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Thermosensitive liposomal cisplatin : formulation development, in vitro characterization and in vivo… Woo, Janet 2005

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THERMOSENSITIVE LIPOSOMAL CISPLATIN: F O R M U L A T I O N D E V E L O P M E N T , IN VITRO C H A R A C T E R I Z A T I O N AND IN VIVO P L A S M A ELIMINATION STUDIES by J A N E T W O O B.Sc. (Biology and Biochemistry), University of Victoria A thesis submitted in partial fulfillment of the requirements for the degree M A S T E R O F S C I E N C E in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Pathology and Laboratory Medicine) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A October, 2005 © Janet Woo, 2005 A B S T R A C T Cytotoxic anticancer drugs lack specificity and for this reason, antitumour effects are often associated with severe and sometimes life threatening toxicities. In an effort to improve the specificity and targeting of anticancer drugs, investigators have developed drug carrier formulations. Among the different drug delivery systems designed for intravenous use, liposomes have demonstrated beneficial properties that translate into meaningful improvements in therapeutic activity for many anticancer drugs. Cisplatin is a commonly employed anticancer drug which is nephrotoxic and myelosuppressive. In an effort to minimize the drug associated toxicities, a liposomal drug formulation of cisplatin has been developed for use in clinical trials. The results of these clinical studies; however, were disappointing as the cisplatin formulation demonstrated insignificant therapeutic activity, due in part, to the insufficient release of encapsulated contents following administration. For this reason, the development of a triggered release liposome formulation is desirable. In this report, cisplatin was encapsulated into lysolipid containing thermosensitive liposomes ( L T S L ) using a novel technique, which relies on the equilibration of cisplatin across the liposomal bilayer at temperatures above the gel-to-liquid phase transition (Tc) of the bulk phospholipid. M i l d heating of the L T S L did not engender the presence of any irregular structures as determined by cryo-transmission electron microscopy and interestingly, the drug loaded liposomes were similar in morphology to the empty liposomes. In vitro data demonstrated that >95% of encapsulated cisplatin was released within 5 minutes following mild heating at 42°C while <5% was released after incubation at 37°C. Plasma elimination studies demonstrated the importance o f regulating the whole i i animal body temperature when injecting thermosensitive liposomes. Under conditions where murine body temperature was well controlled (37° + 0.5°C), plasma elimination profiles indicated that LTSL-cisplat in retained approximately 50% more drug 2 hours following intravenous administration than that observed when animal body temperature was not carefully controlled. Collectively, these results suggest that this thermosensitive liposomal formulation o f cisplatin is suitable for further studies designed to assess therapeutic activity when used in conjunction with mi ld local heating. i i i T A B L E O F C O N T E N T S A B S T R A C T . i i T A B L E O F C O N T E N T S iv LIST O F T A B L E S ' vi LIST O F F I G U R E S v i i A B B R E V I A T I O N S v i i i A C K N O W L E D G E M E N T S ix C H A P T E R 1. I N T R O D U C T I O N 1 1.1 Project overview 1 1.2 Drug delivery 2 1.2.1 Liposomes 3 Therapeutic advantages 3 Components of liposomes 4 Liposome classification 7 Gel-to-liquid crystalline phase transition 8 temperature (Tc) Drug encapsulation methods 10 Advances in liposome technology 13 Triggered drug release 14 1.3 Hyperthermia 14 1.3.1 Combination therapy - hyperthermia and drug delivery 15 1.3.2 Heating techniques 16 1.3.3 Development of lysolipid containing thermosensitive 16 liposomes (LTSL) 1.4 Cisplatin 19 1.4.1 Biological activity 19 1.5 Research objectives and hypotheses 21 C H A P T E R 2. M E T H O D S AND M A T E R I A L S 23 2.1 Materials 23 iv 2.2 Preparation of liposomes 23 2.3 Cisplatin solubility determination 24 2.4 Atomic absorption spectroscopy ( A A S ) 25 2.5 Drug loading methods 25 2.5.1 Cisplatin encapsulation via passive equilibration 25 2.5.2 Loading into cholesterol containing liposomes via passive 26 encapsulation 2.6 Trapped volume determination 27 2.7 Cryo-transmission electron microscopy (Cryo-TEM) 28 2.8 In vitro drug release assay 31 2.9 Plasma elimination of cisplatin loaded liposomes 31 2.10 Statistical analysis 32 C H A P T E R 3 . R E S U L T S 3 3 3.1 Cisplatin encapsulation via passive equilibration 33 3.2 Lactose trapping by passive encapsulation and passive equilibration 37 3.3 Cisplatin loaded thermosensitive liposomes by c r y o - T E M 37 3.4 Temperature dependent release of cisplatin from thermosensitive 40 liposomes 3.5 Plasma elimination studies of cisplatin loaded thermosensitive 42 liposomes C H A P T E R 4 . D I S C U S S I O N A N D C O N C L U S I O N 4 6 C H A P T E R 5 . F U T U R E D I R E C T I O N S 51 R E F E R E N C E S 5 3 v LIST OF TABLES Table 3.1. Cisplatin solubility in 150 m M saline at different temperatures. 36 Table 3.2. Trapped volume determination of L T S L and T S L by the methods 38 of passive encapsulation and passive equilibration. vi L I S T O F F I G U R E S Fig. 1.1. Structure of cholesterol and phospholipids generally used in 6 liposomes. Fig . 1.2. Gel-to-liquid crystalline phase transition temperature (Tc) of 9 membrane lipids. Fig . 1.3. Illustration of the different drug encapsulation methods and drug 11 distribution patterns. Fig . 1.4. Schematic of the mechanism underlying the combination of 18 hyperthermia and liposomal drug delivery in the treatment of solid tumours. Fig. 1.5. (A) Various binding modes of cisplatin to D N A and protein. 20 (B) Fate of cisplatin metabolism in aqueous solution. Fig. 2.1. C r y o - T E M climate chamber and sample preparation technique. 29 Fig. 2.2. 2-D projection of c r y o - T E M images. 30 Fig . 3.1. (A) Time dependent uptake of cisplatin (2mg/mL final drug 34 concentration) via passive equilibration into L T S L at various temperatures. (B) Time dependent uptake of cisplatin (10 mg/mL final drug concentration) via passive equilibration into L T S L , T S L and DPPC/choy D S P E - P E G 2 0 o o liposomes at 70°C. Fig. 3.2. C r y o - T E M electron micrographs of empty and cisplatin-loaded 39 L T S L and T S L prepared using the passive equilibration technique. Fig . 3.3. Drug release assays from three different liposomal formulations at 41 37°C and 42°C as a function of time. Fig. 3.4. Plasma elimination profiles of L T S L and T S L 2 hours following 43 i.v. administration in Rag2-M mice under conditions where animal body temperature was or was not controlled. Fig . 3.5. Plasma elimination profiles of L T S L , T S L , DPPC/cho l / D S P E - 45 PEG2000 cisplatin loaded liposomes and free cisplatin in Rag2 -M mice. vn A B B R E V I A T I O N S A A S atomic absorption spectroscopy [ 1 4 C] carbon 14 radiolabel C H E cholesterylhexadecyl ether chol cholesterol c r y o - T E M cryo-transmission electron microscopy D N A deoxyribonucleic acid D : L drug-to-lipid ratio (wt/wt) D P P C 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine DSPE-PEG2000 l ,2-distearoyl-5«-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt) E P R enhanced permeability and retention [ 3H] tritium radiolabel i.m. intramuscular i.v. intravenous L T S L lysolipid containing thermosensitive liposomes L U V large unilamellar vesicle M L V multilamellar vesicle m P E G - P E methoxy(polyethyleneglycol)-distearoyl-phosphatidylethanolamine M P S mononuclear phagocytic system M S P C l-steoryl-2-hydroxy-,s«-glycero-3-phosphatidylcholine N M R nuclear magnetic resonance P C phosphatidylcholine S E C size exclusion chromatography S U V small unilamellar vesicle Tc gel-to-liquid crystalline phase transition temperature T S L thermosensitive liposomes (non-lysolipid containing) Q E L S quasi-elastic light scattering vin A C K N O W L E D G E M E N T S I would not have been able to complete this thesis without the help of many people. First, I would like to thank Marcel for accepting me as a graduate student in your laboratory and for being such a wonderful supervisor. Your patience and support was much appreciated, especially in M i a m i at the Controlled Release Society meeting, where I continually annoyed you with my many questions. I can't thank you enough for that opportunity - the experience definitely increased my confidence, as a scientist and as an individual. '. . . Special thanks to Kirsten, Don and Hans for introducing me to the world' o f science! Words cannot express how much I appreciate all you have done for me and most of all , for always listening to me talk. Thank you, thank you, and thank you! Gigi and Nancy - thank you for teaching me the art of making liposomes! Thank you for all your help, enthusiasm and discussions about everything and anything (beading!). I am grateful to every member of Advanced Therapeutics as well as to all the other individuals in the research centre I have so fortunately had the opportunity to meet. I would like to take this opportunity to thank each and every one of you. Cheers to research, good times, friendship and laughter. A special thanks also goes to the Dong brothers, the Leung and Lowe families for their continual support and encouragement throughout my entire degree. Lastly, I would like to give my warmest thanks to my parents and my siblings, especially my older brother Richard, for believing in me. Thank you for all the yesterdays, all the todays and all the tomorrows. ix C H A P T E R 1 I N T R O D U C T I O N 1.1 Project overview Liposomes are small, l ipid based structures that have been developed for intravenous drug delivery applications. Encapsulation of anticancer drugs in liposomes has been shown to improve the pharmacokinetic and biodistribution attributes of their associated drugs, such as reducing toxicity and improving therapeutic activity (1). A n ideal and effective liposomal drug carrier would balance circulation longevity and the ability to release their encapsulated contents following localization at the tumour site. A n example where enhanced tumour delivery is not associated with improved therapeutic benefit is a liposomal formulation of the anticancer drug cisplatin (SPI-077 or stealth liposomal cisplatin) (2). It was argued that this formulation did not achieve the correct balance between circulation lifetimes/improved tumour delivery and drug release attributes to provide any therapeutic benefit. Studies with SPI-077 suggested that cisplatin delivery to sites of tumour growth in tumour bearing animals were significantly increased following i.v. administration (3-5). However, lack of therapeutic activity was explained by the fact that cisplatin remained with the liposomal carrier following localization. It was therefore, reasonable to suggest that methods designed to increase drug release from the liposomes following localization would result in improved therapeutic effects. Such "triggered" drug release can be achieved using a number of approaches, but the research within this thesis is focused on the use of mild heating, or hyperthermia to engender drug release from liposomes that have localized in a site of tumour growth. Hyperthermia is a cancer treatment modality that when applied alone is directly cytotoxic (6). In addition, it alters the tumour microenvironment to favour delivery of drugs and as well , provides synergistic effects when combined with chemotherapy and radiation therapy protocols. The combination of local hyperthermia and liposomal drug delivery has been used in the treatment of cancer and has been shown to have an enhanced therapeutic effect compared to either treatment modality alone or the combination of hyperthermia and free drug (7). Currently, a lysolipid containing thermosensitive liposomal formulation ( L T S L ) has been described (8). Studies using this formulation have never been used for the delivery of cisplatin and instead have primarily focused on another anticancer drug, doxorubicin. Data with this drug suggested that doxorubicin is very permeable across the L T S L membrane, even in the absence of heating (9, 10). Therefore, the studies within the context of this research was initiated based on the hypothesis that cisplatin, with its hydrophilic nature and its low affinity for lipid membranes, would be a more suitable candidate for encapsulation within L T S L . The aim of the research described in this thesis was to combine the benefits of hyperthermia with those achieved with liposomes in the development of a thermosensitive liposomal formulation of cisplatin. In the sections to follow, a brief introduction to liposomal drug delivery systems and hyperthermia, as it relates to the intended application described in this research w i l l be provided. 1.2 D r u g delivery The benefits of many chemotherapeutic drugs commonly used in the treatment of cancer are limited by the lack of selectivity to tumour cells. In attempts to address this indiscriminate toxicity, anticancer drugs have been associated with a variety of drug carriers. Among the variety of drug delivery systems designed for intravenous use, 2 liposomes have demonstrated their versatility as carriers to improve the pharmacological effects of a variety of drugs and macromolecules (11-13). Based upon this achievement, liposomes were used for the studies performed within this research. 1.2.1 Liposomes First described by Bangham et al. (14), liposomes are membrane bound vesicles which spontaneously form upon hydration of amphipathic lipids in an aqueous medium. They were initially used as model membrane systems to evaluate the structural and functional roles of lipids in biological systems, such as the processes of permeability and diffusion of molecules (15). However, as it was realized that the unique structural properties of liposomes could be used to effectively entrap hydrophilic drugs in the aqueous interior and l ipid soluble drugs in the hydrophobic moiety ofthe phospholipid bilayer, they were soon considered as an interesting class of delivery vehicles. In fact, liposomes have been evaluated as carriers for the delivery of a wide variety of agents, including anticancer drugs (1, 16-18), antimicrobial agents (19, 20), enzymes (21) and antisense oligonucleotides (22, 23). These research efforts have culminated in the clinical approval of several liposomal formulations that exhibit improved therapeutic profiles relative to the free form of the drug. These consist of DaunoXome® (daunorubicin), DepoCyt® (cytosine arabinoside) and Doxil® (doxorubicin) (24, 25). Therapeutic advantages The therapeutic index of a chemotherapeutic drug can be significantly improved when associated with a liposomal carrier (26, 27). Improvements are a consequence of 3 liposome mediated changes in drug circulation lifetimes and tissue biodistribution characteristics, features that are regulated by two processes: 1) liposome accumulation in target tissues and 2) drug release rates from liposomes within the plasma compartment and/or at the site of liposome accumulation. Liposomes are able to passively localize at sites of increased vascular permeability, such as in regions of solid tumour growth (28) as well as in regions o f infection (29) and inflammation (30). This process is referred to as the E P R (enhanced permeability and retention) effect (31) and results in a local concentration of drug, while decreasing the amount and types of drug associated toxicities. Structurally and physiologically different from their normal counterparts, the tumour microvasculature is typically discontinuous and can contain fenestrations varying in diameter between 380-780 nm (32). It is this heterogeneous and chaotic tumour microenvironment, together with an impaired lymphatic drainage supporting the tumour area which results in selective liposome extravasation and accumulation. Components of liposomes Liposomes are spherical structures o f amphipathic l ipid molecules assembled into bilayers enclosing an aqueous core. Liposomes can be prepared from a variety of lipids and lipid mixtures. However, resembling biomembranes which exist in nature, liposomes are commonly composed of phospholipids and cholesterol. The following sections w i l l introduce the chemical structure and physical properties of the general types of lipids that are commonly used in liposome formulations. 4 Phospholipids Phospholipids constitute the largest class of membrane lipids and are often used as the principal l ipid in liposomes. This group of lipids consists of a hydrophilic phosphate containing head group and hydrophobic acyl chains linked via ester bonds to a glycerol backbone. The distinct properties of each phospholipid are determined both by the nature ofthe head group and by the variability in the length and degree of saturation ofthe hydrocarbon chain. The head groups of phospholipids can be varied and include phosphatidylcholine (PC) and phosphatidylethanolamine (PE), which are zwitterionic and phosphatidylserine (PS), phosphatidylglycerol (PG) and phosphatidylinositol (PI), which are negatively charged. The acyl chain length of phospholipids of physiological membranes typically range from 12-24 carbon atoms (C12-24) and of them, the most commonly used in liposomal drug delivery are myristic (C14), palmitic (C16) and stearic (C18). The general structure of phospholipids is illustrated in F ig . 1.1. Cholesterol (chol) Cholesterol, the major sterol in animal tissues, is a ubiquitous component of cell membranes and is essential in regulating the physical and biological properties of cell membranes (33, 34). One of the specific physical features of cholesterol is its planar steroid nucleus of four fused hydrocarbon rings (Fig. 1.1). The presence o f a hydroxyl group makes this otherwise hydrophobic molecule amphipathic, mediating intercalation into the membrane bilayer. The incorporation of cholesterol into membrane bilayers causes major changes in the properties of liposomes. For example, the interaction between liposomes with plasma 5 General structure of head group glycerol backbone acyl chains Common phospholipid head Zwitterionic phospholipids C H ; — C H 2 — N C H 3 choline (PC) C H 3 C H 2 — C H 2 — N H 3 ethanolamine (PE) Negative phospholipids H I C H 2 — c — N H , serine (PS) coo-O H CH7 C H C H A O H glycerol (PG) O H O H inositol (PI) Common acyl chains Saturated acyl chains Laurie (12:0) CH 3 (CH 2 ) i 0 COOH Myristic (14:0) CH 3 (CH 2 ) i 2 COOH Palmitic (16:0) CH 3(CH 2)i4COOH Stearic (18:0) CH 3 (CH 2 ) 1 6 COOH Unsaturated acyl chains Palmitoleic (16:149) CH 3(CH 2)5CH=CH(CH 2) 7COOH Oleic (18:149) CH 3(CH 2) 7CH=CH(CH 2) 7C00H Linoleic (18:14 9 1 2) CH 3(CH 2) 4CH=CHCH 2CH=CH(CH 2) 7COOH Cholesterol Fig. 1.1. Structure of cholesterol and phospholipids generally used in liposomes. 6 proteins and lipoproteins in vivo often results in extensive l ipid exchange (35, 36). However, the presence of cholesterol can stabilize liposomes in the circulation by reducing this phospholipid transfer and by rigidifying the membrane (37, 38). Cholesterol also has implications in the gel-to-liquid crystalline phase transition temperature (Tc), which w i l l be described in section Polyethylene glycol conjugated lipids ( P E G ) Some have argued that the therapeutic application of i.v. injected liposomes has been limited by the rapid removal and uptake by the cells and macrophages in the mononuclear phagocyte system (MPS) (26, 39). However, it has been shown that the presence of hydrophilic polymers, such as that of polyethyleneglycol (PEG) at the liposome surface provided an extended circulation longevity as well as a decrease in both the rate and extent of uptake by the M P S (40, 41). Due to the presence of these polymers on the liposome surface, these liposomes are often referred to as "sterically stabilized" (41) or "stealth" liposomes (42). It is believed the mechanism of liposome protection offered by the P E G lipids is due to the ability of P E G to provide a steric repulsive barrier which inhibits the binding of plasma proteins, membrane-to-membrane contact and subsequent liposome aggregation (43-45). Liposome classification There are three general types of liposomes which are classified according to the liposome size and the number of lipid bilayers. Upon hydration of a dried lipid film, multiple l ipid bilayers enclosing an equal number of aqueous spaces, referred to as multilamellar vesicles ( M L V s , 1-100 um) are formed. The trapped volumes (the internal 7 aqueous volume in uL/u.mole lipid) are generally small; however, it has been shown that cycling the preparation through freezing and thawing can increase the trapped volume as a result of increases in the interlamellar spacing (46). Due to their large size, M L V s are rapidly removed from the plasma compartment following administration and thus, are of limited value for applications relying on i.v. injections (47). However, the size and lamellarity can be modified by methods such as the extrusion technique (48, 49). The method of extrusion involves passing M L V s through polycarbonate filters of defined pore size, under moderate pressures of an inert gas. This results in a homogenous population of large unilamellar vesicles ( L U V s , 50-200 nm), consisting of a single bilayer membrane enclosing an aqueous core. L U V s , with trapped volumes ranging from 1-2.5 uL/umole lipid, are commonly used for drug delivery purposes due to their stability in circulation, optimal trapped volumes and their ability to extravasate and accumulate in diseased tissues. Small unilamellar liposomes (SUVs, 25-40 nm) can be prepared by sonication and are similar to L U V s in that they comprise a single l ipid bilayer. However, due to their small size, S U V s are relatively unstable and tend to undergo aggregation and fusion (50). Thus, these liposomes are generally not used for i.v. administration routes. Gel-to-liquid crystalline phase transition temperature (Tc) Phospholipids exhibit well defined gel-to-liquid crystalline phase transition temperatures (Tc) that are dependent on the properties of the l ipid head group and the composition of the acyl chains. When phospholipids are fully hydrated, they can exist either within the gel phase, where the acyl chains exhibit a well ordered conformation or within the liquid-crystalline state, where the acyl chains are significantly more disordered. The Tc is the temperature at which the transition occurs between the two phases (Fig. 1.2). 8 chains ordered Gel state Acyl chains disordered Liquid-crystalline state Lipids commonly used in liposomal drug delivery DSPC(c18) Tc = 55°C DPPC (c16) Tc = 41°C DMPC(c14) Tc = 23°C Fig . 1.2. Gel-to-liquid crystalline phase transition temperature (Tc) of membrane lipids. 9 This transition, which arises from melting of the hydrocarbon chains, coincides with a membrane structure that is more permeable (51). The length and saturation ofthe lipid hydrocarbon chains, the nature ofthe head group and the environmental conditions ofthe aqueous phase greatly influence the Tc. In general, lipids with longer and more saturated acyl chains have a higher Tc. The cholesterol content also modulates the Tc of the l ipid membrane. Cholesterol positions itself within the bilayer such that the steroid nucleus is aligned with the phospholipid acyl chain and the hydroxyl group with the carbonyl ester bond, which is oriented toward the aqueous surface. This orientation induces a conformational ordering o f the hydrocarbon chains, which, in turn, affects the fluidity of the membrane. These changes in fluidity are paralleled by changes in membrane permeability (52). A t temperatures above the Tc, cholesterol increases the order and packing density of the acyl chains. In contrast, at temperatures below the Tc, cholesterol helps to decrease the order and packing density within the bilayer. A t concentrations >30 mol% cholesterol, the Tc is virtually eliminated. D r u g encapsulation methods Liposomes can be prepared by a variety of methods, but there are two basic techniques for encapsulating both hydrophilic and hydrophobic drugs within liposomes. Entrapment of candidate drugs may be achieved either during liposome formation (passive encapsulation) or by loading into preformed liposomes (active encapsulation). The latter can be achieved through the use of transmembrane ion gradients (pH gradient, metal ion) as well as the method of drug encapsulation first described in this thesis, termed passive equilibration. A n illustration ofthe different drug encapsulation methods and drug distribution patterns is depicted in Fig . 1.3. 10 Extrude • SEC B) Active Encapsulation (involves preformed liposomes) pH 7.4 O C) Passive equilibration (involves preformed liposomes) o ° o Add drug (cisplatin) SEC Heat >Tc Fig . 1.3. Illustration of the different drug encapsulation methods and drug distribution patterns. Passive encapsulation With passive encapsulation techniques, the candidate drug or small molecule, whether hydrophobic or hydrophilic, is added to dried lipids prior to liposome formation. The passive encapsulation of hydrophobic drugs is entirely dependent on the ability ofthe lipid membrane to solubilize the drug during the rehydration period. Under favourable drug-lipid interactions and drug solubility, high encapsulation efficiencies can be achieved. Passive encapsulation of hydrophilic drugs such as cisplatin, into the liposome interior involves dissolving the drug in the aqueous buffer used to hydrate the l ipid fi lm. A s entrapment and retention of drugs using this procedure depends on the trapped volume of the liposome as well as liposomal lipid concentration, trapping efficiencies <30% are typically achieved. Act ive encapsulation B y contrast, drugs may be encapsulated into the interior of preformed liposomes in response to a transmembrane ion gradient. This is best exemplified by encapsulation methods that depend on establishing a p H gradient (pH 4.0 inside, p H 7.4 outside) across the liposomal bilayer. Prior to encapsulation, the drug is in its uncharged state and as such, is capable of readily diffusing across the liposomal membrane. However, upon reaching the acidic interior o f the liposome, the drug is protonated. Since charged species are often less membrane permeable, the encapsulated drug is thus effectively trapped within the liposome. This method can be applied to drug candidates that contain ionizable amine groups, such as the anthracycline antibiotics. Drug retention by this method is dependent on the liposomal lipid composition, the ability of these liposomes to maintain the p H 12 gradient and the permeability of the encapsulated drug, whether in the protonated or unprotonated form (53). High trapping efficiencies (>90%) can be achieved using this method of encapsulation. ' Passive equil ibration The method of passive equilibration has been designed to encapsulate drug candidates into the interior of preformed thermosensitive liposomes. This technique takes advantage of the inherent Tc of lipids and relies on the equilibration of drug across liposomal membranes at temperatures above the Tc of the bulk phospholipid. The drug concentration entrapped in the liposomes is equivalent to that in the external phase and the amount of the drug which can be encapsulated within liposomes is dependent on the solubility of the drug at the production temperature. Advances in liposome technology With the advancement of liposomal drug delivery systems, the need for specific targeting and mechanisms to trigger release upon arrival at the target site has become apparent. Under the most ideal situation, drug loaded liposomes would circulate for extended periods of time and would only release their encapsulated contents following localization at the tumour site. The rate o f drug release would also need to be regulated in order to achieve optimal therapeutic effects. Interestingly, compositions that readily retain encapsulated drug and circulate for extended time periods often facilitate the greatest improvements in drug accumulation within target tissues. However, enhanced drug delivery does not directly translate into optimal therapeutic activity. It has been argued that i f these formulations could be designed to release encapsulated contents following 13 localization, then they would exhibit improved therapeutic activity and specificity. For these reasons, development of effective triggering mechanisms to allow release of liposomal contents has become an important research topic. Triggered drug release Examples of triggering mechanisms that have been studied include drug release in response to p H changes (54, 55), enzyme mediated processes (56) and mi ld heating, or hyperthermia (57). O f these mechanisms, the latter has received considerable interest, both from a mechanistic (delivery) (58, 59) and a therapeutic (60, 61) perspective. The following sections w i l l provide an overview on the role of hyperthermia as a clinical modality and w i l l also highlight the benefits of combining liposomal drug delivery with hyperthermia treatment. 1.3 Hyper thermia The primary therapeutic approaches of cancer treatment include surgery, chemotherapy and radiation therapy. Hyperthermia is also used as a clinical tool in the treatment of cancer (62). However, as cancer is such a complex disease, a combination of these modalities, depending on the type and stage of disease is generally implemented. Clinically, hyperthermia is defined as the application of a power source to a region of the body purposely to raise the local temperature a few degrees (39° - 42°C) above normal physiological temperature (37°C). The use of hyperthermia offers many benefits and has been used in humans and animals for multimodality treatment of cancer (62). While being directly cytotoxic (63), it is also used as an adjuvant to some radiation (64, 65) and 14 chemotherapy (66, 67) regimens. Application of local hyperthermia modifies the tumour environment, by altering blood flow and tumour physiology (68). These effects improve tumour oxygenation and are the reason why hyperthermia is used in combination with radiation protocols. When used in combination with chemotherapy, hyperthermia increases drug uptake in tumour tissue (7, 67) and when used in conjunction with D N A damaging agents, it can enhance the amount of D N A damage while inhibiting D N A repair (64). The clinical value of hyperthermia in combination with other treatment modalities has been demonstrated in several randomized trials (69-73), where clinical response rates were typically doubled. 1.3.1 Combinat ion therapy - hyperthermia and drug delivery In the context of drug delivery studies, it has been established that hyperthermia can increase the efficiency, both the rate and extent, o f macromolecule delivery to the heated region (74-76). There are numerous studies indicating that the combination of liposomes with hyperthermia increases intratumoural liposome concentration, which in turn, is associated with increased drug delivery compared to that achieved with delivery of free drug or liposomal drug in the absence of heating (77-79). The rationale for the combination of hyperthermia and chemotherapy is further substantiated by the fact that many drugs, including cisplatin and its analogues are potentiated by heat (80). Moreover, it has been demonstrated with many drugs, including cisplatin that the addition of hyperthermia can reverse, or at least partially overcome, drug resistance (81). The mechanisms o f this observation are, however, not well defined. 15 1.3.2 Heating techniques The physical parameters involved in the delivery of heat are extremely complex. The diffusion characteristics of heating are dynamic and as such, results in a lack of uniformity in temperature distribution within the heated tissues. Further, it is difficult to accurately monitor the temperatures within large and geometrically complex tumours. The volume that can be heated is dependent on the physical mechanism of the power source and on the type of array used. There are numerous methods and commercially available equipment to selectively heat tumours (82, 83). These heating methods operate under conditions which do not produce significant side effects and/or damage to healthy tissues. The clinically used modalities deposit energy by different physical mechanisms and can be administered by using either invasive sources involving probes or catheters (84, 85), or non-invasive sources, which involve external heating applicators (86-88). 1.3.3 Development of lysolipid containing thermosensitive liposomes ( L T S L ) Based on the clinical benefits from the combination of hyperthermia and liposomal drug delivery, investigators have pursued the development of thermosensitive liposomes. The primary l ipid component of all previously described thermosensitive liposomes is dipalmitoylphosphatidylcholine (DPPC), a 16 carbon saturated l ipid with a Tc = 41.9°C. With a Tc a few degrees above physiological body temperature, D P P C is the l ipid o f choice for developing thermosensitive liposomes; however, pure D P P C liposomes suffer from several drawbacks that have been only recently addressed. Early DPPC-based thermosensitive liposomes exhibited.non-ideal release attributes and suboptimal tumour localization characteristics (74, 89). The latter was due to the rapid elimination of 16 liposomes, but this problem was solved by the inclusion of P E G lipids (90). Non-ideal drug release attributes were due to a gradual release of encapsulated contents following heating and this meant that tissues, both healthy and diseased, had to be exposed to damaging heat over long periods of time. To address the slow release kinetics, modifications to the l ipid composition resulted in the development of lysolipid containing thermosensitive liposomes ( L T S L ) , composed of DPPC/MSPC/DSPE-PEG2000 (90:10:4 mole ratio) (9, 10). The addition of lysolipids was attempted based on the hypothesis that the lysolipid would enhance the permeability of the bilayer to the encapsulated drug at the Tc (8). The supporting foundation for this hypothesis stemmed from micropipette studies, which demonstrated that lysolipids readily desorbed from lipid bilayers (91). Described as the "gold standard," L T S L facilitated the rapid release of encapsulated contents upon heating to 40-42°C and as well , exhibited pharmacokinetic behaviour that promoted accumulation of the carrier within the tumour. The possible mechanism underlying the combination of hyperthermia and liposomal drug delivery is illustrated in Fig . 1.4. The proposed theory is based upon the capacity of the thermosensitive liposomal carrier to remain relatively stable when circulating at temperatures below the Tc while being capable of releasing their contents as the Tc is attained, either within the bloodstream (Fig. 1.4C) and/or in the tumour matrix (Fig. 1.4D). This, in turn, provides a higher local concentration of the therapeutic agent within the tumour site. Essentially, the strategy is for the liposome to pass through the Tc and undergo a transition from a stable structure during delivery to a less stable, more permeable form when at the site of action. In addition to acting as a triggering mechanism, the application 17 F i g 1.4. Schematic of the mechanism underlying the combination of hyperthermia and liposomal drug delivery in the treatment of solid tumours. (A) Passive targeting of liposomes within tumour vasculature, (B) hyperthermia increases liposome extravasation, (C) hyperthermia triggers liposomal drug release within the tumour vessel, (D) hyperthermia triggers liposomal drug release in the extravascular space where the tumour resides and (E) hyperthermia alone can achieve direct cell k i l l (9). 18 of heat alone can achieve direct cell k i l l (Fig. 1.4E), further substantiating the combination of liposomal drug delivery with hyperthermia. 1.4 Cisplatin (m-dichlorodiammineplatinum II) After an introduction to liposomal drug delivery and hyperthermia and the advantages ofthe combination ofthe two modalities, the following section w i l l discuss cisplatin, the anticancer drug evaluated in this thesis. Cisplatin, a cytostatic drug derived from platinum, is an established anticancer drug widely used in the treatment of solid tumours. It is a heavy metal complex containing two chloride atoms and two amino groups attached in the cis configuration to one atom of platinum in its divalent form. Discovered serendipitously in 1967 by Rosenberg et al. (92), cisplatin is today indicated for the curative treatment of testicular cancer and as well , has demonstrated significant activity against ovarian, lung and head and neck carcinomas (93, 94). 1.4.1 Biological activity The exact mechanism of its action is unknown; however, cisplatin is believed to exert its cytotoxic effect primarily through coordination with D N A , resulting in various plat inum-DNA and platinum-protein adducts and interfering with the D N A repair mechanism. These various binding modes of action are shown in Fig . 1.5 A . It has been reported that cisplatin enters the cell through passive diffusion (95); however, there are also arguments for protein mediated transport (96, 97). Regardless ofthe mode of entry, once inside the cell, cisplatin is rapidly hydrolyzed due to the lower intracellular chloride concentration (4 m M intracellular vs. 100 m M in the plasma), thus forming the positively 19 1,2-intrastrand cross-linking MniersWan8^crbssMm'kirig!' B H 3 N X CI Pt HiN Cl H 3 N O H 2 | K x i A 3 \ / I D N A Pt . i > H 3 N Cl _] N H 3 -I H 3 N — P t - G -Cl G -H J N N G -. . . . / ^ cisplatin 'monoaqua" monofunctional bifunctional Fig . 1.5. (A) Various binding modes of cisplatin to D N A and proteins (113). (B) Fate of cisplatin metabolism in aqueous solution (113). 20 charged mono-aqua species, a more active form of the platinum compound. Within cells, approximately 40% of the platinum is present as this mono-aqua hydrolysis product. This monofunctional adduct reacts further, forming the major bifunctional intrastrand d(GpG) adduct with platinum crosslinking the N7 atoms on neighboring guanine residues of D N A . These cisplat in-DNA complexes can irreversibly bind D N A repair proteins, subsequently distorting the D N A helix and preventing effective repair (98). The fate of cisplatin metabolism is illustrated in Fig. 1.5B. Major obstacles to cisplatin therapy are the development of resistance by the cancer cells and its significant adverse effects. The mechanism accounting for the clinically acquired resistance is unclear. However, it has been suggested that resistance is due to a decreased cellular accumulation (99) and/or enhanced D N A repair (100, 101). The side effects of cisplatin commonly include renal damage, severe nausea and vomiting, myelosuppression, ototoxicity and neurotoxicity. Given that cisplatin exhibits such complications, studies have been directed towards finding less toxic anticancer platinum analogues with improved therapeutic efficacy, such as carboplatin (102, 103). Extensive efforts have also been directed in developing liposomal formulations of cisplatin to alter its toxicity profile, pharmacokinetics and biodistribution (104, 105). 1.5 Research objectives and hypotheses Cisplatin has been encapsulated into non-temperature sensitive, long circulating liposomes (SPI-077 or stealth liposomal cisplatin), comprised of hydrogenated soy-PC, m P E G - P E and cholesterol (51:5:44 mole ratio) (2). This liposomal formulation proved to be clinically uninteresting due to issues related to poor drug dissociation from the 21 liposomes following administration (3-5). Given the potential for a therapeutically improved formulation, it was hypothesized that cisplatin, with its hydrophilic nature and its low affinity for lipid membranes, was an ideal agent to be encapsulated in a thermosensitive liposomal formulation, specifically lysolipid containing thermosensitive liposomes ( L T S L ) . It was anticipated that the combination of liposomal drug delivery and hyperthermia would "trigger" release of cisplatin and result in increased therapeutic activity. The focus of this thesis was to develop and characterize a thermosensitive liposomal formulation of cisplatin. The specific objectives for the research contained in this thesis are listed as follows: 1. To develop and evaluate passive equilibration of cisplatin into L T S L . Passive equilibration is a novel and simple method of drug encapsulation. It is hypothesized that cisplatin w i l l equilibrate across the liposomal membrane at temperatures above the Tc of D P P C , where permeability is enhanced. 2. To evaluate the in vitro drug release attributes of LTSL-cispla t in in the presence and absence o f mild heating. 3. To characterize the in vivo plasma elimination behaviour of LTSL-cisplat in . 22 C H A P T E R 2 M E T H O D S A N D M A T E R I A L S 2.1 Mater ia ls Lipids including dipalmitoylphosphatidylcholine (DPPC) , monostearoylphosphatidylcholine (MSPC) and distearoylphosphatidylethanolamine-poly(ethylene glycol) (DSPE-PEG2000) were obtained from Avanti Polar Lipids (Alabaster, A L , U S A ) . Sephadex G-50 size exclusion gel, cw-diamminedichloroplatinumll (cisplatin), platinum standard and other chemicals (reagent grade) were purchased from Sigma-Aldrich (St. Louis, M O , U S A ) . [ 3H]-cholesterylhexadecyl ether ( [ 3 H]-CHE) and [ 1 4C]-lactose were purchased from N E N Life Sciences Products (Oakville, O N , Canada). [ 1 4C]-methylamine hydrochloride was purchased from Amersham Pharmacia Biotech (Oakville, O N , Canada). Pico-Fluor 15 scintillation fluid was purchased from Packard Bioscience (Groningen, The Netherlands). 2.2 Preparat ion of liposomes The thermosensitive liposome formulations evaluated in this study included a lysolipid containing thermosensitive liposome (LTSL) composed of D P P C / M S P C / D S P E -PEG2000 (90:10:4 mole ratio), a lysolipid free thermosensitive formulation (TSL) composed of DPPC/DSPE-PEG2000 (100:4 mole ratio) and a cholesterol containing formulation composed of DPPC/cho l / DSPE-PEG2000 (55:45:4 mole ratio), which served as a temperature insensitive control. A l l liposome samples were prepared by the extrusion technique (48, 49). Briefly, lipids at the appropriate molar ratios were dissolved in 23 "1 chloroform and combined with [ H ] - C H E as a non-exchangeable and non-metabolizable l ipid marker (106). The preparation was subsequently dried under a stream o f nitrogen gas and the resulting l ipid film was placed under high vacuum overnight to remove all traces of organic solvent. The dried l ipid films were hydrated with gentle mixing in 150 m M N a C l (pH 7.4) at 55°C for 1 hour to form multilamellar vesicles. The resulting preparation was extruded 10 times at 55°C through two stacked 0.1 [am polycarbonate filters (Nucleopore Co., Canada) with an extruding apparatus (Northern Lipids Inc., Vancouver, B C , Canada). The resulting mean diameter of these liposomes was typically 90 + 10 nm, as determined by quasi-elastic light scattering (QELS) using the Nicomp submicron particle sizer model 370/270 operating at 632.8 nm. Liposomal l ipid was quantitated by liquid scintillation 3 • * counting of samples labeled with trace amounts of [ H ] - C H E (Packard 1900TR Liquid Scintillation Analyzer). 2.3 Cispla t in solubility determination The maximum solubility of cisplatin in water when at room temperature is approximately 2 mg/mL. This low solubility results in low D : L ratios, which are of limited pharmaceutical value as this directly translates to an increase in the amount of lipid required to achieve a reasonable injection of drug. It was determined that the solubility of cisplatin increased with an increase in temperature. This allowed cisplatin to be dissolved in an amount greater than the maximum solubility achievable at room temperature. Briefly, 12 mg/mL cisplatin was mixed in 150 m M N a C l with gentle mixing for 30 minutes and incubated at various temperatures. The sample remained at the incubation temperature for a further 30 minutes, without mixing, to allow sedimentation of unsolubilized cisplatin. A n 24 aliquot of the solubilized solution was then transferred to 1 m L of N a C l , at room temperature and subsequently measured against a standard curve by atomic absorption spectroscopy ( A A S ) , as described in section 2.4.. When in aqueous,solutions, the chloride ions of cisplatin may be hydrolyzed to produce a hydrated intermediate, a mono-aqua species. To minimize this ligand substitution and to keep the substitution equilibrium to the planar cisplatin complex, a 150mM N a C l solution, which contains a high chloride ion concentration was used; therefore, all measurements of cisplatin concentration throughout the studies conducted within this thesis were assumed to be of the neutral, planar complex. 2.4 Atomic absorption spectroscopy (AAS) The concentration of cisplatin was assessed using a Varian SpectrAA 300Z spectrometer with a graphite tube atomizer. The instrument operated at a wavelength of 265.9 nm and followed the program of 90°C for 30 seconds, 120°C for 10 seconds, 1100°C for 15 seconds and lastly 2800°C for 8 seconds, where absorption was measured. A l l measurements were made in duplicates and against a standard curve from a commercially available platinum standard (Sigma-Aldrich). Thus, cisplatin values are derived from measurements of platinum and not of cisplatin itself. 2.5 Drug loading methods 2.5.1 Cisplatin encapsulation via passive equilibration The method of passive equilibration described here is a novel and simple drug loading technique. This procedure takes advantage of the Tc of lipids, whereby drug is added to the outside of preformed liposomes at temperatures above the Tc of the bulk lipid. 25 This method was used for both L T S L and T S L . Briefly, following extrusion, l ipid analysis and size determination, liposomes and powdered cisplatin were separately prewarmed at the indicated temperatures for 5 minutes. Liposomes were added to the cisplatin at an initial drug to l ipid ratio of 0.15 (wt/wt). The sample was incubated at various temperatures for 2 hours in a water bath with gentle mixing. The final cisplatin concentration used in these studies was either 2 mg/mL (the maximum solubility achievable at room temperature) or 10 mg/mL (the concentration that could be achieved at 70°C in the absence o f any precipitation). A t various time points (15, 30, 60, 90 and 120 mins) post incubation, aliquots from the liposomal cisplatin preparation were collected and where needed, cooled to room temperature and then subjected to centrifugation (1000 x g, 3 mins) to pellet insoluble cisplatin. Subsequently, all processing and sample handling for these preparations made at 2 mg/mL or 10 mg/mL cisplatin was performed at room temperature. The supernatant was then passed through a 1 m L sephadex G-50 spin column pre-equilibrated with saline. Spin columns were prepared by first adding glass wool to a 1 m L syringe and sephadex G-50 beads were packed by centrifugation (680 x g, 2 mins). The liposome fraction was collected by centrifugation (680 x g, 3 mins) in the void volume and was analyzed for both liposomal lipid and drug content. Liposomal lipid concentration was quantitated by liquid scintillation counting and drug content by graphite furnace A A S . 2.5.2 Loading into cholesterol containing liposomes via passive encapsulation The method of passive equilibration described above does not work with cholesterol containing formulations. Hence, the method of passive encapsulation was used to prepare 26 cisplatin loaded DPPC/chol / DSPE-PEG2000 (55:45:4 mole ratio) liposomes. A cisplatin solution (10 mg/mL) was heated to 70°C to solubilize the drug and was added to a dried lipid film to achieve a final cisplatin-to-lipid ratio of 0.15 (wt/wt). Fol lowing a 1 hour hydration period at 70°C, the sample was extruded and subsequently analyzed as described above. The incubation temperature of 70°C was maintained in all subsequent steps in the preparation of cisplatin loaded liposomes to prevent drug precipitation. 2.6 Trapped volume determination Trapped volume determination was ascertained by using [ 1 4C]-lactose as a marker loaded into the liposomes using the passive encapsulation methods. These values were compared to methods where [ 1 4C]-lactose was trapped using the passive equilibration method. To determine the trapped volume by passive encapsulation, [ 3 H ] - C H E labeled lipid films were prepared as described above and hydrated in saline containing [ 1 4 C]-lactose. The methods used were essentially identical to those used when preparing the cisplatin loaded liposomes. For example, DPPC/cho l /DSPE-PEG 2 ooo (55:45:4 mole ratio) formulations were incubated and extruded at 70°C prior to separation of the free lactose from liposomally associated lactose. The sample was passed down sephadex G-50 spin columns equilibrated with saline to remove unencapsulated [ 1 4C]-lactose. Trapped volume was calculated using the following equation: Trapped volume = M (uL) / L (umole lipid) (equation 1) where M = [ 1 4C]-lactose dpm per uL eluted from the column / [ 1 4C]-lactose dpm per uL of the initial liposome suspension prior to separation on the column and L = [ 3 H ] - C H E dpm 27 per uL eluted from the column in dpm / specific activity of the liposomal preparation in dpm/umole total lipid. Using the technique of passive equilibration, [ 1 4C]-lactose was added to the outside of preformed liposomes and the level of liposome associated [ 1 4C]-lactose was monitored over time. When the level of liposome associated [ 1 4C]-lactose saturated (at time points > 8 hours), the value was used to estimate the trapped volume using equation 1, as described above. 2.7 Cryo-transmission electron microscopy ( C r y o - T E M ) C r y o - T E M is a specialized technique that can be used for the imaging and visualization of liposomes and other amphiphilic aggregates (107). Sample preparation avoids staining, drying and chemical fixation, which minimizes artifacts and distortion of sample. The cisplatin loaded liposomes developed in this research were analyzed by cryo-T E M . The method used and the interpretation of liposome images have been previously described (108). Briefly, in a climate controlled chamber, a drop of the liposomal sample was placed onto a copper grid coated with a cellulose acetate butyrate polymer film and blotted, forming a thin aqueous layer on the membrane. The sample was flash frozen in liquid ethane, allowing the film to vitrify and then immediately transferred to liquid nitrogen so as to maintain the temperature below -165°C. This step was to minimize formation of ice crystals and associated sample perturbation. The grid containing the sample was transferred to a Zeiss E M 9 0 2 transmission electron microscope at a temperature of -165°C for analysis in a zero-loss bright field mode and an accelerating voltage o f 80 k V . The sample preparation stage is illustrated in Fig . 2.1. The interpretation ofthe projected 2-D images acquired from the 3-D liposomal sample is depicted in F ig . 2.2. 28 Fig 2.1. C r y o - T E M climate chamber and sample preparation technique (108). 29 e l e c t r o n rad ia t ion • t f t • • • • f vi t r i f ied s a m p l e 2 -D i m a g e Fig 2.2. 2-D projection o f c r y o - T E M images. Photomicrographs are acquired from all angles during image recording of the 3-D liposomal sample. In the 2-D projections, liposomes appear spherical and have a dense outline. Depending on the orientation, circular disks w i l l either appear circular without a dense outline or as a dense line (108). 30 2.8 In vitro drug release assay To assess the thermosensitivity of the cisplatin encapsulated liposomal preparations, drug loaded liposomes were divided into two aliquots, adjusted to a 2 mg/ml final liposomal l ipid concentration and incubated at either 37°C or 42°C. Changes in D : L ratio (wt/wt) were followed over time, where decreases in D : L ratio indicates cisplatin release. Liposomal lipid concentrations were determined using [ 3 H ] - C H E as a marker and cisplatin concentrations were determined using A A S . 2.9 Plasma elimination of cisplatin loaded liposomes The in vivo behaviour of cisplatin encapsulated in L T S L , T S L and cholesterol containing liposomes was evaluated. A l l animal studies were conducted according to procedures approved by the University of British Columbia's Animal Care Committee and in accordance with the current guidelines of the Canadian Council o f An ima l Care. Free cisplatin and [ H ] - C H E radiolabeled liposomes containing cisplatin were administered i.v. as a single bolus injection into the lateral tail vein of female Rag2 -M mice at 2 mg/kg cisplatin in an injection volume of 200 uL. Dilation of the tail vein was achieved by submerging the tail of the mice in a 37°C water bath for 1-2 minutes. A t various times post administration, mice were terminated by CO2 asphyxiation. For free cisplatin, blood was collected at 5, 15, 30 mins, 1, 2 and 4 hours post administration. For cisplatin loaded liposomes, time points were collected at 15, 30 mins, 1, 2, 4, and 8 hours. Blood was collected by cardiac puncture, placed into E D T A microtubes (Becton-Dickinson) and kept at room temperature until ready for centrifugation (1000 x g, 15 mins). Plasma liposomal l ipid and cisplatin concentrations were quantified as described above. 31 For studies that required the control of animal body temperature, mice were anesthetized i.m. with 6 mg/kg acepromazine and kept in a custom designed temperature controlled chamber ( B C Cancer Agency workshop) prior to administration o f the liposomal formulations. Mice were kept in the holding chamber, where the temperature of the animals was maintained at 37° + 0.5°C throughout the entire time course of the study. This minimized fluctuations in mice body temperature due to handling. The temperature of the chamber and the temperature o f the tranquillized mice were monitored at regular time intervals on a multi-channel Model 46 T U C Tele-Thermometer using Y S I 400-thermistor probes (Yellow Springs Inc., Ye l low Springs, O H , U S A ) . Plasma elimination profiles of free cisplatin and [ 3 H ] - C H E radiolabeled liposomal cisplatin were completed as described above. 2.10 Statistical analysis A standard one way analysis of variance ( A N O V A ) was used to compare plasma cisplatin concentrations for both L T S L and T S L formulations under conditions which were/were not temperature controlled. The Newman Keuls test for the post-hoc comparison of means was employed. A p<0.05 was considered significant. 32 CHAPTER 3 RESULTS 3.1 Cisplatin encapsulation via passive equilibration The customary method of passive encapsulation of cisplatin into conventional, non-thermosensitive liposomes involves significant manufacturing challenges, including the necessity of passing 70°C cisplatin solutions through the extruder apparatus. Even when using extensive precautions, the processing resulted in a visible and permanent black deposit on the extruder. For this reason, a new loading method to achieve cisplatin loading into preformed liposomes was evaluated. The novel passive equilibration technique bypassed the need for cisplatin to pass through the extruder and resulted in a simple method to prepare drug loaded liposomes, albeit the efficiency of loading was no better than that achieved using standard passive encapsulation methods. The first objective was to assess the method o f passive equilibration o f cisplatin into L T S L as a function of time, at various temperatures both above and below the Tc of D P P C , the bulk phospholipid in the formulation. Cisplatin uptake into liposomes after the addition of cisplatin (final concentration of 2 mg/mL) to the outside of preformed liposomes was monitored as a function o f time. These results are summarized in Fig . 3.1 A . Cisplatin association with liposomes was negligible when the incubation temperature was 37°C (Fig. 3.1 A , triangles). Rapid equilibration of cisplatin across the liposomal membrane was achieved when the sample was incubated at temperatures of 45°C, 55°C and 70°C, temperatures above the Tc of D P P C . The rate of equilibration of cisplatin across the 33 0.004 •o time (minutes) F i g . 3.1. (A) Time dependent uptake of cisplatin (2 mg/mL final drug concentration) via passive equilibration into L T S L at various temperatures. Cisplatin encapsulation was determined at (A) 37°C, (•) 45°C, (•) 55°C and (•) 70°C. (B) Time dependent uptake of cisplatin (10 mg/mL final drug concentration) via passive equilibration into (•) L T S L , (•) T S L and (A) DPPC/chol /DSPE-PEG 2 0 oo l iposomes at 70°C. A t the indicated time points during the incubation, aliquots of sample were taken. L ip id content was analyzed by [ 3 H]-C H E label as described in the methods. Cisplatin levels were determined on the basis of platinum equivalents, as measured by A A S . Each point represents mean + SD ( n ^ ) . 34 liposomal membrane was comparable at'these temperatures. At temperatures above the Tc, the majority of drug was encapsulated within 15 minutes, the first time point measured. Loading was stable over the 2 hour time course. Under the conditions used, the final D : L ratio was approximately 0.0025 (wt/wt) when encapsulation was completed at temperatures of 45°C or greater. The loading efficiency was <10% under the conditions used. A strategy to increase the final D : L ratio was based on enhancing cisplatin solubility by using increased temperatures when preparing the formulation. A s shown in Table 3.1, the solubility of cisplatin increased as temperature was increased. A t room temperature, the maximum solubility o f cisplatin in saline was found to be approximately 1.49 mg/mL. In comparison, cisplatin solubility at 70°C increased to approximately 10.20 mg/mL, an improvement over the maximum solubility of cisplatin when at room temperature. When the passive equilibration method was used at a cisplatin concentration of 10 mg/mL and an incubation temperature of 70°C, there was an approximately equivalent increase in final D : L ratio (wt/wt), as shown in Fig. 3 . IB (circles) for L T S L . Temperatures greater than 70°C were not considered in these studies. Consistent with results from Fig . 3.1 A , the majority of cisplatin was encapsulated within 15 minutes and the encapsulation level was stable over the 2 hour incubation. This loading procedure was also evaluated in T S L and a cholesterol containing formulation. A difference in the loading rate between L T S L and T S L was observed, where maximum encapsulation was achieved by 15 minutes for L T S L (Fig. 3 . IB, circles) as compared to 2 hours for the T S L formulation (Fig. 3 . IB, squares). The extent of cisplatin loading into L T S L was; however, comparable to that observed for T S L following a 2 hour incubation. B y contrast, negligible loading was observed when the 35 Table 3.1. Cisplatin solubility in 150 m M saline at different temperatures3. Temperature (°C) cisplatin (mg/mL) 1.49 + 0 .21 b 3.71 ± 0 . 1 8 6.69 ± 0 . 5 7 10.20 + 0.36 Room temperature 37°C 55°C 70°C a In order to determine the solubility, 12 mg/mL cisplatin was mixed in 150 m M N a C l at the indicated temperatures with gentle mixing for 30 minutes. The sample remained at the indicated temperature for a further 30 minutes, without mixing to allow sedimentation. A n aliquot ofthe solubilized solution was then transferred to 1 m L of N a C l , at room temperature and measured against a standard curve by atomic absorption spectroscopy (see chapter 2.4). b Data represents mean ± SD (n = 3). 36 liposomes used contained cholesterol (Fig. 3 . IB, triangles). These findings indicate that the method of passive equilibration w i l l not work with liposomes with 45 mole% cholesterol. 3.2 Lactose trapping by passive encapsulation and passive equilibration methods To address the question whether there were differences in cisplatin trapping efficiencies depending on whether passive encapsulation and passive equilibration was used, trapped volume assessments were determined using radiolabeled lactose as a trapped marker (Table 3.2). Wi th passive encapsulation methods, where the l ipid f i lm was hydrated in a solution containing the radiolabeled lactose, L T S L and T S L exhibited a mean trapped volume of 1.88 + 0.15 uL/umole lipid and 2.4 + 0.15 ul/umole lipid, respectively. In contrast, passive equilibration methods, where liposomes were incubated for > 8hours at 70°C, measured trapped volumes of 1.92 + 0.22 uL/umole lipid and 1.79 ± 0 . 1 7 uL/umole lipid, respectively were obtained. Differences in lactose association were not statistically different for L T S L (p>0.05), but significantly lower (p<0.05) when using the passive equilibration method for T S L . 3.3 Evaluation of cisplatin loaded thermosensitive liposomes by Cryo-TEM C r y o - T E M studies can be used to provide information on liposome morphology as well as the encapsulated content (109). In our studies, c r y o - T E M analysis suggested that there were no substantial differences between cisplatin loaded L T S L , T S L and empty liposomes (Fig. 3.2). Further, substantial changes in the liposome structure were not observed when the liposomes were incubated at 42°C. Regardless of the temperature and of the encapsulated content, L T S L and T S L exhibited atypical surface features consistent of 37 Table 3.2. Trapped volume determination of L T S L and T S L by the methods of passive encapsulation and passive equilibration. Liposome formulation Trapped volume (uL/umole lipid) Passive encapsulation a Passive equilibration b Liposome size (nm) c L T S L 1.88 ± 0.15 d 1.92 ± 0 . 2 2 88 ± 5 T S L 2 . 4 ± 0 . 1 5 1 . 7 9 ± 0 . 1 7 e 86 ± 5 a Measurements determined by trapped [ 1 4C]-lactose added during sample rehydration. b Measurements determined by trapped [ 1 4C]-lactose into preformed liposomes over time. c Liposome size determined by quasi-elastic light scattering following extrusion. d Data represents mean + S D (n = 3). e Significantly different than data obtained for passive encapsulation (p < 0.05). 38 LTSL TSL empty drug loaded 37°C drug loaded 42°C HHHHHHHHH jjj^jl^j^jjjj^jjl^^ •••••••• Fig . 3.2. C r y o - T E M electron micrographs of empty and cisplatin-loaded L T S L and T S L prepared using the passive equilibration technique. The bar represents 1 OOnm and all images are shown at the same magnification. 39 liposomal formulations that lack cholesterol (110). Drug precipitation was not observed in the liposome core.despite the prediction that at least 75% of the solubilized cisplatin at the optimized temperature of 70°C would be in a precipitated form when at room temperature. 3.4 Temperature dependent release of cisplatin from thermosensitive liposomes Considering that the loading method used involved incubation temperatures of 70°C, it was important to demonstrate that the thermosensitivity of the formulation was not compromised. To assess the temperature dependent drug release for various liposome formulations, cisplatin release from liposomes was compared at 37°C and 42°C. A s shown in Fig . 3.3, cisplatin was well retained in all formulations evaluated when incubated at 37°C. The L T S L released <5% of the encapsulated cisplatin when incubated at 37°C (Fig. 3.3A, open symbols), a release rate that was comparable to that observed for DPPC/cho l / DSPE-PEG2000 liposomes (Fig. 3.3C, open symbols). However, - 9 5 % cisplatin release was observed from L T S L within 5 minutes following incubation at 42°C, with 100% dissociation of drug by 30 minutes (Fig. 3.3A, filled symbols). B y comparison, T S L released only 70% and 85% o f the encapsulated cisplatin at the 5 minutes and 30 minutes time points at 42°C, respectively (Fig. 3.3B, filled symbols). A s demonstrated in Fig. 3.3C, negligible release was noted from the cholesterol containing formulation when incubated at 42°C. These data are consistent with previous publications suggesting that the lysolipid ( M S P C ) enhances the rate and extent of drug release from thermosensitive liposomes (9, 10). 40 A 0.020 I 0.015 CO E I 0.010 CL CO 0.005-I E 0.000 10 20 30 40 50 60 B 0.020 S 0.015 D) E 0.010+ J5 Q. 0.005 0.000 10 20 30 40 50 60 0.015n = 0.010+ E o. 52 'o oi E 0.005 0.000 10 20 30 40 50 60 time (minutes) F i g . 3.3. Drug release assays from three different liposomal formulations at 37°C (open symbols) and 42°C (filled symbols) as a function o f time. Release o f cisplatin from (A) L T S L , (B) T S L and (C) D P P C / c h o l / D S P E - P E G 2 0 0 o liposomes. At the indicated time points during the incubation, aliquots of sample were taken. L ip id content was analyzed by [ 3 H]-C H E label as described in the methods. Cisplatin levels were determined on the basis of platinum equivalents, as measured by A A S . Each point represents mean + SD (n=3). 41 3.5 Plasma elimination studies of cisplatin loaded thermosensitive liposomes In consideration of further preclinical development of the thermosensitive liposomal formulation, the in vivo stability and drug release attributes of the thermosensitive liposome formulations was evaluated. Since animal body temperature can fluctuate as a consequence of handling, the plasma elimination behaviour under conditions where animal body temperature was carefully controlled as well as under standard animal handling methods typically required for i.v. injection of test articles (in the absence of temperature monitoring and/or control) was assessed. To determine whether animal handling had an impact on the cisplatin loaded thermosensitive formulations, the plasma concentrations of cisplatin and liposomal lipid, as well as the calculated D : L ratio (wt/wt) in the plasma compartment was assessed at a single time point (2 hours) following i.v. injection (Fig. 3.4). Differences in liposomal lipid concentration (Fig. 3.4B) were not statistically different (p > 0.05), albeit a higher level o f liposomal l ipid was observed for T S L under temperature controlled conditions. In contrast, the levels of cisplatin in the plasma compartment at 2 hours were significantly higher (p < 0.05) under temperature controlled conditions (Fig. 3.4A). The calculated D : L ratio (wt/wt) provides a true indication of the amount of drug released from the liposomes within the plasma compartment following i.v. injection (Fig. 3.4C). In the absence of temperature control, >95% of the encapsulated cisplatin was released from liposomes within the plasma compartment 2 hours after administration. Cisplatin retention in the liposomes was markedly enhanced under conditions where animal body temperature was regulated to be 37° + 0.5°C. These results clearly demonstrate the importance of regulating the body temperature of mice prior to and following injection of cisplatin loaded thermosensitive liposomes. 42 W i t h o u t Temperature temperature controlled control F i g . 3.4. Plasma elimination profiles of L T S L (solid bars) and T S L (open bars) 2 hours following i.v. administration in Rag2-M mice under conditions where animal body temperature was or was not controlled. (A) Plasma drug levels, (B) plasma liposomal lipid levels, and (C) plasma D : L ratios (wt/wt) were determined as described in the methods. Cisplatin levels were determined on the basis o f platinum equivalents, as measured by A A S . Each bar represents mean + SD (n=3). Regulation of animal body temperature was found to be important and statistically significant as determined by Newman Keuls test where * = p < 0.005 compared to that without temperature control and ** = p < 0.01 compared to that without temperature control. 43 Subsequent plasma elimination studies were conducted over a time course of 8 hours and the results summarized in Fig . 3.5. Free cisplatin is rapidly eliminated from the plasma compartment, with <1% of the injected dose remaining after 15 minutes (Fig. 3.5A, diamonds). However, when cisplatin is encapsulated within liposomes, higher drug levels are achieved over extended periods of time for all formulations evaluated. Fol lowing injection of DPPC/chol/DSPE-PEG2ooo cisplatin containing liposomes, the level of cisplatin in the plasma compartment was substantially greater than that observed following injection of either L T S L or T S L . A t 2 hours post injection, the cholesterol containing formulation released - 1 5 % of the entrapped cisplatin in the plasma compartment (Fig. 3.5A, squares). In comparison to DPPC/CI10I/DSPE-PEG2000 liposomes, both L T S L and T S L exhibited faster cisplatin and liposome elimination rates. L T S L exhibited the fastest liposome elimination rates, with - 8 0 % of the injected dose eliminated 8 hours following i.v. administration. In contrast, less than 40% of the T S L injected dose was eliminated when assessed at the same time point. When evaluating cisplatin levels, the cisplatin circulation half-life following administration of L T S L was approximately 1 hour. A t the same time point, - 8 5 % of the injected cisplatin dose was still remaining following injection of the DPPC/chol/DSPE-PEG2ooo formulation. Changes in D : L ratio following i.v. injection (Fig.3.5C) highlight the fact that the fastest drug release occurs from L T S L . A t the 8 hour time point, >95% drug release is observed from these liposomes as compared to - 7 0 % and - 3 0 % release from T S L and DPPC/CI10I/DSPE-PEG2000 liposomes, respectively. 44 A 0.05 B 0.000 2 3 4 5 time (hours) F i g . 3.5. Plasma elimination profiles of (•) L T S L , (A) T S L , (•) DPPC/cho l /DSPE-PEG2000 cisplatin loaded liposomes and (•) free cisplatin in Rag2 -M mice. The mice were tranquilized with 6 mg/kg acepromazine and kept in a custom built temperature controlled mouse chamber where body temperature was maintained at 37° + 0.5°C throughout the entire time course o f the study. (A) Plasma drug levels, (B) plasma liposomal l ipid levels, and (C) plasma D : L ratios (wt/wt) were measured as described in the methods. Cisplatin levels were determined on the basis of platinum equivalents, as measured by A A S . Each point represents mean + SD (n=3, except T S L n=6). 45 f C H A P T E R 4 D I S C U S S I O N A N D C O N C L U S I O N Recently, treatment with L T S L encapsulating the anticancer drug doxorubicin has, in combination with hyperthermia, resulted in complete tumour regression in 100% of the animals treated, lasting up to 60 days post treatment in a human squamous cell carcinoma model (10). Despite this exciting result, it was interesting to note that this formulation exhibited a low doxorubicin encapsulation capacity. Moreover, approximately 50% of the encapsulated contents were released within 1 hour following i.v. injection, suggesting that doxorubicin was inherently permeable across the L T S L membrane (111). The goal of the studies described here was to evaluate i f cisplatin, an alternate anticancer drug, was more suitable for encapsulation within L T S L and to assess whether cisplatin retention in these liposomes was improved relative to that obtained with LTSL-doxorubicin. The results suggested that LTSL-cisplat in can be prepared relatively easily using a novel passive equilibration method. Although the liposomes were thermosensitive, cisplatin release rates in vivo were comparable to those obtained for doxorubicin loaded L T S L . Further development of LTSL-cisplat in is warranted; however, their use w i l l l ikely focus on strategies where heating is applied to the tumour prior to injection, a strategy that is comparable to that used for thermosensitive formulations of doxorubicin (10). To improve and avoid the technical challenges of loading cisplatin into liposomes by passive encapsulation, a simple drug loading method was developed. This process takes advantage of the inherent increased permeability of the l ipid membrane near the Tc. Within the context of this thesis, it was demonstrated that cisplatin was able to equilibrate across the membrane of thermosensitive liposomes. Addition of cholesterol to the D P P C based 46 liposomes inhibited this equilibration event (see Fig . 3. IB) . The results further suggested that the rate of cisplatin equilibration across the liposomal membrane is faster for L T S L as compared to T S L . However, it should be noted that equilibration attributes across the liposome membrane w i l l l ikely be compound dependent. For example, lactose took in excess of 8 hours at 70°C to equilibrate across the L T S L and T S L formulations. Using a 10 mg/mL cisplatin solution and incubation temperatures of 70°C, the D : L ratio of the thermosensitive liposomes could be adjusted to achieve a final D : L ratio of 0.020 + 0.002 (wt/wt). This final D : L ratio was comparable to that achieved with the conventional stealth formulation of cisplatin, SPI-077 (2, 4). Since l ipid membranes act as selective permeability barriers, allowing the bidirectional flow of molecules, such as drugs, it was expected that an increased permeability would be observed at or above the Tc of the bulk phospholipid (DPPC). When the temperatures were below the Tc, the acyl chains of phospholipids are less mobile and this contributes to reduced membrane permeability, as exemplified by the data shown in Fig. 3.1A for cisplatin loading at 37°C. However, when temperatures were above the Tc, rapid equilibration of drug across the liposomal membrane was achieved. The rate and extent of loading was comparable when the incubation temperature was 45°C, 55°C or 70°C. It is anticipated that other therapeutic agents, in addition to cisplatin could also be encapsulated into cholesterol free liposomes using this methodology. Further, it can be suggested that this method could be applied to liposomal formulations prepared of D S P C , when incubated at temperatures above 60°C. D S P C liposomes, prepared without cholesterol, have been previously shown to have improved drug retention attributes (112); however, these formulations w i l l not be appropriate for use in combination with mild heating. 47 C r y o - T E M analysis demonstrated no morphological differences between empty and drug loaded liposomes at 37°C and 42°C for both L T S L and T S L (see Fig . 3.2). This was somewhat surprising given that the trapped internal concentration of cisplatin theoretically should be approximately 10 mg/mL and that the storage conditions of the liposomes following cisplatin encapsulation but prior to c r y o - T E M analysis was at 4°C. It was anticipated that a drug precipitate would form within the liposome core under these conditions and i f the cisplatin precipitate was electron dense, it would easily be resolved by c r y o - T E M analysis. However, in all photomicrographs, the liposome interior did not appear different from the controls prepared without cisplatin. It is possible that cisplatin precipitates are not visible by this method or that cisplatin did not form a precipitate. However, observations reported here are consistent with a previous study which demonstrated via nuclear magnetic resonance that cisplatin exists in a soluble form within the liposomal aqueous core of the stealth liposomal cisplatin formulation, with amounts of cisplatin encapsulated similar to those of LTSL-cisplat in (113). Differences in drug loading rate for L T S L and T S L are consistent with the idea that the lysolipid could enhance membrane permeability at the Tc. This was also reflected by the in vitro release data (see F ig . 3.3), where drug release from L T S L at temperatures above the Tc was more rapid as compared to cisplatin release from T S L . The function of the lysolipid is not well understood; however, it has been suggested that its presence imparts an increased fluidity to the liposomal membrane and helps to reduce the Tc into a range more clinically attainable using available hyperthermia methods (8). The greater fluidity correlates to an enhanced permeability which, in part is reflected by an increased release of cisplatin. Lysolipids incorporate well within the vesicle bilayer (91) and biophysical 48 studies have suggested that lysolipids concentrate within grain boundaries, which appear as structural defects in the membrane (110, 111). Given the importance of the lysolipid in the behaviour of the L T S L formulation, it w i l l be critical to establish whether the lysolipid is stably retained in the liposomes following administration. Our data suggested that L T S L are eliminated from the plasma compartment more rapidly than T S L (see Fig . 3.5B). It is conceivable that loss of lysolipid from the liposomes following i.v. injection has a negative impact on circulation longevity (114). Initial plasma elimination studies suggested substantial release o f entrapped cisplatin in the plasma compartment following injection of the L T S L and T S L formulations. This result is likely due to temperature fluctuations in the mice caused by handling and stress. These findings indicated that the body temperature of the mice needed to be better controlled when assessing the pharmacokinetic behaviour o f the thermosensitive formulations. When animal body temperature was controlled to be 37° + 0.5°C, results indicated LTSL-cisplat in exhibited a circulation half-life of 1 hour (see Fig . 3.5). The poor drug retention and short circulation half-life observed with LTSL-cisplat in are attributes which require improvement. Short circulation half-lives especially affect the therapeutic potential o f a drug candidate as an increase in circulation longevity is believed to mediate improved drug uptake into tumours. In conclusion, the studies summarized have focused on the development and characterization of cisplatin loaded L T S L , to be used in combination with hyperthermia. The results give new insights in the development of a versatile pharmaceutical formulation that could provide improved therapeutic effects, particularly in the treatment o f head and neck cancers where cisplatin is an active single agent (93, 115) and hyperthermia is 49 commonly used (69, 70). The described thermosensitive liposomal cisplatin formulation has the potential to be an efficacious, highly tumour selective anticancer drug with significantly reduced side effects. 50 C H A P T E R 5 F U T U R E DIRECTIONS Consistent with previously reported results, a principle limitation of the L T S L carrier was in vivo drug release shortly following i.v. administration (111, 116). This effect was hypothesized to be attributed to the high permeability of the L T S L membrane to the drug evaluated, as with cisplatin in the context of this research. The mechanism of drug release and the roles played by the individual formulation components, specifically of the lysolipids, are not yet fully characterized and further research is required to evaluate drug delivery attributes in the absence and presence of mild heating. For this reason, it would be useful i f the roles played by the lysolipid and P E G lipid components were better understood so that composition changes could be made to improve drug retention, while maintaining thermosensitivity. Optimization and modification ofthe existing lipid composition of the "gold standard" to enhance retention properties and circulation longevity would be advantageous. Suggested modifications to the L T S L carrier include decreasing the lysolipid concentration and/or increasing the P E G lipid concentration with the attempt of reducing the amount of in vivo drug leakage and increasing the circulation lifetime of L T S L , respectively. Adjustment ofthe lysolipid concentration in the formulation may compromise the rapid release effect observed when these formulations are heated to 42°C. Similarly, incorporation of P E G lipids at high concentrations may disrupt the membrane bilayer and lead to the formation of non-bilayer structures. However, it is important to stress the balance necessary between drug retention and drug release from any liposomal carrier. 51 Another alternative would be to consider the encapsulation of alternative drug compounds with a less intrinsic membrane permeability in the thermosensitive formulation. It is anticipated that large, high molecular weight compounds w i l l exhibit minimal drug leakage from the L T S L carrier, even after i.v. administration. It would be particularly interesting to determine whether these methods could be used to deliver thermosensitive formulations of antisense oligonucleotides or s i R N A . Optimizations as well as characterization of the physicochemical properties of the high molecular weight compounds in the attempt to better understand the mechanism of drug release is important. 52 R E F E R E N C E S 1. Mayer, L . D . 1998. Future developments in the selectivity of anticancer agents: drug delivery and molecular target strategies. 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