COPPER-TOPOTECAN COMPLEXATION: DEVELOPMENT OF A NOVEL LIPOSOMAL FROMULATION OF TOPOTECAN
by
AMANDEEP S. TAGGAR
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
THE FACULTY OF GRADUATE STUDIES
(PATHOLOGY AND LABORATORY MEDICINE)
The University of British Columbia April, 2006
© Amandeep S. Taggar, 2006 ABSTRACT
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 Mn2+, copper(II) (Cu2+), zinc(II)
(Zn2+) or cobalt(II) (Co2+) ion gradients (metal inside) were prepared. Subsequently, 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 Mn2+-containing 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 Co2+ or Zn2+. However, Cu2+-containing liposomes, in the presence 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 x
DEDICATIONS xi
CHAPTER 1 - INTRODUCTION 1 1.1 Project Overview 1
1.2 Transition metals in medicine 3 1.2.1 Transition metal pharmaceuticals 4 1.2.2 Influence of transition metals on therapeutic 6 outcome
1.3 Liposomes 7 1.3.1 Liposomes as drug carriers 7 1.3.2 Components of Liposomes 11 1.3.2.1 Phospholipids 11 1.3.2.2 Cholesterol 15 1.3.2.3 Non-conventional excipients used in bilayers 16 1.3.3 Liposome Classification 17 1.3.4 Formation of liposomes: thermodynamic considerations 19 1.3.5 Gel-to-liquid crystalline phase transition 21 1.3.6 Drug Encapsulation and retention 23 1.3.6.1 Encapsulation methods 24 1.3.6.1.1 Passive encapsulation 26 1.3.6.1.2 Active Encapsulation (remote loading) 26 1.3.6.1.2.1 Ionophores 27
1.4 Complexation reactions with active anticancer agents: their use in 28 liposome formulations
1.5 Camptothecins 31 1.5.1 Camp to thecin Chemistry 32 1.5.2 Camptothecins in liposomes 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 OF TABLES
Table 1.1 List of FDA approved liposomal formulations of some therapeutic 8 agents Table 1.2 Melting points of some saturated alkanes and their 1-unsaturated 14 derivatives Table 3.1 Conversion of topotecan from ring closed to ring open form at 60°C 58 in pH 7.4 buffer
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 13 groups, acyl chains, and other excipients found in lipid bilayers
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 pre- 45
entrapped MnS04 at 40°C or 60°C, (A) in the presence of ionophore A23187, (B) in the absence of ionophore
Figure 3.2 Topotecan encapsulation into DSPC/CH (55:45) liposomes with pre- 47
entrapped MnS04, C0SO4, CuS04 and ZnS04 at 60°C
Figure 3.3 Determination of transmembrane pH gradient before and after 49 addition of drug/ionophore to DSPC/CH (55:45) liposomes
formulated with unbuffered MnS04
Figure 3.4 Topotecan encapsulation into DSPC/CH (55:45) liposomes with pre- 51
entrapped MnS04, CoS04, CuS04 and ZnS04 at 60°C in the presence of ionophore Nigericin
Figure 3.5 Topotecan encapsulation into DSPC/CH (55:45) liposomes with pre- 53
entrapped CuS04 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 CuS04 solution. Inset: representative Cryo-TEM images of liposomes loaded with topotecan under various conditions
Figure 3.6 NaOH induced precipitation of copper in the presence or absence of 56 topotecan
vii Figure 3.7 Influence of lactone ring hydrolysis on topotecan loading into 59
DSPC/Ch liposomes prepared in unbuffered CuS04 solution
Figure 4.1 Possible coordination sites in topotecan 67
vm ABBREVIATIONS
ao area of phospholipid head group CH Cholesterol Co2+ cobalt(II) ions CPP critical packing parameter CPT camptothecin Cryo-TEM cryogenic-transmission electron microscopy Cu2+ copper(II) ions DNA deoxyribonucleic acid dsDNA double strand DNA DOTAP 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 electron spin resonance FATMLV freeze and thawed multilamellar vesicles FDA Food and Drug Administration HBS HEPES buffered saline HEPES 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 Mn2+ manganese(II) ions 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 transition temperature Tm Topo-I topoisomerase-I enzyme UV ultra-violet
V volume of a phospholipid molecule V volume trapped within liposomes (ul/umol) Vis visible light Zn2+ zinc(II) ions 3H-CHE 3H-cholesterylhexadecyl ether 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 AA, HPLC 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 GGW.. .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
1 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 metal-
drug 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.
2 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].
3 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 of free radical 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 pre•
existing 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.
4 OH O OH °o H3C-0
CH2COOH
anthracycline - Doxorubicin anti-inflammatory drug Indomethacin
Cl / CH2-CH2 /
H3C—N \ CHo-CHo \ Cl OH O
Nitrogen Mustard Camptothecin
Figure 1.1 Examples of chemical structures of some drugs that can complex with transition metal ions.
5 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 of free radicals [12],
ii) catalysis of free radical 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].
6 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 long- circulating 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 bio-
distribution characteristics. These properties are regulated via two processes: 1)
liposomal accumulation in the target tissue, and 2) drug retention and release rate from
7 Table 1.1 List of FDA approved Liposomal formulations of some therapeutic agents [65]
Liposomal FDA Therapeutic Disease treated Product Company Name approval agent name year Ambelcet® Enzon 1995/1996 Systemic Amphotericin- B Fungal Ambiosome® Gilead Sciences 1997 Infections Amphotec® Alza Corp. 1997 Lymphomatous Cytarabine DepoCyt® SkyePharma/Enzon 1999 Meningitis AIDS-related Daunorubicin Kaposi's DaunoSome® Gilead Sciences 1996 Sarcoma Ovarian and Caelyx®* Schering-Plough 1999 Breast Cancer AIDS-related Doxorubicin Kaposi's Doxil®* Alza Corp. 1995 Sarcoma Metastatic Myocet® Elan Corp. 2000 Breast Cancer Age-related QLT/Novartis macular Visudyne® 2000 Verteporfin Opthalmics degeneration
*Caelyx® and Doxil® are the same products but approved under different names in different countries
8 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
9 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 adapted fromPH D thesis of Gigi NC Chiu.
10 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
11 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.
12 Head group Glycol Fatty Acyl Chains
Common Head groups Common Acyl Chains
Zwitterionic Saturated Melting point CH3
Laurie (12:0) CH3(CH2)i0COOH 43-44°C
Choline (PC) H3C- -CH, N* CH, CH (CH ) COOH 52-54 °C 1 Myristic (14:0) 3 2 12 CH3 Palmitic (16:0) CH3(CH2)14COOH 59-63°C
Ethanolamine (PE) H3C- —CH2 N*H3 Stearic (18:0) CH3(CH2)16COOH 69-72 °C
Positive Unsaturated CH3
A9 Palmitoleic(16:1 ) CH3(CH2)5CH=CH(CH2)7COOH + Trimethylammonium < ! N CH3
A9 (DOTAP) Oleic(18:1 ) CH3(CH2)7CH=CH(CH2)7COOH
CH3
Negative H I Serine (PS) + H3C N CH3
COOH
HO OH
Inositol (PI) H,C OH
HO OH Cholesterol
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.
13 Table 1.2 Melting points of some saturated alkanes and their 1-unsaturated derivatives
Alkane chain Melting Points
number of double bonds = 0 1*
-35°C Dodecane (Ci2H26) -10°C -23°C Tridecane (Ci3H28) -5°C -12°C Tetradecane (C14H30) 5°C
Pentadecane (C15H32) 10°C -4°C
Hexadecane (C16H34) 18°C 4°C
Heptadecane (C17H36) 21°C 10°C 18°C Octadecane (Ci8H38) 30°C
*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, 1- pentadecene, 1-hexadecene, 1-heptadecene and 1-octadecene.
14 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
15 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 trigger-
release 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 removed frombloo d after i.v.
administration [107, 108]. Although, the entrapped volume and consequently entrapment
efficiency can be increased by freeze-thawing the 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 = V 1.1
where v = volume of the phospholipid
^ = area of the head group
lc = critical chain length
If the CPP
such as Hn phase [75, 112]. However, if CPP is between lA and 1, then bilayers are
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/(aolc)). (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 adopted from[75 ] and [112].
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 transform from a 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 (Tm), the liquid-
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 Tm. On the other hand the pH and ionic strength of the aqueous
solution surrounding the bilayer have direct impact on the Tm, (i.e. increasing the pH
and/or salt concentration increases Tm). The presence of alkaline or transition metal
cations also increases Tm. Acyl chains also have a profound effect on the membrane
phase transition as an increase in the alkane chain length increases the Tm, due to an
increase in chain melting temperature (Figure 1.3 and Table 1.2).
21 Below phase transition Above phase transition temperature; lipid temperature; lipid chains are ordered 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 liquid- crystalline state.
22 On the other hand, the presence of double bonds decreases the Tm, due to a decrease in chain melting temperature (see Table 1.2). The presence of macromolecules, such as
proteins and cholesterol also influences Tm. For example, incorporation of cholesterol in 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, Tm gradually broadens
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 re• hydration 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 well-
established 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 [15-
17] show that doxorubicin can be loaded into Mn2+-containing liposomes even in the
absence of ionophore and these investigators propose that this encapsulation is due to
interaction between doxorubicin molecules and Mn2+ 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 Mn2+-containing liposomes the drug permeated 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 doxorubicin-
Mn2+ complexation, the loading reaction was shown to occur in the absence of a 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 anti• inflammatory 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 extracted from a 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 (Cu2+) was formed and irradiated with UV-light to
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 the right and 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
open ring form 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-ring from lactone 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 (Mn2+) ions [158, 159].
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. Mn2+) was essential for topotecan accumulation. This method was, therefore,
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 work from Thomas 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 E-
ring (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 (Mn2+) and the ionophore A23187 to generate a pH 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 Cu2+ ions could drive the accumulation of
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 Cu2+ 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 as free base. 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 (3H-CHE) and Pico-Fluor 15 scintillation cocktail was purchased fromPerkinElme r Life Sciences Products (Woodbridge, ON, Canada). [14C]- methylamine hydrochloride was purchased from GE Healthcare (Piscatatway, NJ).
Cholesterol (CH), ionophores A23187 & Nigericin, Sephadex G-50 size exclusion gel
and other chemicals (reagent grade) were purchased from Sigma-Aldrich (Oakville, ON,
Canada).
37 2.2 Liposome Preparation
DSPC and CH were dissolved in chloroform at a 55:45 molar ratio. 3H-CHE was 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 polycarbonate filters(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 [3H]-
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 ions from the 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-to-
lipid 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 3H-CHE, by liquid scintillation counting
(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 increased from 10% 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, [14C]-methylamine. Briefly, [14C]-
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 [,4C]-methylamine. The
concentration of liposomal lipid and [14C-]-methylamine in the sample prior to and
following fractionation on the spin column was determined by measuring the [3H] and
[14C] radioactivity on a Packard 1900TR Liquid Scintillation Analyzer using a calibrated
dual label counting program. The transmembrane pH gradient was then calculated by
equation 2.1:
+ + ApH = log{[H ]inSide/[H ]outSjde} = log{[methylamine]inside/[methylamine]outSide} 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 [14C]-lactose. The trapped volume (V) was then
calculated using measured values of [3H] (lipid) and [I4C] (lactose) before and after
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 Mn2+-containing liposomes since results with the Cu2+-containing liposomes clearly
suggested that the Cu2+ was binding the pH probe methylamine. Thus it should be noted that results obtained using the Mn2+-containing liposomes would be comparable to
liposomes containing other metal ions.
2.6 pH titrations of Topotecan and/or copper containing solutions
Samples containing CUSO4, 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 Cu2+ and topotecan containing solutions were added to eight different
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 titrated from 0 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 3H-CHE lipid marker. Drug loaded and 'empty' liposomes were 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 Mn2+ 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 Mn2+ 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 Cu2+ [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-to- lipid ratio was 0.2 (mol/mol) and the final liposomal 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 -\
— # t —I 1 1 1 T 20 30 40 50 60 Time (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) MnS04 gradient. The outer solution was exchanged with pH 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 drug- to-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.
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 Mn2+-containing liposomes was dependent on the presence of a pH gradient. Three reasons could explain the absence of
drug loading under ionophore free conditions: (i) topotecan can not form a complex with
Mn2+ under the conditions used; (ii) Mn2+ is not the appropriate metal; (iii) the belief that
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, CuS04, MnS04 and ZnS04 (pH ~ 3.5)
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 CoS04 (•) and MnS04 (A)
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 ZnS04 (O),
but the extent of encapsulation was low (0.04 mokmol, <20% efficiency) and the
resulting formulation was not stable, i.e. by 60 minutes measured topotecan-to-lipid ratios
were equivalent to those observed for the CoS04 and MnS04 containing liposomes. In
contrast to these results, efficient and stable uptake was observed when topotecan was
added to CuS04 containing liposomes (Figure 3.2; •). Maximum loading (-0.15 drug to
lipid ratio (mokmol)) was achieved within 5 minutes and this drug-to-lipid ratio was
46 0 10 20 30 40 50 60 Time (minutes)
Figure 3.2: Topotecan encapsulation in four DSPC/CH (55:45) liposome formulations containing four different transition metal ions. Liposomes were prepared using C0SO4
(•), CuS04 (•), MnS04 (A), and ZnS04 (O) as described in the methods. The pH of all transition metal ion solutions was adjusted to 3.5 using dilute HC1. In order to create ion gradient the outer solution was exchanged with pH 7.5 HBS using column chromatography. Topotecan was added to the outside of liposomes to achieve a final drug-to-lipid ratio of 0.2 (mol/mol) and incubated at 60 °C for 60 minutes. At 5, 10, 30 and 60 minutes aliquots were fractionated on 1 ml spin columns (packed with Sephadex G-50) to separate encapsulated from unencapsulated drug. Lipid and drug concentrations were quantitated. Each data point represents the mean drug-to-lipid ration of at least three replicate experiments and the error bars indicate the standard deviation.
47 maintained throughout the 60 minute incubation period.
The results in Figure 3.2 demonstrate that topotecan accumulaton into preformed liposomes is facilitated by the presence of encapsulated Cu2+, but not Mn2+, Co2+, or Zn2+.
Further studies were conducted to investigate the potential influence of pH on the formation of metal-topotecan complexes. These studies initially focused on the MnSC^ containing formulations where the magnitude of the transmembrane pH gradient could be determined using the pH gradient probe [14C]-methylamine. Methylamine distributes across the lipid membrane according to the magnitude of the pH gradient. This behaviour is represented by the log of the ratio of methylamine inside/methylamine outside, and was determined under various conditions outlined in Figure 3.3.
Figure 3.3A shows that liposomes prepared in unbuffered MnSCU (pH ~ 3.5) and
subsequently suspended in HBS buffer pH 7.5 possess a transmembrane pH gradient
(ApH) of approximately 2 units (Figure 3.3A; left bar). Previous studies have demonstrated that upon addition of doxorubicin to MnSCM containing liposomes, drug
encapsulation was associated with collapse of the transmembrane pH gradient [16]. This
is illustrated in Figure 3.3A (middle bar) which was included here as a positive control to
demonstrate that doxorubicin encapsulation through use of Mn complexation resulted in complete dissipation of the pH gradient. This does not appear to be the case following
addition of topotecan (Figure 3.3A - right bar), although there was a significant (p<0.05)
decrease in the ApH. This partial collapse of the pH gradient, however, is not associated with any topotecan accumulation inside the liposome (Figure 3.1 and 3.2).
48 2.5 (A)
1.5
P 0.5 0 X No drug Doxorubicin Topotecan 2.5 (B)
1.5 H
0.5 -\
0 + Nigericin/- Topo + Nigericin/+ Topo
Figure 3.3: Determination of the transmembrane pH gradient before and after addition of drug to DSPC/CH (55:45) liposomes formulated with unbuffered MnS04. The pH gradient was estimated using 14C-methylamine as a probe, as described in the Methods, where the log ([methylamine]inside/[methylamine]0ustside) was calculated. This value is reflective of the magnitude of the pH gradient. Panel A - the estimated pH gradient (acidic inside) in the absence of any drug (Bar 1), in the presence of doxorubicin (Bar 2), or in the presence of topotecan (Bar 3). Panel B - the estimated pH when the MnS04 liposomes were pre-incubated (10 min) with the ionophore nigericin: in the absence of any drug (Bar 1), or in the presence of topotecan (Bar 2). Data points represent mean results obtained from at least three replicate experiments and the error bars indicate the standard deviation.
49 It is known that formation of transition metal ion coordination complexes is dependent on the protonation/deprotonation state of the ligands, which is dictated by the pH of the solution [165, 166]. These reactions are, therefore, highly pH dependent. In the case of doxorubicin, complexation was favored at neutral pH [16] and considering that
the addition of doxorubicin to MnS04 containing liposomes resulted in a collapse of the pH gradient, the conditions inside the liposomes were ideal for complex formation. Since it has been reported that the pKa for the hydroxyl group of the A-ring of topotecan is between 6.5 and 7.5 [167] we felt it was important to determine topotecan encapsulation into metal containing liposomes that exhibited an internal pH of 7.5. One strategy used to
achieve this involved pre-incubating MnS04 loaded liposomes with nigericin prior to addition of topotecan. Nigericin is a monovalent cation/proton exchanger that can shuttle one Na+ ion inside the liposome in exchange for one proton, which is moved outside, thus collapsing any pre-existing pH gradient that may be present prior to addition of topotecan. As shown in Figure 3.3B, the addition of nigericin collapses the transmembrane pH gradient that is present across the MnS04 containing liposomes.
Nigericin mediated collapse of the pH gradient was also observed in the presence of topotecan (Figure 3.3B; right bar). The influence of pre-incubation with nigericin on the topotecan loading capacity of liposomes formulated with unbuffered 300 mM solutions
(pH ~ 3.5) of MnS04 (A), C0SO4 (•), CuS04 (•) and ZnS04 (O) suspended in HBS
(pH 7.5) was then determined and the results are summarized in Figure 3.4. Similar to the results obtained in the absence of nigericin (Figure 3.2), topotecan loading was only observed in the formulation prepared with CUSO4 (Figure 3.4, •).
50 Figure 3.4: Topotecan accumulation in DSPC/CH (55:45) liposome formulations prepared in unbuffered C0SO4 (•), CuS04 (•), MnS04 (A), and ZnS04 (O). After preparation the external solution surrounding the liposomes was replaced with HBS (pH 7.5) and the ionophore nigericin was added prior to heating the liposomes to 60°C. Subsequently, topotecan was added to the liposomes and the amount of liposome associated topotecan was measured as a function of time. Each point represents the mean ± standard deviation determined for at least three separate samples.
51 The role of the transmembrane pH gradient was further characterized in the
CuSC>4 containing liposomal formulations. The results of these studies, summarized in
Figure 3.5, address two specific questions. First, how is topotecan loading influenced by
addition of the ionophore A23187? As illustrated in Figure 3.5 A, in the presence of
ionophore topotecan uptake occurs, but only when the incubation temperature is 60 °C
(Figure 3.5 A, O) and the rate of drug loading is slower than that observed in the absence
of A23187. Figure 3.5B indicates that in the absence of A23187, topotecan accumulation
occurs more rapidly both at 40 °C and 60 °C with maximum drug-to-lipid ratios being
achieved within 5 minutes. In the presence of A23187, drug loading would be in response
to a pH gradient, whereas we make the assumption that in the absence of A23187, drug
loading is due to metal complexation. It is not clear why drug loading rates would be
slower in response to a pH gradient, but this result draws a clear distinction between the
two drug loading methods.
The second question assessed whether topotecan would accumulate into
liposomes comprised of an internal CUSCM pH 7.5 buffered solution. The CUSCM buffer
was prepared with HEPES and adjusted to pH 7.5 with triethyleneamine to mimimize
copper precipitation. The resulting solution was stable, as judged by the lack of visible
precipitates and changes in pH, for approximately 24 hours at room temperature and 2
hours at 60 °C. Liposomes prepared with this meta-stable CuSC>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) CuS04 plus A23187
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) CUSO4 (pH 3.5) inside plus transmembrane ionophore A23187, (B) CUSCM (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 TEA. 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 CuS04 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 CUSO4 with/without ionophores as described previously in the methods. Panel A shows the empty liposomes; Panel B shows the CuS04 containing liposomes loaded with topotecan; Panel C shows topotecan loaded liposomes containing CUSO4 and nigericin.
53 loading was remarkably similar to that observed in liposomes prepared in unbuffered
CuS04 (Figure 3.5B). At 20 °C (Figure 3.5C, A) there was no appreciable drug uptake.
Given our assumption that Cu2+ ions were forming a complex with topotecan
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 CuS04 in the absence of
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 CuS04,
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 Cu2+-topotecan interaction. The rational for 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
Figure 3.6: NaOH induced precipitation of Cu2+ 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.
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
Cu2+-containing liposomes, we pre-incubated the drug in acidic or basic pH buffers prior
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 <0.001 (Figure 3.7A; middle bar)).
Consistent with the results in Table 1, at pH 7.5 >85% of the drug exists in carboxylate
form, i.e. the lactone/total drug <0.20 (Figure 3.7A; right bar). Figure 3.7B illustrates the
encapsulation efficiency (drug-to-lipid ratio) of liposomes (prepared in unbuffered
300mM CuSCu) following addition of topotecan that has been pre-incubated at pH 3.5 or
pH 10.0. The pre-incubated drug was added to liposomes suspended in HBS pH 7.5 and
the amount of liposome-associated drug was calculated after further 30 minute incubation
at 60 °C. When topotecan was pre-incubated at pH 3.5, drug-to-lipid ratio of 0.15
57 Table 3.1: Conversion of topotecan from ring closed (lactone) to ring open (carboxy) form at 60 °C in ph 7.4 SHE buffer.
Incubation time (min) (lactone/total drug)3 (carboxy/total drug)3
5 0.30 0.70
10 0.18 0.82
30 0.15 0.85
60 0.15 0.85 atopotecan was analyzed using an HPLC method (see Methods) that could resolve the lactone and carboxylate forms of the drug. The peak AUC for the two forms were combined to define total drug levels and the ratio of the lactone or carboxylate to total drug was calculated.
58 1 (A) 0.8
03 o 0.6
c 0.4 o o ro 0.2
pH 3.5 pH 10 pH 7.5
0.2 (B) O E 0.15 o E -a 0.1 Q.
D) 3 0.05
Loaded liposomes (pre- Loaded liposomes (pre- incubated pH 3.5) incubated pH 10)
(C)
i_ 0.8 •o ro 0.6
0) 0.4 c o TJ 0.2 H ro
Loaded liposomes (pre- Loaded liposomes (pre- incubated pH 3.5) incubated pH 10)
Figure 3.7: The influence of lactone ring hydrolysis on topotecan loading into DSPC/CH liposomes prepared in unbuffered 300 mM CuS04. Topotecan was analysed using an HPLC method that was designed to qualitatively measure the proportion of topotecan in the lactone form or the carboxylate form (see Methods). Peak AUC for lactone and carboxylate forms were added to define total drug levels and the ratio of lactone to total
59 drug was determined under various conditions. Panel A - Unencapsulated topotecan was analyzed following 30 minute incubation at 60°C under different pH conditions: (i) pH 3.5, (ii) pH 10.0 and (iii) pH 7.5. Panel B - Topotecan was pre-incubated for 30 minutes at 60 °C in: (i) pH 3.5 buffer, and (ii) pH 10.0 buffer prior to addition to liposomes. The exterior buffer was adjusted to pH 7.5 and the lactone to total drug ratio for encapsulated topotecan was calculated after 30 minutes. Panel C - The final drug-to-lipid ratio for the topotecan-loaded liposomes described in Panel B. Sephadex G-50 size exclusion column chromatography was used to separate encapsulated drug (collected in the eluted volume) from unencapsulated drug. Lipid and topotecan concentrations were measured as described in the Methods. Each data point represents the mean value determined from three different replicate experiments and error bars indicate standard deviations. Panel C shows the drug-to-lipid ratio of encapsulated drug under various conditions.
60 (mohmol) was obtained (Figure 3.7B; left bar), suggesting a loading efficiency of 75%.
In contrast, pre-incubation of topotecan at pH 10 significantly inhibited drug loading.
After 30 minute incubation with the liposomes, a drug-to-lipid ratio of < 0.02 was observed (Figure 3.7B; right bar). The integrity of the liposome associated topotecan was also assessed and as indicated in Figure 3.7C. > 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 Mn2+ 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 Cu2+ ions and is not dependent on use of a transmembrane pH gradient or an ionophore such as A23187. Evidence is provided to suggest that topotecan can complex with Cu2+ and HPLC analysis of the drug loaded liposomes suggest that the encapsulated drug exists primarily in its active lactone form. This result supports previous conclusions developed on the basis of a complexation reaction between Mn2+ 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 Cui+ containing liposomes and this effort proved successful (Figure 3.2). Additionally, I evaluated topotecan uptake into liposomes containing Co2+ and Zn2+, but only Cu2+ containing 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 Co2+, Mn2+ or Zn2+ solutions (pH ~ 3.5) were not ideal for 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 Co2+, Mn2+ or Zn2+, 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 protons from MnSCU containing liposomes for Na+
from the external HBS resulting in the complete collapse of any pre-existing pH gradient.
When liposomal formulations containing Cu2+, Co2+, Mn2+ or Zn2+ 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 Co2+, Mn2+ or Zn containing liposomes. However, from the 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 Nl 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 Cu2+ is preferred over that with Co2+, Mn2+ or Zn2+. This preference is consistent with the well established Irving-
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 (pn) for the four metals used in this study would be: Mn < Co < Cu >Zn .
Briefly, the overall formation constant (pn) is an equilibrium constant of a reaction in 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 M2+ reacts with nL, representing ligand molecules:
2+ 2+ [M(H20)x] + nL U [MLn] + xH20 3.1
B = [MU]2+ 32 P 2+ n " [M(H20)x] [L]
Therefore, if pn is large then ML, is thermodynamically preferred, and is a stable
complex.
65 Regardless of where the complexation occurs and the value of (3n, the results presented in Figure 3.7 demonstrate that topotecan, when loaded into liposomes prepared
in CuS04, is primarily in its active lactone (ring closed) form. Thus it can be concluded that complexation with copper may protect topotecan from hydrolysis 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 Cu2+ ions interact with lipid molecules in monolayers and bilayers. These
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 Cu2+ 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.
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 release fromliposome s 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 release from these 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
(Cu2+) and topotecan can be utilized to drive the accumulation of the drug into the
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 with free drug 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 benefits from combination 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 results fromDr . Bally's laboratory (unpublished data) show that vincristine can also be encapsulated into liposomes using a Cu2+ 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.
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