards (Uppsala University, Sweden) could not be radiolabeled and for these samples the lipid concentration was determined by the Fiske and Subbarow phosphate assay [161].

38

2.3

Ion gradient Preparation and topotecan loading

The LUVs formed by extrusion were sequentially passed through columns packed with Sephadex G-50 beads equilibrated with 300 mM sucrose/20 mM Hepes/15 mM EDTA (SHE) pH 7.5 to remove metal ionsfromthe liposome exterior and to facilitate dissociation of any metals bound to the outer leaflet of the liposomal membrane. Subsequently, these liposomes were exchanged into 150 mM NaCl/25 mM Hepes (HBS) solution to remove EDTA. The resulting liposomal formulations contained the indicated metal ion solution (typically an unbuffered solution that was at pH 3.5) inside and were suspended in HBS at pH 7.5. To facilitate drug loading, the liposomes and topotecan solution were pre-incubated at the indicated temperatures for 10-15 minutes. Topotecan was then added to the liposomes (5mM) with vortex mixing to achieve a final drug-tolipid ratio (mol/mol) of 0.2. Following the addition of topotecan to the liposomes, the pH of the resulting solution was measured and adjusted to pH 7.5 with NaOH if necessary. The resulting mixture was incubated at the indicated temperature and at specified time points, encapsulated and unencapsulated drug were separated by placing lOOpl aliquots onto 1 ml Sephadex G-50 spin columns pre-equilibrated with HBS. Liposomes were collected in the eluate following centrifuging of the columns at 680 g for 3 minutes. In experiments where A23187 was used, the ionophore (0.2 pg/pmol lipid) was added to the liposomes 10 minutes prior to drug loading, at the same temperature at which loading was performed. In the experiments where pH gradient was collapsed by the addition of nigericin, the ionophore was added at a final concentration of 0.1 pg/umol of lipid.

39

2.4

Topotecan assays

The concentration of encapsulated topotecan was measured by an absorption at its A.max = 382nm using the Hewlett Packard UV-Vis spectrophotometer (model 8453). Briefly, a portion of the samples collected from the spin columns was adjusted to a final volume of 100 uL with HBS. Subsequently 900 uL Triton X-100 1% was added and the samples were heated in a water bath at >90 °C until the cloud point of the detergent was observed. The samples were then cooled to room temperature, and the absorbance was determined against a freshly prepared topotecan standard curve. Lipid concentration was determined by measuring the amount of H-CHE, by liquid scintillation counting 3

(Packard 1900TR Liquid Scintillation analyzer), in a portion of the sample collected from the column that was added to 5 ml of Pico-Fluor 15. Where indicated, HPLC analysis of topotecan was preformed in order to measure the amount of drug in the ring open (carboxy) and ring closed (lactone) form. HPLC analysis was performed by using the Waters Chromatograph model 6475. The samples were prepared by diluting samples into 100% ice cold methanol. The two forms of topotecan were separated using 75 x 3.5 mm symmetry-C18 column using 3% triethyleneamine as aqueous mobile phase and mixture of methanohacetonitrile (30%:70%) as the organic phase. The sample temperature was kept at 4 °C and the column temperature was adjusted to 40 °C. Each sample was run for 15 minutes using a gradient method, where the amount of organic phase was increasedfrom10% to 25% over 9 minutes. The two analytes were detected using a Waters 675 fluorescence detector using an excitation wavelength (k) of 380 nm and emission wavelength of 525nm.

40

2.5

pH Gradient and Trapped Volume Determination

The pH gradient across the membranes of selected liposomal formulations was determined by using the radioactive probe, [ C]-methylamine. Briefly, [ C]14

14

methylamine (0.3uCi/umol lipid) was added to liposomes and allowed to equilibrate for 30 minutes at 60 °C. Aliquots of samples were then passed through 1 mL Sephadex G-50 spin columns to separate the trapped and untrapped [ C]-methylamine. The ,4

concentration of liposomal lipid and [ C-]-methylamine in the sample prior to and 14

following fractionation on the spin column was determined by measuring the [ H] and 3

[ C] radioactivity on a Packard 1900TR Liquid Scintillation Analyzer using a calibrated 14

dual label counting program. The transmembrane pH gradient was then calculated by equation 2.1:

ApH

= log{[H ]in ide/[H ] +

+

S

outS

j } de

= log{[methylamine]i ide/[methylamine] id } ns

outS

e

2.1

To calculate the concentration of methylamine trapped inside, the trapped volume of the liposomes was estimated based on trap volume assessments completed using liposomes prepared under comparable conditions. Briefly, lipid films were hydrated with a buffer containing trace levels of [ C]-lactose. The trapped volume (V) was then 14

calculated using measured values of [ H] (lipid) and [ C] (lactose) before and after 3

I4

removing encapsulated lactose as described elsewhere [162]. It was assumed that under the conditions described, the exterior pH did not change. It should be noted that transmembrane pH gradient studies were completed only

41

with Mn -containing liposomes since results with the Cu -containing liposomes clearly 2+

2+

suggested that the Cu was binding the pH probe methylamine. Thus it should be noted 2+

that results obtained using the Mn -containing liposomes would be comparable to 2+

liposomes containing other metal ions.

2.6

pH titrations of Topotecan and/or copper containing solutions

Samples containing C U S O 4 , topotecan and CuSC^ plus topotecan were titrated with NaOH and the amount of each reagent remaining in the solution, after separation of precipitated material by centrifugation at 10,000 g in a microfuge, was measured by either UV-Vis spectroscopy (topotecan) or atomic absorption (AA) spectroscopy (Cu ). Briefly, aliquots of C u and topotecan containing solutions were added to eight different 2+

microcentrifuge tubes to achieve a final concentration of ImM of the indicated reagent. To each tube NaOH was added incrementally, such that the final concentration of NaOH would be titratedfrom0 to 2.4 mM. The samples were incubated at room temperature for 30 minutes prior to being centrifuged at 10,000 g for 30 minutes. The supernatant was measured for: (i) pH, (ii) topotecan concentration using spectrophotmetric analysis as described above and (iii) copper concentration using AA spectroscopy (Perkin Elmer AAnalyst 600 operating at 324nm).

42

2.7

Cryogenic Transmission Electron Microscopy (Cryo-TEM)

DSPC/CH liposomes for Cryo-TEM were prepared by using methods described earlier without the H-CHE lipid marker. Drug loaded and 'empty' liposomes were 3

visualized with cryo-TEM methods using a Zeiss EM 902A Transmission Electron Microscope. Briefly, a portion of the sample was equilibrated at 25 °C within a climate chamber and a small drop (1-2 uL) of sample was then deposited on a copper grid covered with perforated cellulose acetate butyrate. Excess liquid was removed by means of blotting with a filter paper, leaving a thin film of solution on the grid. Immediately after blotting the samples were vitrified in liquid ethane, held just above its freezing point (108K), to minimize the sample perturbation and formation of ice crystals. The grid was then transferred to the microscope, where observations were made at 80kV in zero loss bright-field mode.

43

Chapter 3 RESULTS

Previously, it was well established that doxorubicin [17, 163, 164], vincristine [125], and topotecan [51, 159, 160] can be loaded into liposomes using pH gradient techniques, however some of the methods also utilized encapsulated M n

2+

in combination

with an ionophore to generate the transmembrane pH gradient. In the case of doxorubicin, it was demonstrated that the pH gradient is not actually required to achieve efficient loading of the drug, a result that was explained, in part, by the fact that M n

2+

could form a transition metal complex with doxorubicin. A primary objective of this study was to establish whether topotecan encapsulation into liposomes could also be accomplished through the use of an entrapped metal ion capable of forming a complex with topotecan. This objective was pursued in part because of published reports demonstrating that camptothecins are able to form complexes with selected transition metal ions, in particular C u

2+

[14, 42, 43].

The initial study assessed topotecan accumulation into DSPC/CH liposomes prepared in unbuffered 300mM MnSC>4 (pH ~ 3.5) in the presence and absence of the ionophore A23187. Topotecan was added to the liposomes such that the final drug-tolipid ratio was 0.2 (mol/mol) and thefinalliposomal lipid concentration was 5mM. The results, summarized in Figure 3.1, indicated that topotecan encapsulation was evident when the drug was added to liposomes exhibiting a MnSC^ gradient plus A23187 and when the incubation temperature was 60 °C (Figure 3.1 A; O). The loading efficiency

44

0.05 -\ — —I

20

#

t

1

1

1

T

30

40

50

60

T i m e (minutes)

Figure 3.1: Topotecan encapsulation into DSPC/CH (55:45) liposomes with the MnSCu loading procedure. Liposomes were prepared using (A) MnS04 (pH 3.5) with added transmembrane ionophore A23187, and (B) M n S 0 gradient. The outer solution was exchanged with p H 7.5 HBS using column chromatography in order to create ion gradients. A23187 was added to MnSC>4 containing liposomes prior to adding the drug and incubated for 10 minutes. Topotecan was added to liposomes to achieve a final drugto-lipid ratio of 0.2 (mol/mol) and incubated at 60 °C (O) and 40 °C (•) for 60 minutes. At 5, 10, 30 and 60 minutes aliquots were fractionated on 1ml spin columns to separate encapsulated drug (collected in the eluted volume) from unencapsulated drug. Lipid and topotecan concentrations were quantitated. Data points represent mean drug-to-lipid ratios of at least three replicate experiments and the error bars indicate the standard deviation. 4

45

was approximately 75% and this was achieved within 30 minutes of adding the drug to the liposomes. At a lower incubation temperature of 40 °C (Figure 3.1 A; •), or in the absence of A23187 (Figure 3.IB), no drug accumulation was observed. These results indicate that topotecan loading into Mn -containing liposomes was 2+

dependent on the presence of a pH gradient. Three reasons could explain the absence of drug loading under ionophorefreeconditions: (i) topotecan can not form a complex with Mn under the conditions used; (ii) Mn is not the appropriate metal; (iii) the belief that 2+

2+

topotecan could be encapsulated into liposomes through use of a metal complexation reaction was incorrect. To address the first two explanations, two experiments were completed. The first assessed topotecan uptake into four liposome formulations prepared with different transition metal ions (see Figure 3.2). DSPC/CH lisposomes were prepared so that unbuffered 300 mM solutions of C0SO4, CuS0 , MnS0 and ZnS0 (pH ~ 3.5) 4

4

4

were trapped inside, while the external solution was HBS (pH 7.5). Topotecan was added (0.2 drug to lipid ratio, mol:mol) to these liposomes which were pre-eqilibrated at 60 °C. The results, summarised in Figure 3.2, indicated that for the CoS0 (•) and MnS0 (A) 4

4

containing liposomes there was no appreciable drug uptake over the entire course of time. Some topotecan encapsulation was noted for liposomes prepared in 300 mM ZnS0 (O), 4

but the extent of encapsulation was low (0.04 mokmol, 4 solution were subsequently exchanged into a pH 7.5 HBS solution to create a transmembrane Cu gradient in the absence of accompanying pH gradient. Topotecan accumulation into these liposomes after incubation at 20 °C, 40 °C and 60 °C is summarized in Figure 3.5C. The results indicated that at 40 °C (•) and 60 °C ( O ) the rate and extent of drug

52

(A) C u S 0 plus A23187 4

10

20

30

40

50

60

Time (minutes) Figure 3.5: Topotecan encapsulation in DSPC/CH (55:45) liposomes using three different loading procedures. Liposomes were prepared using (A) C U S O 4 (pH 3.5) inside plus transmembrane ionophore A23187, (B) C U S C M (pH 3.5) inside, and (C) CuSCu (pH 7.5) inside. The pH of CuSC»4 was adjusted to 3.5 by dilute HC1, and to 7.5 using 20mM Hepes and T E A . A23187 was added prior to the drug and liposomes were incubated for 10 minutes to facilitate ionophore incorporation into the bilyar. Topotecan was added to achieve final drug-to-lipid ratio of 0.2 (mol/mol) and incubated at 60 ° C (O), 40 °C (•), and 20 °C (•) (only for C u S 0 7.5 system). At 5, 10, 30 and 60 minutes 100 ul aliquots were fractionated on 1 ml spin columns to separate encapsulated drug form unencapsulated drug. The data points represent the mean of three different replicate experiments and the error bars indicate the standard deviation. Inset: Cryo-TEM images of liposomes loaded with topotecan under various conditions. Liposomes were prepared with C U S O 4 with/without ionophores as described previously in the methods. Panel A shows the empty liposomes; Panel B shows the C u S 0 containing liposomes loaded with topotecan; Panel C shows topotecan loaded liposomes containing C U S O 4 and nigericin. 4

4

53

loading was remarkably similar to that observed in liposomes prepared in unbuffered CuS0 (Figure 3.5B). At 20 °C (Figure 3.5C, A ) there was no appreciable drug uptake. 4

Given our assumption that C u ions were forming a complex with topotecan 2+

following the permeation of the drug from outside of the liposome to the copper containing interior, we wanted to determine whether the drug loaded liposomes exhibited any changes in morphology and we wanted to obtain some evidence in support of copper topotecan complexation. We assessed the morphology of topotecan loaded liposomes by cryo-TEM analysis and representative micrographs have been provided in the Figure 3.5 inset. Panel A shows liposomes prepared in unbuffered 300 mM CuS0 in the absence of 4

topotecan. The liposome size is consistent with the QELS analysis data and suggests that the liposomes are between 100 and 120 nm in size. There is no apparent difference in the morphology of the liposome interior when compared to the liposome exterior. In contrast, the drug loaded liposomes appear heterogeneous. Some liposomes appear comparable to liposomes which do not contain drug and some liposomes contain a visible electron dense aggregate within the core (Figure 3.5 Cryo-TEM Panel B, arrows). Those liposomes with the aggregate are deformed, adopting the 'coffee bean' morphology that has been observed previously for doxorubicin containing liposomes [16]. Panel C is a cryo-TEM micrograph of topotecan loaded liposomes prepared using unbuffered CuS0 , 4

however, prior to drug loading the liposomes were exposed to nigericin to collapse the pH gradient, as described in Figure 3.4. The drug loaded liposomes appear comparable to those prepared in the absence of nigericin, but where a precipitate was observed (arrows) the size of the aggregate appeared smaller.

54

Studies were conducted to assess the behaviour of copper and topotecan in solution to establish some direct evidence of Cu -topotecan interaction. The rational for 2+

these studies was based on the well established behaviour of transition metal ions to form insoluble precipitates upon addition of NaOH. The precipitate is due to formation of copper hydroxide. It was postulated that if copper interacted with topotecan this precipitation event would be inhibited/prevented. The results of these studies are summarized in Figure 3.6. In the absence of topotecan, the addition of sufficient NaOH to engender an increase in solution pH to values >6.2 caused 95% of the copper to precipitate (Figure 3.6A, O ) . The presence of topotecan limited the formation of the copper hydroxide precipitate to less than 5% at pH 6.0 to pH 6.5 (Figure 3.6, • ) . Further, significant copper precipitation ( > 20%) was only observed when the solution pH reached values of 6.7. Interestingly, topotecan concentrations in solution were not significantly effected by addition of NaOH unless copper was present (Figure 3.6B). For example, at pH 6.7, almost 60% of the topotecan precipitated from solution when copper was present (Figure 3.6B, A ) as opposed to the less than 5% loss noted in the absence of copper (Figure 3.6B, A ) . The results thus far indicate that the presence of copper ions within liposomes can mediate the accumulation of topotecan and this accumulation does not appear to be dependent on the presence of a transmembrane pH gradient. The results in Figure 3.6 suggest topotecan can form a complex with copper in solution, as judged by an assay that measured inhibition of copper hydroxide formation. When considering this formulation method, however, the integrity of the topotecan lactone ring needs to be confirmed to ensure therapeutic activity.

55

Q.

il

20.0 0.0

-I

1

1

1

1

1

1

5.2

5.5

5.8

6.1

6.4

6.7

7.0

pH of the solution NaOH induced precipitation of C u in the presence or absence of topotecan. Panel A - pre-determined amounts of NaOH were added to a solution that contained 1 mM copper ions alone (O), or 1 mM copper plus 1 mM topotecan (•). The pH of the resulting solution was measured and the amount of copper remaining in solution was quantified by atomic absorption spectroscopy. Panel B - the concentration of topotecan in solution in the presence (A) or absence ( A ) of copper ions was also measured as a function of increasing pH. Topotecan in solution was determined using the spectrophotometric assay described in the Methods. Each data point represents the mean value determined from three different replicate experiments and error bars indicate the standard deviation. Figure 3.6:

2+

56

The question about the integrity of lactone ring is particularly relevant given the drug loading conditions where the external pH was adjusted to pH 7.5 after the addition of topotecan. Equilibrium favours the inactive membrane-impermeant carboxylate form of the drug at pH 7.5 and this may explain, in part, the variable topotecan loading efficiencies (50% to 75% encapsulation) observed in Figures 3.1, 3.2, 3.4 and 3,5. Therefore, HPLC analysis was performed to monitor the stability of topotecan in the loading buffer at 60 °C. The results shown in Table 3.1 indicate that within 5 minutes approximately 70% of the drug was converted to the ring open form. An apparent equilibrium was reached after 30 minutes, where 85% of the drug existed in the carboxylate form. To address whether the lactone or carboxylate form of topotecan could load into Cu -containing liposomes, we pre-incubated the drug in acidic or basic pH buffers prior 2+

to addition to liposomes. Figure 3.7A shows that following a 30 minute incubation at 60 °C, at pH 3.5, > 98% of the drug is in the lactone form, i.e. the lactone to total drug ratio was 0.98 (Figure 3.7A; left bar). In contrast, >99.9% of the drug exists in the carboxylate form at pH 10.0 (lactone-to-total drug ratio was 85% of the drug exists in carboxylate form, i.e. the lactone/total drug 90% of the encapsulated topotecan was in the active lactone form (lactone to total drug ratio was >0.90; Figure 3.7C; left bar).

61

Chapter 4 DISCUSSION

Previously, topotecan has been successfully encapsulated into liposomes using an equilibration method that involved drug partitioning into the liposomal lipid bilayer and encapsulation within the aqueous liposomal compartments [130]. So-called 'remote' loading methods have also been described, where topotecan is added to preformed liposomes exhibiting a transmembrane pH gradient created by use of an encapsulated ammonium sulphate buffer [160], a low pH buffer [158] or an encapsulated metal ion such as M n

2+

in combination with an ionophore, A23187, [159]. In this thesis a novel

method to prepare topotecan loaded lipoosmes is described. This method relies on the presence of encapsulated C u ions and is not dependent on use of a transmembrane pH 2+

gradient or an ionophore such as A23187. Evidence is provided to suggest that topotecan can complex with C u and HPLC analysis of the drug loaded liposomes suggest that the 2+

encapsulated drug exists primarily in its active lactone form. This result supports previous conclusions developed on the basis of a complexation reaction between M n

2+

ions and the anticancer drug doxorubicin which promotes drug accumulation into preformed liposomes. It is apparent that metal complexation chemistry can be used as a tool to prepare liposomally encapsulated drugs, however the results here indicate that the choice of drug and metal may have to be determined on a case-by-case basis. Although I first attempted to establish that Mn

ions could be used to engender topotecan loading in

the absence of a pH gradient, this effort proved unsuccessful (Figure 3.1). However, guided by previous reports that provided evidence to suggest that copper ions were more

62

likely to complex with camptothecins, I also considered the use of C u containing i+

liposomes and this effort proved successful (Figure 3.2). Additionally, I evaluated topotecan uptake into liposomes containing Co and Zn , but only C u containing 2+

2+

2+

liposomes exhibited rapid (maximum loading in 5 minutes), efficient (greater than 75% under the conditions used here) and stable (no drug loss over a 60 minute incubation at 60 °C) loading attributes. Although this discussion could focus on points related to the further development and therapeutic assessment of topotecan loaded liposomes prepared using copper ions to promote drug encapsulation, I believe that this is not warranted in the context of this thesis, which focused more on characterization of the loading reaction itself. Hence the primary discussion points to be addressed here concern the nature of the metal - ligand interaction and the role of internal pH and pH gradients in facilitating drug uptake into copper containing liposomes. If it is assumed that all metal ions are capable of interacting with camptothecins, then the type of interaction (or complex formed) would likely dictate which metal ions are capable of complexing topotecan in a manner that results in stable loading. Based on the results shown in Figure 3.2, only those complexes formed with copper were sufficiently stable to drive accumulation of topotecan into the liposomes. It is worth noting, however, that the results in Figure 3.2 suggest that encapsulation of topotecan is achievable with encapsulated zinc ions, but the efficacy of loading and the stability of this preparation were not comparable to the copper containing liposomes. It is known that the interactions between transition metal ions and ligands are highly pH dependent [165, 166, 168]. For doxorubicin, interactions with transition metal

63

ions are favoured at neutral pH. Since topotecan has a neutral pKa, it was possible that the encapsulated unbuffered Co , M n 2+

2+

or Zn solutions (pH ~ 3.5) were not ideal for 2+

promoting strong interactions between metal and drug molecules. For this reason, I attempted to prepare buffered solutions of these metal ions at pH 7.5. Liposomes formulated with an entrapped metastable 300 mM CuSC^ solution prepared using HEPES (20mM) and buffered to pH 7.5 with TEA were able to accumulate topotecan (Figure 3.5 C) in a manner that was comparable to liposomes prepared in unbuffered pH 3.5

CUSCM

solutions (Figure 3.5 B). Similar solution could not be prepared with Co , M n or Zn , 2+

2+

2+

all of which precipitated as hydroxides rapidly from solution as the pH was increased to 7.5. An alternative approach to facilitate an internal liposome environment that would favour metal-topotacan complexation involved the use of the monocarboxylate ionophore, nigericin. Figure 3.3 panel B, demonstrated that nigericin mediated the electroneutral exchange of internal protonsfromMnSCU containing liposomes for Na

+

from the external HBS resulting in the complete collapse of any pre-existing pH gradient. When liposomal formulations containing Cu , Co , M n 2+

2+

2+

or Z n

2+

ions were incubated

with nigericin to collapse the pH gradient, the only formulation that was able to encapsulate topotecan was the one prepared with copper. It could still be argued that the right conditions were not established for stable topotecan accumulation into Co , Mn 2+

or Zn

2+

containing liposomes. However,fromthe results it can be concluded that copper

is a unique metal that is able to mediate stable liposomal accumulation of topotecan. Therefore, if all the metals studied here are capable of forming a complex then copper complexation results in a most stable complex.

64

Kuwahara et al. and Brezova et al. suggested that copper could form a coordination complex with the oxygen atoms present on the E-ring of camptothecin [14, 43]. However, it should be noted that topotecan has additional sites where metal ions can interact (Figure 4.1). Notably, these are quinoline N (at N l site), tertiary amine N (at C9 site) and phenolic O (at CIO site). Although the quinoline N is able to interact with metal ions at any given pH, at neutral pH the interaction between metal ions and tertiary amine N and phenol O at the A-ring would be preferred. The complex resulting from such an interaction would be a 6-membered metallacycle as shown in Figure 4.1. Regardless of the site, the results clearly indicate that an interaction with C u is preferred over that 2+

with Co , M n 2+

2+

or Zn . This preference is consistent with the well established Irving2+

Williams effect based on the observation that for the +2 oxidation states of the first transition series, complexes become more stable roughly as the size of the metal ion decreases [169, 170]. Accordingly, the general trend of overall, complex formation constants (p ) for the four metals used in this study would be: Mn n

< Co

< Cu

>Zn .

Briefly, the overall formation constant (p ) is an equilibrium constant of a reaction in n

which water molecules attached to the metal ions are replaced by the ligand molecules. This is illustrated in the following generic equations 3.1 and 3.2, where M

2 +

reacts with

nL, representing ligand molecules: [M(H 0) ] 2

2+

x

+

nL

U

[MLn]

B = " [M(H 0) ] [L]

2+

+

xH 0 2

[ M U ] 2 +

P

2+

2

n

3.1

32

x

Therefore, if p is large then M L , is thermodynamically preferred, and is a stable n

complex.

65

Regardless of where the complexation occurs and the value of (3 , the results n

presented in Figure 3.7 demonstrate that topotecan, when loaded into liposomes prepared in CuS0 , is primarily in its active lactone (ring closed) form. Thus it can be concluded 4

that complexation with copper may protect topotecanfromhydrolysis at neutral pH. Alternatively, the pH inside the liposomes may, for unknown reasons, not be neutral. We believe that the latter is unlikely, since formation of the ring open form would be readily detected at pH 6.5, an internal pH that would be measurable using the methods defined in this report. The work developed here focused on drug metal interactions, but the behaviour of the formulations, including drug loading and retention attributes, may involve more then just complexation between the topotecan and copper. For example, the phospholipid used in these studies (DSPC) has a polarizable phosphate head group. This phosphate head group has pKa = 1.5-2 (value obtained via personal communication with manufacturer, Avanti Polar Lipids), therefore at pH 3.5 and above the phosphate group will be negatively charged while the choline group will be positively charged to form a zwitterionic head group. It is, therefore, likely that the divalent transition metal cations may bind to the phosphate moiety. For example, previous studies [79-82] have shown that Cu ions interact with lipid molecules in monolayers and bilayers. These 2+

interactions may have a direct impact on liposome permeability [118, 121] and could play a role in the stable retention of topotecan. It will be important to characterize these interactions between the selected metal ions and lipids and/or drugs in order to better define what conditions favour drug loading and retention.

66

Figure 4.1 Possible C u coordination sites in topotecan. Interactions at ring A would be stronger and result in six-membered metallacyclic coordination complex, whereas interaction at ring B would be weaker. 2+

67

Ultimately, however, the utility of these formulations will be defined by their biological activity, and in the case of topotecan, questions regarding its therapeutic activity in vivo. In this case it is well established that the effectiveness of liposomal anticancer drug formulations is often governed by the rate of drug releasefromliposomes following parenteral administration [125, 171, 172]. Part of the enthusiasm for using the metal complexation reaction to mediate the accumulation of drugs into liposomes is based on the belief that drug releasefromthese formulations will be controlled, in part, by dissociation of the drug from the transition metal complex [15, 16]. In this regard, Dr. Bally's laboratory has completed studies evaluating the retention of topotecan and another water soluble derivative of camptothecin, irinotecan, in liposomes prepared using metal complexation methods. This work is supported by comprehensive assessments of antitumor effects in murine models of cancer which clearly demonstrate that improved drug retention is associated with improved therapeutic activity (unpublished data).

68

Chapter 5 FUTURE DIRECTIONS

It is well established that for a liposome formulation to be successful it must (i) be able to efficiently and stably encapsulate the selected agent, (ii) have adequate drug retention attributes and release rates, (iii) have sufficient blood circulating time and (iv) be able to evade the clearance mechanisms of the body to successfully deliver the drug at the target tissue, such that efficacy of the entrapped agent can be improved. In this thesis I attempted to address the first question and have reported a novel method to develop a liposomal formulation of topotecan, a promising anti-tumour agent. I have demonstrated that the complexation reaction between the transition metal ions (Cu ) and topotecan can be utilized to drive the accumulation of the drug into the 2+

liposome aqueous core. However, this accumulation is not optimal, as illustrated by variability in encapsulation efficiency. Therefore, further experiments must be conducted where the loading process can be optimized such that >90% of the drug can be efficiently encapsulated. The loading procedure has a number of variables that play critical roles in drug permeation across the bilayer: the pH of the solution in which liposomes are suspended, the temperature at which the loading is performed and/or the types of phospholipids used to form the bilayer. Controlling all these variables to achieve efficient loading is crucial for developing a successful formulation. For example, the pH of the solution in which liposomes are suspended can be adjusted such that a minimum amount of topotecan is converted to bilayer impermeable carboxylate form. The temperature of

69

the loading reaction can be adjusted such that conversion rate of topotecan is slowed but the rate of drug loading is unaffected. After optimization of the loading process, studies can be designed to address other questions, directly related to the clinical development of the formulation. The retention and the release rate of drug can be assessed by an in vitro assay, where drug loaded liposomes are placed in plasma and incubated at 37 °C (body temperature). The release of drug from liposomes into plasma can be measured either via UV/Vis absorption assay or by HPLC techniques. To understand the in vivo behaviour of liposome formulation, animal studies assessing the pharmacokinetics and bio-distribution characteristics can be conducted. Furthermore, animal studies can be conducted to investigate the efficacy of this formulation in mice bearing human xenograft tumours and the results can be compared with those obtained withfreedrug and/or other liposomal formulations of topotecan. Only then will we be able to evaluate fully the impact of liposomal encapsulation of topotecan achieved via complexation reaction with a transition metal ion. 2+

Parallel studies can be designed to investigate the interactions between Cu and topotecan, which can be characterized by conventional physical and chemical techniques such as IR, ESR, cyclic voltammetry and/or NMR. With adequate understanding of the drug-metal interactions, this methodology can be applied to other chemotherapeutic agents possessing chemical groups capable of forming coordination complexes with transition metal ions. Furthermore, this technology can be applied to co-encapsulate two or more chemotherapeutic agents that exhibit synergy in the clinic, and are able to form coordination complexes with transition metal ions. Thus, a fixed dose combination

70

product can be developed that would be more effacious that individual agents. Evidence shows that cancer chemotherapy benefitsfromcombination regimens, when two or more agents are mixed appropriately. Examples of drugs that have shown synergy and possess coordination groups include: vincristine, vinorelbine, irniotecan, topotecan and doxorubicin. Preliminary resultsfromDr. Bally's laboratory (unpublished data) show that vincristine can also be encapsulated into liposomes using a C u

2+

gradient. At the same

time clinical trials of vincrsitine and topotecan combination regimens have shown positive synergistic results [173]. Therefore, one could pursue a fixed-dose combination liposomal product of vincristine and topotecan that is more effective against solid pediatric tumours.

71

References

[I] [2] [3] [4]

[5]

[6]

[7] [8]

[9] [10] [II] [12] [13]

[14]

[15]

D. Hanahan and R.A. Weinberg, The hallmarks of cancer. Cell, 100(1) (2000) 5770. M.M. Gottesman, MECHANISMS OF CANCER DRUG RESISTANCE. Annual Review of Medicine, 53(1) (2002) 615-627. B. Tan, D. Piwnica-Worms, and L. Ratner, Multidrug resistance transporters and modulation. Curr Opin Oncol, 12(5) (2000) 450-8. R. Garcia-Carbonero and J.G. Supko, Current perspectives on the clinical experience, pharmacology, and continued development of the camptothecins. Clin Cancer Res, 8(3) (2002) 641-61. D.L. Emerson, J.M. Besterman, H.R. Brown, M.G. Evans, P.P. Leitner, M.J. Luzzio, J.E. Shaffer, D.D. Sternbach, D. Uehling, and A. Vuong, In vivo antitumor activity of two new seven-substituted water-soluble camptothecin analogues. Cancer Res, 55(3) (1995) 603-9. S.M. Klein, G.J. Hauser, B.D. Anderson, A.T. Shad, J.E. Gootenberg, H.J. Dalton, and J.H. Hertzog, Comparison of intermittent versus continuous infusion of propofol for elective oncology procedures in children. Pediatr Crit Care Med, 4(1) (2003) 78-82. H. Kelly and R.M. Goldberg, Systemic therapy for metastatic colorectal cancer: current options, current evidence. J Clin Oncol, 23(20) (2005) 4553-60. T. Lam, J.W. Hetherington, J. Greenman, and A. Maraveyas, From total empiricism to a rational design of metronomic chemotherapy phase I dosing trials. Anticancer Drugs, 17(2) (2006) 113-21. S. Gately and R. Kerbel, Antiangiogenic scheduling of lower dose cancer chemotherapy. Cancer J, 7(5) (2001) 427-36. T.M. Allen and P.R. Cullis, Drug delivery systems: entering the mainstream. Science, 303(5665) (2004) 1818-22. J.R. Sorenson, Copper chelates as possible active forms of the antiarthritic agents. J Med Chem, 19(1) (1976) 135-48. S.D. Aust, L.A. Morehouse, and C E . Thomas, Role of metals in oxygen radical reactions. J Free Radic Biol Med, 1(1) (1985) 3-25. M. Jezowska-Bojczuk, W. Szczepanik, W. Lesniak, J. Ciesiolka, J. Wrzesinski, and W. Bal, DNA and RNA damage by Cu(II)-amikacin complex. Eur J Biochem, 269(22) (2002) 5547-56. V. Brezova, M. Valko, M. Breza, H. Morris, J. Telser, D. Dvoranova, K. Kaiserova, L. Varecka, M. Mazur, and D. Leibfritz, Role of Radicals and Singlet Oxygen in Photoactivated DNA Cleavage by the Anticancer Drug Camptothecin: An Electron Paramagnetic Resonance Study. J. Phys. Chem. B, 107(10) (2003) 2415-2425. G.N. Chiu, S.A. Abraham, L.M. Ickenstein, R. Ng, G. Karlsson, K. Edwards, E.K. Wasan, and M.B. Bally, Encapsulation of doxorubicin into thermosensitive liposomes via complexation with the transition metal manganese. J Control Release, 104(2) (2005) 271-288.

72

[16]

[17]

[18]

[19] [20] [21]

[22]

[23] [24]

[25]

[26]

[27]

[28]

[29] [30] [31]

S.A. Abraham, K. Edwards, G. Karlsson, S. Macintosh, L.D. Mayer, C. McKenzie, and M.B. Bally, Formation of transition metal-doxorubicin complexes inside liposomes. Biochim Biophys Acta, 1565(1) (2002) 41-54. B.C. Cheung, T.H. Sun, J.M. Leenhouts, and P.R. Cullis, Loading of doxorubicin into liposomes by forming Mn2+-drug complexes. Biochim Biophys Acta, 1414(1-2) (1998) 205-16. D.R. Williams, Transition Metals involved in Man and his Medicines, in: Trans. Met. Chem. Proc. Workshop, Inst. Sci. Technol., Univ. Wales, Cardiff, UK: Verlag Chem, 1980. D.R. Williams, Metals, ligands, and cancer. Chem Rev, 72(3) (1972) 203-13. K.H. Thompson and C. Orvig, Boon and Bane of Metal Ions in Medicine. Science, 300(5621) (2003) 936-939. S. Kirschner, Y.K. Wei, D. Francis, and J.G. Bergman, Anticancer and potential antiviral activity of complex inorganic compounds. J Med Chem, 9(3) (1966) 369-72. M. Jezowska-Bojczuk, W. Bal, and K.S. Kasprzak, Copper(II) interactions with an experimental antiviral agent, I-deoxynojirimycin, and oxygen activation by resulting complexes. J Inorg Biochem, 64(4) (1996) 231-46. J. Reedijk, Medicinal applications of heavy-metal compounds. Curr Opin Chem Biol, 3(2) (1999) 236-40. P.J. Blower, J.R. Dilworth, R.I. Maurer, G.D. Mullen, C A . Reynolds, and Y. Zheng, Towards new transition metal-based hypoxic selective agents for therapy and imaging. J Inorg Biochem, 85(1) (2001) 15-22. M. Navarro, E.J. Cisneros-Fajardo, T. Lehmann, R.A. Sanchez-Delgado, R. Atencio, P. Silva, R. Lira, and J. A. Urbina, Toward a Novel Metal-Based Chemotherapy against Tropical Diseases. 6. Synthesis and Characterization of New Copper(II) and Gold(I) Clotrimazole and Ketoconazole Complexes and Evaluation of Their Activity against Trypanosoma cruzi. Inorg. Chem., 40(27) (2001) 6879-6884. LB. Afanas'eva, E.A. Ostrakhovitch, E.V. Mikhal'chik, G.A. Ibragimova, and L.G. Korkina, Enhancement of antioxidant and anti-inflammatory activities of bioflavonoid rutin by complexation with transition metals. Biochem Pharmacol, 61(6) (2001) 677-84. S.A. Akman, J.H. Doroshow, T.G. Burke, and M. Dizdaroglu, DNA base modifications induced in isolated human chromatin by NADH dehydrogenasecatalyzed reduction of doxorubicin. Biochemistry, 31(13) (1992) 3500-6. Z. Hartman and P.E. Hartman, Copper and cobalt complexes of carnosine and anserine: production of active oxygen species and its enhancement by 2mercaptoimidazoles. Chem Biol Interact, 84(2) (1992) 153-68. C.X. Zhang and S.J. Lippard, New metal complexes as potential therapeutics. Curr Opin Chem Biol, 7(4) (2003) 481-9. C. Metcalfe and J.A. Thomas, Kinetically inert transition metal complexes that reversibly bind to DNA. Chem Soc Rev, 32(4) (2003) 215-24. M. Galanski, V.B. Arion, M.A. Jakupec, and B.K. Keppler, Recent developments in the field of tumor-inhibiting metal complexes. Curr Pharm Des, 9(25) (2003) 2078-89.

73

[32] [33]

[34]

[35]

[36]

[37] [38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

E. Raymond, S.G. Chaney, A. Taamma, and E. Cvitkovic, Oxaliplatin: a review of preclinical and clinical studies. Ann Oncol, 9(10) (1998) 1053-71. G.E. Jackson, P.M. May, and D.R. Williams, Metal-ligand complexes involved in rheumatoid arthritis~I: Justifications for copper administration. Journal of Inorganic and Nuclear Chemistry, 40(6) (1978) 1189. G.E. Jackson, P.M. May, and D.R. Williams, Metal-ligand complexes involved in rheumatoid arthritis. VII. Formation of binary and ternary complexes between 2,3-diaminopropionic acid, histidine, and copper(II), nickel(II), and zinc(II). J Inorg Nuc Chem., 43(4) (1981) 825-9. R. Paschke, C. Paetz, T. Mueller, H.J. Schmoll, H. Mueller, E. Sorkau, and E. Sinn, Biomolecules linked to transition metal complexes—new chances for chemotherapy. Curr Med Chem, 10(19) (2003) 2033-44. Z. Afrasiabi, E. Sinn, S. Padhye, S. Dutta, S. Padhye, C. Newton, C E . Anson, and A.K. Powell, Transition metal complexes of phenanthrenequinone thiosemicarbazone as potential anticancer agents: synthesis, structure, spectroscopy, electrochemistry and in vitro anticancer activity against human breast cancer cell-line, T47D. J Inorg Biochem, 95(4) (2003) 306-14. P. Kopf-Maier, Complexes of metals other than platinum as antitumour agents. Eur J Clin Pharmacol, 47(1) (1994) 1-16. P.M. May, G.K. Williams, and D.R. Williams, Speciation studies of adriamycin, quelamycin and their metal complexes. Inorganica Chimica Acta, 46(5) (1980) 221-8. M.M. Fiallo and A. Garnier-Suillerot, Metal anthracycline complexes as a new class of anthracycline derivatives. Pd(II)-adriamycin and Pd(II)-daunorubicin complexes: physicochemical characteristics and antitumor activity. Biochemistry, 25(4) (1986) 924-30. L.L. Parker, S.M. Lacy, L.J. Farrugia, C. Evans, D.J. Robins, C C O'Hare, J.A. Hartley, M. Jaffar, and I.J. Stratford, A novel design strategy for stable metal complexes of nitrogen mustards as bioreductive prodrugs. J Med Chem, 47(23) (2004) 5683-9. C T . Dillon, T.W. Hambley, B.J. Kennedy, P.A. Lay, Q. Zhou, N.M. Davies, J.R. Biffin, and H.L. Regtop, Gastrointestinal toxicity, antiinflammatory activity, and superoxide dismutase activity of copper and zinc complexes of the antiinflammatory drug indomethacin. Chem Res Toxicol, 16(1) (2003) 28-37. J. Kuwahara, T. Suzuki, and Y. Sugiura, Studies on antitumor drugs targeting DNA: photosensitive DNA cleavage of copper-camptothecin. Nucleic Acids SympSer, (16) (1985) 201-4. J. Kuwahara, T. Suzuki, K. Funakoshi, and Y. Sugiura, Photosensitive DNA cleavage and phage inactivation by copper(II)-camptothecin. Biochemistry, 25(6) (1986) 1216-21. X.F. Liu, Y.M. Xia, and Y. Fang, Effect of metal ions on the interaction between bovine serum albumin and berberine chloride extracted from a traditional Chinese Herb coptis chinensis franch. J Inorg Biochem, 99(7) (2005) 1449-57. I. El-Gibaly, Oral delayed-release system based on Zn-pectinate gel (ZPG) microparticles as an alternative carrier to calcium pectinate beads for colonic drug delivery. Int J Pharm, 232(1-2) (2002) 199-211.

74

[46] [47] [48] [49] [50] [51] [52]

[53]

[54] [55]

[56]

[57]

[58] [59] [60]

[61] [62]

[63]

A.D. Bangham, M.M. Standish, and J.C. Watkins, Diffusion of univalent ions across the lamellae of swollen phospholipids. J Mol Biol, 13(1) (1965) 238-52. A.D. Bangham, Membrane models with phospholipids. Prog Biophys Mol Biol, 18 (1968) 29-95. J.H. Fendler and A. Romero, Liposomes as drug carriers. Life Sciences, 20(7) (1977) 1109. A.D. Bangham, M.W. Hill, and G.A. Miller, in: E.D. Korn, (Ed.), Methods in membrane biology, Plenum Press, New York,, 1974, pp. 1-68. G. Gregoriadis, E.J. Wills, CP. Swain, and A.S. Tavill, Drug-carrier potential of liposomes in cancer chemotherapy. Lancet, 1(7870) (1974) 1313-6. D. Subramanian and M.T. Muller, Liposomal encapsulation increases the activity of the topoisomerase I inhibitor topotecan. Oncol Res, 7(9) (1995) 461-9. Y. Sadzuka, S. Hirotsu, and S. Hirota, Effect of liposomalization on the antitumor activity, side-effects and tissue distribution of CPT-11. Cancer Lett, 127(1-2) (1998) 99-106. A.A. Gabizon, Selective tumor localization and improved therapeutic index of anthracyclines encapsulated in long-circulating liposomes. Cancer Res, 52(4) (1992) 891-6. J.H. Senior, Fate and behavior of liposomes in vivo: a review of controlling factors. Crit Rev Ther Drug Carrier Syst, 3(2) (1987) 123-93. R. Sadasivan, R. Morgan, C. Fabian, and R. Stephens, Reversal of multidrug resistance in HL-60 cells by verapamil and liposome-encapsulated doxorubicin. Cancer Lett, 57(2) (1991) 165-71. S. Oudard, A. Thierry, T J . Jorgensen, and A. Rahman, Sensitization of multidrug-resistant colon cancer cells to doxorubicin encapsulated in liposomes. Cancer Chemother Pharmacol, 28(4) (1991) 259-65. A. Mori, A.L. Klibanov, V.P. Torchilin, and L. Huang, Influence of the steric barrier activity of amphipathic poly(ethyleneglycol) and ganglioside GM1 on the circulation time of liposomes and on the target binding of immunoliposomes in vivo. FEBS Lett, 284(2) (1991) 263-6. M . C Woodle and D.D. Lasic, Sterically stabilized liposomes. Biochim Biophys Acta, 1113(2) (1992) 171-99. T.M. Allen and A. Chonn, Large unilamellar liposomes with low uptake into the reticuloendothelial system. FEBS Lett, 223(1) (1987) 42-6. M.B. Yatvin, J.N. Weinstein, W.H. Dennis, and R. Blumenthal, Design of liposomes for enhanced local release of drugs by hyperthermia. Science, 202(4374) (1978) 1290-3. D. Papahadjopoulos and A. Gabizon, Targeting of liposomes to tumor cells in vivo. Ann N Y Acad Sci, 507 (1987) 64-74. K. Maruyama, SJ. Kennel, and L. Huang, Lipid composition is important for highly efficient target binding and retention of immunoliposomes. Proc Natl Acad Sci U S A , 87(15) (1990) 5744-8. I. Ahmad and T.M. Allen, Antibody-mediated specific binding and cytotoxicity of liposome-entrapped doxorubicin to lung cancer cells in vitro. Cancer Res, 52(17) (1992) 4817-20.

75

[64] [65]

[66] [67] [68]

[69]

[70] [71]

[72]

[73]

[74] [75] [76] [77] [78]

[79] [80]

[81]

P. Sapra, P. Tyagi, and T.M. Allen, Ligand-targeted liposomes for cancer treatment. Curr Drug Deliv, 2(4) (2005) 369-81. E.C. Ramsay, N. Dos Santos, W.H. Dragowska, J.J. Laskin, and M.B. Bally, The formulation of lipid-based nanotechnologies for the delivery of fixed dose anticancer drug combinations. Curr Drug Deliv, 2(4) (2005) 341-51. W.G. Roberts and G.E. Palade, Neovasculature induced by vascular endothelial growth factor is fenestrated. Cancer Res, 57(4) (1997) 765-72. L. W. Seymour, Passive tumor targeting of soluble macromolecules and drug conjugates. Crit Rev Ther Drug Carrier Syst, 9(2) (1992) 135-87. H.F. Dvorak, J.A. Nagy, J.T. Dvorak, and A.M. Dvorak, Identification and characterization of the blood vessels of solid tumors that are leaky to circulating macromolecules. Am J Pathol, 133(1) (1988) 95-109. F. Yuan, M. Dellian, D. Fukumura, M. Leunig, D.A. Berk, V.P. Torchilin, and R.K. Jain, Vascular permeability in a human tumor xenograft: molecular size dependence and cutoff size. Cancer Res, 55(17) (1995) 3752-6. H. Maeda and Y. Matsumura, Tumoritropic and lymphotropic principles of macromolecular drugs. Crit Rev Ther Drug Carrier Syst, 6(3) (1989) 193-210. N.Z. Wu, D. Da, T.L. Rudoll, D. Needham, A.R. Whorton, and M.W. Dewhirst, Increased microvascular permeability contributes to preferential accumulation of Stealth liposomes in tumor tissue. Cancer Res, 53(16) (1993) 3765-70. A. Gabizon, A. Dagan, D. Goren, Y. Barenholz, and Z. Fuks, Liposomes as in vivo carriers of adriamycin: reduced cardiac uptake and preserved antitumor activity in mice. Cancer Res, 42(11) (1982) 4734-9. F. Olson, E. Mayhew, D. Maslow, Y. Rustum, and F. Szoka, Characterization, toxicity and therapeutic efficacy of adriamycin encapsulated in liposomes. Eur J Cancer Clin Oncol, 18(2) (1982) 167-76. D. Papahadjopoulos, in: A.S. Janoff, (Ed.), Liposomes : rational design, M. Dekker, New York, 1999, pp. 1-11. J.N. Israelachvili, S. Marcelja, and R.G. Horn, Physical principles of membrane organization. Q Rev Biophys, 13(2) (1980) 121-200. J.N. Israelachvili and D.J. Mitchell, A model for the packing of lipids in bilayer membranes. Biochim Biophys Acta, 389(1) (1975) 13-19. C. Tanford, Amphiphile orientation: physical chemistry and biological function. Biochem Soc Trans, 15 Suppl (1987) 1S-7S. R.L. Juliano and D. Stamp, The effect of particle size and charge on the clearance rates of liposomes and liposome encapsulated drugs. Biochemical and Biophysical Research Communications, 63(3) (1975) 651. M. Suwalsky, B. Ungerer, L. Quevedo, F. Aguilar, and CP. Sotomayor, Cu2+ ions interact with cell membranes. J Inorg Biochem, 70(3-4) (1998) 233-8. S.-H. Park, S.-G. Oh, J.-Y. Mun, and S.-S. Han, Effects of silver nanoparticles on the fluidity of bilayer in phospholipid liposome. Colloids and Surfaces B: Biointerfaces, 44(2-3) (2005) 117. V.S. Lebedev, L.A. Volodina, E. Deinega, and I. Fedorov lu, [Structural modifications of the surface of Escherichia coli bacteria and copper-induced permeability of plasma membrane]. Biofizika, 50(1) (2005) 107-13.

76

[82]

[83]

[84] [85]

[86] [87] [88] [89] [90] [91]

[92]

[93]

[94] [95]

[96]

G.B. Khomutov, S.A. Yakovenko, E.S. Soldatov, V.V. Khanin, M.D. Nedelcheva, and T.V. Yurova, Interaction of copper ions with stearic acid Langmuir monolayers and formation of cluster structures in monolayers and LangmuirBlodgett films. Membr Cell Biol, 10(6) (1997) 665-81. O. Bittner, S. Gal, I. Pinchuk, D. Danino, H. Shinar, and D. Lichtenberg, Copperinduced peroxidation of liposomal palmitoyllinoleoylphosphatidylcholine (PLPC), effect of antioxidants and its dependence on the oxidative stress. Chem Phys Lipids, 114(1) (2002) 81-98. T.J. Mcintosh, S.A. Simon, and R.C. MacDonald, The organization of n-alkanes in lipid bilayers. Biochim Biophys Acta, 597(3) (1980) 445-63. J.A. Urbina, S. Pekerar, H.B. Le, J. Patterson, B. Montez, and E. Oldfield, Molecular order and dynamics of phosphatidylcholine bilayer membranes in the presence of cholesterol, ergosterol and lanosterol: a comparative study using 2H-, 13C- and 31P-NMR spectroscopy. Biochim Biophys Acta, 1238(2) (1995) 16376. R.A. Demel and B. De Kruyff, The function of sterols in membranes. Biochim Biophys Acta, 457(2) (1976) 109-32. O.G. Mouritsen and M.J. Zuckermann, What's so special about cholesterol? Lipids, 39(11) (2004) 1101-13. P.L. Yeagle, Lanosterol and cholesterol have different effects on phospholipid acyl chain ordering. Biochim Biophys Acta, 815(1) (1985) 33-6. D.M. Engelman and J.E. Rothman, The planar organization of lecithin-cholesterol bilayers. J Biol Chem, 247(11) (1972) 3694-7. D. Chapman, Fluidity and phase transitions of cell membranes. Biomembranes, 7 (1975) 1-9. P.L. Yeagle, R.B. Martin, A.K. Lala, H.K. Lin, and K. Bloch, Differential effects of cholesterol and lanosterol on artificial membranes. Proc Natl Acad Sci U S A , 74(11) (1977) 4924-6. P.R. Cullis and M.J. Hope, The bilayer stabilizing role of sphingomyelin in the presence of cholesterol: a 31P NMR study. Biochim Biophys Acta, 597(3) (1980) 533-42. S.C. Semple, R. Leone, J. Wang, E.C. Leng, S.K. Klimuk, M.L. Eisenhardt, Z.N. Yuan, K. Edwards, N. Maurer, M.J. Hope, P.R. Cullis, and Q.F. Ahkong, Optimization and characterization of a sphingomyelin/cholesterol liposome formulation of vinorelbine with promising antitumor activity. J Pharm Sci, 94(5) (2005) 1024-38. N. Haran and M. Shoporer, Study of water permeability through phospholipid vesicle membranes by 170 NMR. Biochim Biophys Acta, 426(4) (1976) 638-46. D. Papahadjopoulos, M. Cowden, and H. Kimelberg, Role of cholesterol in membranes. Effects on phospholipid-protein interactions, membrane permeability and enzymatic activity. Biochim Biophys Acta, 330(1) (1973) 8-26. J. Damen, J. Regts, and G. Scherphof, Transfer and exchange of phospholipid between small unilamellar liposomes and rat plasma high density lipoproteins. Dependence on cholesterol content and phospholipid composition. Biochim Biophys Acta, 665(3) (1981) 538-45.

77

[97]

[98]

[99]

[100] [101]

[102] [103]

[104] [105]

[106]

[107]

[108]

[109] [110] [Ill]

[112]

C. Kirby, J. Clarke, and G. Gregoriadis, Cholesterol content of small unilamellar liposomes controls phospholipid loss to high density lipoproteins in the presence of serum. FEBS Lett, 111(2) (1980) 324-8. S.C. Semple, A. Chonn, and P.R. Cullis, Influence of cholesterol on the association of plasma proteins with liposomes. Biochemistry, 35(8) (1996) 25215. C. Kirby and G. Gregoriadis, The effect of the cholesterol content of small unilamellar liposomes on the fate of their lipid components in vitro. Life Sci, 27(23) (1980) 2223-30. D. Needham, D.V. Zhelev, and T J . Mcintosh, in: A.S. Janoff, (Ed.), Liposomes : rational design, M. Dekker, New York, 1999, pp. 13-62. J.K. Mills and D. Needham, Lysolipid incorporation in dipalmitoylphosphatidylcholine bilayer membranes enhances the ion permeability and drug release rates at the membrane phase transition. Biochim Biophys Acta, 1716(2) (2005) 77-96. M.C. Sandstrom, L.M. Ickenstein, L.D. Mayer, and K. Edwards, Effects of lipid segregation and lysolipid dissociation on drug release from thermosensitive liposomes. J Control Release, 107(1) (2005) 131-42. V.C. Anderson and D.H. Thompson, Triggered release of hydrophilic agents from plasmalogen liposomes using visible light or acid. Biochim Biophys Acta, 1109(1) (1992) 33-42. V.P. Torchilin and V. Weissig, Liposomes : a practical approach, 2nd, Oxford University Press, Oxford ; New York, 2003. S.M. Gruner, R.P. Lenk, A.S. Janoff, and M.J. Ostro, Novel multilayered lipid vesicles: comparison of physical characteristics of multilamellar liposomes and stable plurilamellar vesicles. Biochemistry, 24(12) (1985) 2833-42. L.D. Mayer, M.J. Hope, P.R. Cullis, and A.S. Janoff, Solute distributions and trapping efficiencies observed infreeze-thawedmultilamellar vesicles. Biochim Biophys Acta, 817(1) (1985) 193-6. P.R. Cullis, A. Chonn, and S.C. Semple, Interactions of liposomes and lipid-based carrier systems with blood proteins: Relation to clearance behaviour in vivo. Adv Drug Deliv Rev, 32(1-2) (1998) 3-17. R.L. Richards, R.C. Habbersett, I. Scher, A.S. Janoff, H.P. Schieren, L.D. Mayer, P.R. Cullis, and CR. Alving, Influence of vesicle size on complement-dependent immune damage to liposomes. Biochim Biophys Acta, 855(2) (1986) 223-30. Y. Barenholzt, S. Amselem, and D. Lichtenberg, A new method for preparation of phospholipid vesicles (liposomes) - French press. FEBS Lett, 99(1) (1979) 210-4. C. Huang, Studies on phosphatidylcholine vesicles. Formation and physical characteristics. Biochemistry, 8(1) (1969) 344-52. M J . Hope, M.B. Bally, G. Webb, and P.R. Cullis, Production of large unilamellar vesicles by a rapid extrusion procedure. Characterization of size distribution, trapped volume and ability to maintain a membrane potential. Biochimica et Biophysica Acta (BBA) - Biomembranes, 812(1) (1985) 55. P.R. Cullis and B. de Kruijff, The polymorphic phase behaviour of phosphatidylethanolamines of natural and synthetic origin. A 3 IP NMR study. Biochim Biophys Acta, 513(1) (1978) 31-42.

78

[113] P.A. Forsyth, Jr., S. Marcelja, D.J. Mitchell, and B.W. Ninham, Phase transition in charged lipid membranes. Biochim Biophys Acta, 469(3) (1977) 335-44. L114] A.G. Lee, Lipid phase transitions and phase diagrams. II. Mictures involving lipids. Biochim Biophys Acta, 472(3-4) (1977) 285-344. 115] R.E. Jacobs, B. Hudson, and H.C. Andersen, A theory of the chain melting phase transition of aqueous phospholipid dispersions. Proc Natl Acad Sci U S A , 72(10) (1975) 3993-7. T16] G. Cevc, Isothermal lipid phase transitions. Chem Phys Lipids, 57(2-3) (1991) 293-307. 117] C H . Huang, Mixed-chain phospholipids: structures and chain-melting behavior. Lipids, 36(10) (2001) 1077-97. 118] H. Hauser, Effect of inorganic cations on phase transitions. Chem Phys Lipids, 57(2-3) (1991) 309-25. [119] D.C. Lee and D. Chapman, The effects of temperature on biological membranes and their models. Symp Soc Exp Biol, 41 (1987) 35-52. [120] B. Maggio, G.D. Fidelio, F.A. Cumar, and R.K. Yu, Molecular interactions and thermotropic behavior of glycosphingolipids in model membrane systems. Chem Phys Lipids, 42(1-3) (1986) 49-63. 121] G. Cevc, A. Watts, and D. Marsh, Titration of the phase transition of phosphatidylserine bilayer membranes. Effects of pH, surface electrostatics, ion binding, and head-group hydration. Biochemistry, 20(17) (1981) 4955-65. T22] P.R. Cullis, P.W. van Dijck, B. de Kruijff, and J. de Gier, Effects of cholesterol on the properties of equimolar mixtures of synthetic phosphatidylethanolamine and phosphatidylcholine. A 3 IP NMR and differential scanning calorimetry study. Biochim Biophys Acta, 513(1) (1978) 21-30. [123] M.J. Johnston, S.C. Semple, S.K. Klimuk, K. Edwards, M.L. Eisenhardt, E . C Leng, G. Karlsson, D. Yanko, and P.R. Cullis, Therapeutically optimized rates of drug release can be achieved by varying the drug-to-lipid ratio in liposomal vincristine formulations. Biochim Biophys Acta, 1758(1) (2006) 55-64. T24] X.M. Zhang, A.B. Patel, R.A. de Graaf, and K.L. Behar, Determination of liposomal encapsulation efficiency using proton NMR spectroscopy. Chem Phys Lipids, 127(1) (2004) 113-20. [125] I.V. Zhigaltsev, N. Maurer, Q.F. Akhong, R. Leone, E. Leng, J. Wang, S.C. Semple, and P.R. Cullis, Liposome-encapsulated vincristine, vinblastine and vinorelbine: a comparative study of drug loading and retention. J Control Release, 104(1) (2005) 103-11. [126] A. Roos, Intracellular pH and distribution of weak acids across cell membranes. A study of D- and L-lactate and of DMO in rat diaphragm. J Physiol, 249(1) (1975) 1-25. [127] L.D. Mayer, M.B. Bally, M.J. Hope, and P.R. Cullis, Uptake of antineoplastic agents into large unilamellar vesicles in response to a membrane potential. Biochim Biophys Acta, 816(2) (1985) 294-302. [128] M.B. Bally, L.D. Mayer, H. Loughrey, T. Redelmeier, T.D. Madden, K. Wong, P.R. Harrigan, M.J. Hope, and P.R. Cullis, Dopamine accumulation in large unilamellar vesicle systems induced by transmembrane ion gradients. Chem Phys Lipids, 47(2) (1988) 97-107.

79

[129] L.D. Mayer, M.B. Bally, and P.R. Cullis, Uptake of adriamycin into large unilamellar vesicles in response to a pH gradient. Biochim Biophys Acta, 857(1) (1986) 123-6. "130] T.G. Burke and X. Gao, Stabilization of topotecan in low pH liposomes composed of distearoylphosphatidylcholine. J Pharm Sci, 83(7) (1994) 967-9. 131] E. Maurer-Spurej, K.F. Wong, N. Maurer, D.B. Fenske, and P.R. Cullis, Factors influencing uptake and retention of amino-containing drugs in large unilamellar vesicles exhibiting transmembrane pH gradients. Biochim Biophys Acta, 1416(12) (1999) 1-10. 132] P.R. Harrigan, K.F. Wong, T.E. Redelmeier, JJ. Wheeler, and P.R. Cullis, Accumulation of doxorubicin and other lipophilic amines into large unilamellar vesicles in response to transmembrane pH gradients. Biochim Biophys Acta, 1149(2) (1993) 329-38. 133] D. Papahadjopoulos, S. Nir, and S. Ohki, Permeability properties of phospholipid membranes: Effect of cholesterol and temperature. Biochimica et Biophysica Acta (BBA) - Biomembranes, 266(3) (1972) 561. 134] M.A. Alonso and L. Carrasco, Molecular basis of the permeabilization of mammalian cells by ionophores. Eur J Biochem, 127(3) (1982) 567-9. [135] P.H. Stern, Ionophores. Chemistry, physiology and potential applications to bone biology. Clin Orthop Relat Res, (122) (1977) 273-98. [136] B.C. Pressman and N.T. deGuzman, Biological applications and evolutionary origins of ionophores. Adv Exp Med Biol, 84 (1977) 285-300. [137] M. Tachibana, M. Iwaizumi, and S. Tero-Kubota, EPR studies of copper(II) and cobalt(II) complexes of adriamycin. J Inorg Biochem, 30(2) (1987) 133-40. [138] B. Halova-Lajoie, V. Brumas, M.M. Fiallo, and G. Berthon, Copper(II) interactions with non-steroidal anti-inflammatory agents. Ill - 3Methoxyanthranilic acid as a potential ()OH-inactivating ligand: A quantitative investigation of its copper handling role in vivo. J Inorg Biochem, (2006). [139] X. Li, D J . Hirsh, D. Cabral-Lilly, A. Zirkel, S.M. Gruner, A.S. Janoff, and W.R. Perkins, Doxorubicin physical state in solution and inside liposomes loaded via a pH gradient. Biochim Biophys Acta, 1415(1) (1998) 23-40. [140] L.D. Mayer, L.C. Tai, M.B. Bally, G.N. Mitilenes, R.S. Ginsberg, and P.R. Cullis, Characterization of liposomal systems containing doxorubicin entrapped in response to pH gradients. Biochim Biophys Acta, 1025(2) (1990) 143-51. 141] P.G. Tardi, N.L. Boman, and P.R. Cullis, Liposomal doxorubicin. J Drug Target, 4(3) (1996) 129-40. [142] J.C. Dabrowiak, F.T. Greenaway, and R. Grulich, Transition-metal binding site of bleomycin A2. A carbon-13 nuclear magnetic resonance study of the zinc(II) and copper(II) derivatives. Biochemistry, 17(19) (1978) 4090-6. [143] R.P. Sheridan and;R.K. Gupta, A IH nuclear relaxation study of the Mn2+ . bleomycin complex. Proximity of the metal to the DNA-binding site. J Biol Chem, 256(3) (1981) 1242-7. [144] Y.H. Hsiang, R. Hertzberg, S. Hecht, and L.F. Liu, Camptothecin induces proteinlinked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem, 260(27) (1985)14873-8.

80

"145] Y.H. Hsiang and L.F. Liu, Identification of mammalian DNA topoisomerase I as an intracellular target of the anticancer drug camptothecin. Cancer Res, 48(7) (1988) 1722-6. T46] J.F. Pizzolato and L.B. Saltz, The camptothecins. Lancet, 361(9376) (2003) 223542. 147] L.F. Liu, S.D. Desai, T.K. Li, Y. Mao, M. Sun, and S.P. Sim, Mechanism of action of camptothecin. Ann N Y Acad Sci, 922 (2000) 1-10. [148] P.K. Turner, L.C. Iacono, and C.F. Stewart, Topoisomerase I interactive agents. Cancer Chemother Biol Response Modif, 21 (2003) 69-101. "149] J. Fassberg and V J . Stella, A kinetic and mechanistic study of the hydrolysis of camptothecin and some analogues. J Pharm Sci, 81(7) (1992) 676-84. [150] T.G. Burke and Z. Mi, Preferential binding of the carboxylate form of camptothecin by human serum albumin. Anal Biochem, 212(1) (1993) 285-7. "151] Z. Mi and T.G. Burke, Differential interactions of camptothecin lactone and carboxylate forms with human blood components. Biochemistry, 33(34) (1994) 10325-36. [152] V.M. Herben, W.W. ten Bokkel Huinink, and J.H. Beijnen, Clinical pharmacokinetics of topotecan. Clin Pharmacokinet, 31(2) (1996) 85-102. [153] P. Stano, S. Bufali, C. Pisano, F. Bucci, M. Barbarino, M. Santaniello, P. Carminati, and P.L. Luisi, Novel camptothecin analogue (gimatecan)-containing liposomes prepared by the ethanol injection method. J Liposome Res, 14(1-2) (2004) 87-109. [154] S.S. Daoud, M.I. Fetouh, and B.C. Giovanella, Antitumor effect of liposomeincorporated camptothecin in human malignant xenografts. Anticancer Drugs, 6(1)(1995) 83-93. [155] B.B. Lundberg, Biologically active camptothecin derivatives for incorporation into liposome bilayers and lipid emulsions. Anticancer Drug Des, 13(5) (1998) 453-61. [156] D.S. Chow, L. Gong, M.D. Wolfe, and B.C. Giovanella, Modified lactone/carboxylate salt equilibria in vivo by liposomal delivery of 9-nitrocamptothecin. Ann N Y Acad Sci, 922 (2000) 164-74. [157] W.J. Loos, D. Kehrer, E. Brouwer, J. Verweij, P. de Bruijn, M. Hamilton, S. Gill, K. Nooter, G. Stoter, and A. Sparreboom, Liposomal lurtotecan (NX211): determination of total drug levels in human plasma and urine by reversed-phase high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl, 738(1) (2000) 155-63. [158] S.A. Abraham, K. Edwards, G. Karlsson, N. Hudon, L.D. Mayer, and M.B. Bally, An evaluation of transmembrane ion gradient-mediated encapsulation of topotecan within liposomes. J Control Release, 96(3) (2004) 449-61. [159] P. Tardi, E. Choice, D. Masin, T. Redelmeier, M. Bally, and T.D. Madden, Liposomal encapsulation of topotecan enhances anticancer efficacy in murine and human xenograft models. Cancer Res, 60(13) (2000) 3389-93. [160] J.J. Liu, R.L. Hong, W.F. Cheng, K. Hong, F.H. Chang, and Y.L. Tseng, Simple and efficient liposomal encapsulation of topotecan by ammonium sulfate gradient: stability, pharmacokinetic and therapeutic evaluation. Anticancer Drugs, 13(7) (2002) 709-17. 81

161] C H . Fiske and Y. Subbarow, The Colorimetric Determination of Phosphorus. J Biol Chem, 66(2) (1925) 375-400. 162] W.R. Perkins, S.R. Minchey, P.L. AM, and A.S. Janoff, The determination of liposome captured volume. Chem Phys Lipids, 64(1-3) (1993) 197-217. 163] S.A. Abraham, D.N. Waterhouse, L.D. Mayer, P.R. Cullis, T.D. Madden, and M.B. Bally, The liposomal formulation of doxorubicin. Methods Enzymol, 391 (2005) 71-97. 164] S.A. Abraham, C. McKenzie, D. Masin, R. Ng, T.O. Harasym, L.D. Mayer, and M.B. Bally, In vitro and in vivo characterization of doxorubicin and vincristine coencapsulated within liposomes through use of transition metal ion complexation and pH gradient loading. Clin Cancer Res, 10(2) (2004) 728-38. 165] L.A. Finney and T.V. O'Halloran, Transition Metal Speciation in the Cell: Insightsfromthe Chemistry of Metal Ion Receptors. Science, 300(5621) (2003) 931-936. 166] R.H. Crabtree, The organometallic chemistry of the transition metals, 3rd, John Wiley, New York, 2001. 167] S. Streltsov, V. Oleinikov, M. Ermishov, K. Mochalov, A. Sukhanova, Y. Nechipurenko, S. Grokhovsky, A. Zhuze, M. Pluot, and I. Nabiev, Interaction of clinically important human DNA topoisomerase I poison, topotecan, with doublestranded DNA. Biopolymers, 72(6) (2003) 442-54. 168] R. Andreoli, G.B. Gavioli, L. Benedetti, G. Grandi, G. Marcotrigiano, L. Menabue, and G.C Pellacani, Complex formation of zinc(II) ion with glycine, Nacetyl- and N-benzoyl-glycine anions in aqueous and ethanolic solution by polarographic method. Inorganica Chimica Acta, 46 (1980) 215. 169] A.E. Martell and R.M. Smith, Critical stability constants, Plenum Press, New York, 1974. 170] H.M. Irving and R.J.P. Williams, Stability of transition metal complexes. J Chem Soc, Abstracts, (1953) 3192-3210. 171] I.V. Zhigaltsev, N. Maurer, K. Edwards, G. Karlsson, and P.R. Cullis, Formation of drug-arylsulfonate complexes inside liposomes: A novel approach to improve drug retention. J Control Release, (2005). 172] E. Ramsay, J. Alnajim, M. Anantha, H. Yan, and M. Bally, Enhanced liposomal retention of irinotecan is correlated to increases in plasma irinotecan AUC and improved efficacy against a model of colorectal cancer in: AACR annual meeting, Anaheim, 2005. 173] J. Thompson, E.O. George, C A . Poquette, P.J. Cheshire, L.B. Richmond, S.S. de Graaf, M. Ma, C.F. Stewart, and P.J. Houghton, Synergy of topotecan in combination with vincristine for treatment of pediatric solid tumor xenografts. Clin Cancer Res, 5(11) (1999) 3617-31.

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