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Systemic administration of liposomal encapsulated [beta]- galactosidase: a model to investigate the development.. Mok, Wilson Wing Ki 1997

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S Y S T E M I C A D M I N I S T R A T I O N O F L I P O S O M A L E N C A P S U L A T E D pG A L A C T O S I D A S E : A M O D E L TO INVESTIGATE T H E D E V E L O P M E N T OF THERAPEUTIC PROTEIN D R U G by WILSON WING KI M O K B . S c , The University o f British Columbia, 1992 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF  M A S T E R OF SCIENCE  in  T H E F A C U L T Y OF G R A D U A T E STUDIES (Department o f Biochemistry and Molecular Biology) We accept this  theses as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A December 1997 © Wilson W i n g K i M o k , 1997  In  presenting  degree freely  at  this  the  thesis  in  partial  fulfilment  of  University  of  British  Columbia,  I agree  available for  copying  of  department publication  this or of  reference  thesis by  this  for  his thesis  and  scholarly  or for  her  of  The University of British Vancouver, Canada  Date  DE-6 (2/88)  Columbia  I further  purposes  gain  requirements that  agree  may  representatives.  financial  permission.  Department  study.  the  It  shall not  be is  that  the  permission  granted  allowed  an  advanced  Library shall  by  understood be  for  for  the that  without  make  it  extensive  head  of  my  copying  or  my  written  ABSTRACT W i t h advances i n recombinant protein technology, a growing number o f therapeutic protein products have become available for clinical applications. But their wide use is limited by poor biodistribution, limited circulation time and the generation o f immune responses to the drug by the patient. The specific aim o f the work described i n this thesis is to characterize the pharmacokinetic behaviour o f an immunogenic protein encapsulated in a liposomal delivery system.  Liposomes have been demonstrated to increase the therapeutic index o f drugs by ameliorating toxicity and enhancing biodistribution to sites o f disease. The therapeutic index o f a broad spectrum o f drugs has been improved following encapsulation, including antibiotics, antimalarials, antifungals, antineoplastics, cytokines and antisense oligonucleotides. However, many studies have shown that liposomes can enhance the immune response against an encapsulated protein because they naturally target the antigen to phagocytic, antigen presenting cells such as macrophages and dendritic cells. This raises the question as to whether liposomal delivery o f therapeutic proteins is feasible, particularly i f repeated systemic administration is being considered. In order to test this we employed the enzyme p-galactosidase (P-gal), which was encapsulated in liposomes and administered intravenously to mice. The generation o f P-gal antibodies and the pharmacokinetics o f both protein and lipid vehicle were monitored following multiple, weekly injections.  A n overview of liposomes and their role as drug delivery systems and vaccine adjuvants is given in Chapter 1. Chapter 2 describes the protein encapsulation procedures employed and the physical characterization of the resulting liposomes. A n assay was developed to measure P-gal latency and the stability of free and encapsulated P-gal was measured in vitro in buffer and plasma prior to initiating in vivo studies. It is demonstrated that vesicles can be made reproducibly with a latency > 95% and that encapsulated P-gal is protected from serum induced inactivation at 37 °C.  In the first part of Chapter 3, the pharmacokinetics of free protein and protein encapsulated in phospholipid/cholesterol vesicles ± polyethylene glycol (PEG) are investigated in normal mice and mice pre-immunized against P-gal protein. These experiments demonstrate that in the absence of p-gal antibodies, free protein and protein encapsulated in PEG-free vesicles are cleared from the circulation at similar rates, whereas protein formulated in PEG-coated vesicles is cleared much more slowly. The data are consistent with the known ability of P E G to protect the vesicle surface from opsonization. In the presence of P-gal antibodies (immunized mice), free protein was cleared from the blood immediately but the rate of clearance for protein protected inside PEG-free vesicles was unchanged from that measured in non-immunized animals. However, protein encapsulated inside PEG-coated vesicles was removed from the circulation as fast as free protein, indicating the formation of antigen-antibody complexes.  The difference in biodistribution between normal and PEG-coated vesicles to macrophages, and other antigen presenting cells, may influence the nature or magnitude of an immune response to repeated i.v. administrations to normal mice. This was also investigated in C h a p t e r 3. Normal mice were subjected to five weekly injections o f encapsulated P-gal in vesicles ± P E G . The results show that both preparations elicit P-gal antibodies at the same rate and to the same level during the course o f administration. Despite this, the rate o f clearance for (3-gal-containing, PEG-coated liposomes is increased dramatically by the second injection compared to the naked vesicles. After the full course o f five weekly injections, both types o f protein delivery system exhibit similar blood clearance kinetics. These results suggest that a progressive immune response is mounted by these animals, which can recognize the protein carrier system and that P E G coated vesicles are recognized more readily than naked vesicles. Possible reasons for the pharmacokinetic differences observed are discussed in C h a p t e r 4. These include the physical characteristics o f the vesicles and the exposure o f protein epitopes at the surface of the vesicles, as well as the nature o f the immune response and the possibility that antibodies are raised against the P E G anchor.  iv  TABLE OF CONTENTS  ABSTRACT  :  ..  TABLE OF CONTENTS  n v  LIST O F FIGURES  vn  LIST O F T A B L E S  vm  ABBREVIATIONS  IX  ACKNOWLEDGEMENTS DEDICATION CHAPTER 1  x xii 1  INTRODUCTION  1  1.1  LIPOSOMES AS CARRIERS  1  1.2  LIPOSOMES  2  1.2.1 Classification and preparation of liposomes 1.2.1.1 1.2.1.2 1.2.1.3 1.3  Multimellar vesicles (MLV) Small unilamellar vesicles (SUV) Large unilamellar vesicles (LUV)  2 2 5 5  PROPERTIES OF LIPOSOMES INFLUENCING THEIR CIRCULATION LIFETIME IN v i v o . . . . 8  1.3.1 Chemistry and physics of lipids 1.3.1.1 1.3.1.2  Phospholipid Cholesterol  1.3.2 In vivo behavior of liposomes 1.3.2.1 1.3.2.2  Interaction of liposomes with plasma protein Interaction with the mononuclear phagocyte system (MPS)  1.3.3 Liposomes coated by Poly(ethylene glycol) (PEG)  10 10 15  16 17 19  20  1.4  T H E P-GALACTOSIDASE  24  1.5  THESIS OBJECTIVES  26  C H A P T E R 2...  28  F O R M U L A T I O N OF LIPOSOMAL P-GALACTOSIDASE  28  2.1  INTRODUCTION  28  2.2  M A T E R I A L S A N D METHODS  29  2.2.1 2.2.2 2.2.3 2.2.4 2.3  Materials Measurement of /3-galactosidase activity Encapsulation of /3-galactosidase in liposomes Latency of /3-gal and in vitro stability study  RESULTS  2.3.1 Assay for measuring /3-galactosidase activity 2.3.2 Characterization of encapsulated /3-gal 1.1.3 In vitro stability of /3-gal and liposomal /3-gal in buffer and plasma 1.4  SUMMARY  CHAPTER 3  29 30 30 31 32  33 33 38 42 44  PHARMACOKINETICS OF LIPOSOMAL P - G A L A C T O S I D A S E IN N O R M A L A N D IMMUNIZED  v  MICE 3.1 INTRODUCTION 3.2 MATERIALS AND METHODS  3.2.1 3.2.2 3.2.3 3.2.4 3.3  44 44 45  Materials , Measurement of anti ffgal antibodies in mouse serum Immunization of mice against j3-galactosidase Pharmacokinetic studies  RESULTS  '.  :  45 45 47 48 48  3.3.1 Pharmacokinetics offree and encapsulated ffgalactosidase in normal mice 48 3.3.2 Pharmacokinetics of free and encapsulated 0-galactosidase in immunized mice 51 1.1.3 Pharmacokinetics and humoral immune response to encapsulated /3galactosidase following repeated weekly administration 57 1.1.4 Phenotypic response of mice to ffgalactosidase formulations 60 3.4  SUMMARY  CHAPTER 4  62 64  DISCUSSION  64  4.1 E N Z Y M E REPLACEMENT AND PROTEIN THERAPEUTICS 4.2 FORMATION OF ANTIGEN-ANTIBODY COMPLEXES AND VESICLE CLEARANCE 4.3 REPEATED ADMINISTRATION AND VESICLE CLEARANCE 4.4 O N THE DIFFERENCE BETWEEN UNCOATED AND PEG-COATED VESICLES BIBLIOGRAPHY  64 66 67 68 71  vi  LIST OF FIGURES FIGURE 1.1  (A) AMPHIPATHIC LIPIDS IN A BILAYER CONFIGURATION, (B) FREEZE-  FRACTURE ELECTRON MICROSCOPY OF MULTIMELLAR VESICLES ( M L V ) , LARGE UNILAMELLAR VESICLES ( L U V ) , AND SMALL UNILAMELLAR VESICLES ( S U V ) . T H E BAR REPRESENTS 200 N M FIGURE 1.2  4  FREEZE FRACTURE ELECTRON MICROGRAPHS OF L U V PRODUCED BY  EXTRUSION FIGURE 1.3  7  BIOPHYSICAL PROPERTIES OF LIPOSOMES THAT INFLUENCE STABILITY AND  CLEARANCE IN VIVO FIGURE 1.4  9  GENERAL STRUCTURE OF A PHOSPHOLIPID SHOWING COMMONLY OCCURRING  HEADGROUPS AND FATTY ACID MOIETIES FIGURE 1.5  11  LIPID POLYMORPHISM - ORGANIZATION OF LIPID MOLECULES IN MICELLE,  BILAYER, AND HEXAGONAL PHASES FIGURE 1.6  14  T H E STRUCTURE OF CHOLESTEROL (A) AND ITS EFFECT ON THE STRUCTURE OF  LIPID BILAYERS (B)  16  FIGURE 1.7  P E G - C O A T E D VESICLE AND THE CHEMICAL STRUCTURE OF D S P E - P E G  23  FIGURE 1.8  RIBBON REPRESENTATION OF THE P-GALACTOSIDASE TETRAMER  25  FIGURE 2.1  STANDARD CURVE FOR P-GAL ACTIVITY  33  FIGURE2.2  COMPARISON OF PROTEIN SPECIFIC ACTIVITY BEFORE AND AFTER EXTRUSION. 35  FIGURE 2.3  SEPARATION OF LIPOSOMAL P-GAL FROM FREE PROTEIN  36  FIGURE 2.4  LATENCY MEASUREMENT  37  FIGURE 2.5  P N P G ASSAY VALIDATION  40  FIGURE 2.6  IN VITRO STABILITY OF P-GAL, LIPOSOMAL P-GAL, AND P E G - C O A T E D  LIPOSOMAL P-GAL FIGURE 3.1  MEASUREMENT OF ANTI P-GAL I G G BY INDIRECT TWO-STEP E L I S A .  41 PRIMARY  ANTIBODY REACTS WITH BOUND ANTIGEN AND A LABELED SECONDARY ANTIBODY REACTS WITH THE PRIMARY ANTIBODY FIGURE 3.2  46  PHARMACOKINETICS OF FREE AND ENCAPSULATED P-GALACTOSIDASE IN  NORMAL MICE  50  FIGURE 3.3  GENERATION OF ANTI P-GAL I G G UPON REPEAT S.C. INJECTION  FIGURE 3.4  A COMPARISON OF THE CIRCULATION LIFE-TIME OF FREE P-GAL IN IMMUNIZED  AND NON-IMMUNIZED MICE FIGURE 3.5  51 53  CLEARANCE KINETICS OF LIPOSOMAL P-GAL  54  FIGURE 3.6  CLEARANCE KINETICS OF PEG-COATED LIPOSOMAL P-GAL  55  FIGURE 3.7  IMMUNIZATION WITH PROTEIN FREE VESICLES HAS NO EFFECT ON THE  PHARMACOKINETICS OF VESICLE CLEARANCE FIGURE 3.8  57  CORRELATION BETWEEN LIPOSOMAL P-GAL CLEARANCE KINETICS AND THE  GENERATION OF ANTI P-GAL I G G FOLLOWING WEEKLY I.V. ADMINISTRATION  59  vii  list of tables T A B L E 1.1 TRANSITION TEMPERATURE (Tc)  OF VARIOUS COMBINATIONS OF A C Y L CHAIN  L E N G T H , D E G R E E OF SATURATION, A N D HEADGROUP MOIETY. T H E HIGHLIGHTED PHOSPHOLIPID, P O P C WAS USED IN THIS THESIS  12  T A B L E 1 . 2 C E L L S OF M O N O N U C L E A R P H A G O C Y T I C S Y S T E M ( M P S )  20  T A B L E 2.1 C A L C U L A T I O N OF T H E N U M B E R OF - G A L M O L E C U L E S E N C A P S U L A T E D INSIDE P  P O P C : C H O L (55:45) LIPOSOMES  38  T A B L E 3.1PHENOTYPIC RESPONSE FOLLOWING BOLUS I.V. ADMINISTRATION TO MICE PREIMMUNIZED AGAINST P - G A L (SECTION 3.2.2) T A B L E 3.2PHENOTYPIC RESPONSE FOLLOWING FIVE W E E K L Y B O L U S I.V. TO N O R M A L MICE  60 ADMINISTRATIONS 61  Vlll  ABBREVIATIONS aa Ab Ag P-gal BSA CaCl Choi H-CHE Da D S P E - P E G 2000 2  3  EDTA ELISA G  M 1  HEPES H S0 HPI IgG i.v. LSC LUV MPS MgCl MLV 2  4  2  M W  NaOH NaHCOj Na C0 NaN POPC PNPG RES SM s.c. S.D. SUV Tris 2  3  3  amino acid antibody antigen P-galactosidase bovine serum albumin calcium chloride cholesterol [ H]-cholesterylhexadecylether Dalton l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol)-2000] ethylenediaminetetraacetic acid enzyme-linked immunosorbent assay Monosialo-ganglioside [4-(2-hydroxyethyl)]-piperazine ethane sulfonic acid sulfuric acid Hydrogenated soy phosphatidylinositol immunoglobulin G intravascular liquid scintillation counting large unilamellar vesicles mononuclear phagocyte system magnesium chloride multilamellar vesicles molecular weight sodium hydroxide sodium bicarbonate sodium carbonate sodium azide l-palmitoyl-2-01eyl-sn-Glycero-3-Phosphocholine para-nitrophenyl-P-D-galactoside reticuloendothelial system sphingomyelin subcutaneous standard deviation small unilamellar vesicles tris(hydroxymethyl)aminomethane half-life 3  IX  A C K N O W L E D G E M E N T S  I would like to take this opportunity to thank you M i c k (&) for accepting me into the Skin Barrier Research Lab and providing an inspiring project and enjoyable working environment. I am deeply appreciated your generosity and guidance throughout this study, and your effort i n correcting and refining all my writing. I ' l l never forget all the memorable parties, enjoyable jfc trips, and the non-destructible extruder. Thanks Lipex. In addition, I am also appreciative to all members o f the lab. Specifically Wendi who had assisted me to get established i n this lab.  Members o f the C u l l i s ' lab have also contributed numerous helpful input and support. Barb (your hard working inspiration and informative discussion), Conrad (H games), Norbert (the Friday seminar), A n g e l (thanks for proofreading all m y writing, v), and Loren (®—DORA—>  ©I). Thank you for the laughs, warmth and hospitality. To K i m  ( © © ! ? ) , Dave (m- review), and Pieter (Friday beer and chips!), m y sincerest thanks for your patients for putting up with me. K e n , I ' m always proud o f you. Keep up the graveyard shift!  A l s o , thank you  0 for providing a real life  ,NE>  o f business i n science:  Conventional Formulation team (Payday, leave early!); Murray (for your patients and suggestions), Diane (for processing all my request); Steven (continuous supply o f D S P E P E G 2 0 0 0 ) ; r (solving all my (21H^O# ). In addition, m y thesis would not have made 4  it this far i f it had not been for the support from all member o f the vivarium.  Last but not least, I would like to acknowledge the financial assistance from the Science Council o f British Columbia for providing me with a studentship.  xi  DEDICATION  To my Daddy & Mommy Ken & Edwin  CHAPTER 1 INTRODUCTION  1.1  Liposomes as carriers Liposomes have been employed as delivery vehicles for many small molecular  weight drugs, as well as macromolecules. Drugs can be encapsulated i n their aqueous core or solubilized i n the membrane and administered by most routes including s.c, i.v., and i.p. It has been shown that the toxicity o f many o f these agents is reduced when they are formulated i n liposomes [Chonn and Culllis, 1995; Sharma, et al., 1997]. This reduction i n toxicity allows higher doses o f the therapeutic agents to be administered resulting in improved efficacy [Gabizon et al., 1986; B a l l y et al., 1990]. It has also been suggested that liposomes can serve as circulating reservoirs for slow release o f the entrapped agents i n the blood compartment as well as at sites o f disease [Mayer et al., 1990; A l l e n et al., 1992]. Furthermore, the circulation lifetime o f entrapped drug is greatly enhanced over that observed for free drug. Recent applications o f liposomes as carriers for pharmaceutical agents have been described i n several reviews [Hope et al., 1986; Cullis et al., 1989; Wasan and Lopez-Berestein, 1995].  1  1.2  1.2.1  Liposomes  Classification and preparation of liposomes  When bilayer forming lipids are dispersed in aqueous media, they spontaneously form structures called liposomes where multiple bilayers are usually configured in an onion skin arrangement o f concentric lamellae (Figure 1.1). The bilayer structure arises as a result o f the amphipathic nature o f lipids. The combination o f a hydrophilic head group and hydrophobic tail within the same lipid molecule results i n an orientation o f the lipid head group toward the aqueous environment and the acyl tails facing each other so that they are sequestered from water (Figure 1.1a). Liposome is a generic term that is commonly used to describe many different types o f model membrane systems formed by lipids. Throughout this thesis more descriptive terminology is used to describe the three most common types o f liposome used: Multilamellar vesicles ( M L V ) , large unilamellar vesicles ( L U V ) and small unilamellar vesicles ( S U V ) . i  1.2.1.1  Multimellar  vesicles (ML V)  M o d e l membranes that exhibit the classical liposome structure with morphology o f alternating concentric spheres o f lipid bilayers and aqueous compartments are referred to as M L V . They are formed spontaneously by mechanical dispersion o f a lipid film i n an aqueous solution as first described by Bangham et al. [1965]. Typical diameters o f M L V are on the order o f 1000 nm but preparations are heterogeneous i n size. In a typical structure o f this type o f liposome, the majority o f the lipid is present as internal lamellae and only 10% or less o f the total lipid is present in the outermost bilayer. M L V  2  containing neutral lipids usually have an aqueous trapped volume o f about 0.5 p i / umol lipid. This can be improved by a freezing and thawing procedure [Mayer et al., 1985], which generate frozen and thawed M L V ( F A T M L V ) . This technique increases the interbilayer spacing and trapped volumes i n excess o f 2 p i / umol lipid can be achieved. Another method to increase the trapped volume o f M L V is to incorporate charged lipids,  i which cause charge repulsion between the internal lamellae, and increase the interbilayer spacing [Hope et al., 1986]. M L V are relatively large, heterogenous structures, and are rapidly cleared from the circulation by the reticuloendothelial (RE) organs and mononuclear phagocyte system ( M P S ) , consequently these vesicles are rarely used for drug delivery.  3  F i g u r e 1.1  (a) A m p h i p a t h i c lipids i n a bilayer configuration, (b) Freeze-fracture electron microscopy of multimellar vesicles ( M L V ) , large unilamellar vesicles ( L U V ) , and small unilamellar vesicles ( S U V ) . T h e bar represents 200 n m .  a.  hydrophilic  hydrophobic bilayer structure b.  4  1.2.1.2 Small unilamellar vesicles (SUV)  S U V contain a small internal aqueous compartment (< 0.5 p i / umol) surrounded by a single lipid bilayer 20 to 50 n m i n diameter. They represent the lower physical limit o f liposome size and are generally produced by sonicating M L V [Huang, 1969] or by utilizing the French press or another type o f high pressure homogenization technique [Barenholz et al., 1979]. Since S U V are unilamellar and uniform i n size, they have been used extensively for in vitro and in vivo studies. However, because o f their small size, the bilayer is highly curved [Lichtenberg et al., 1981], resulting i n an unstable vesicle prone to fusion [Wong et al., 1982], as well as attack by phospholipases [Gillett et al., 1980] and high density lipoproteins ( H D L ) in vivo (see section 1.3.2.1) [Scherphof and Morselt, 1984].  1.2.1.3 Large unilamellar vesicles (LUV)  Typical L U V range from 50 to 400 n m i n diameter. They can be prepared by reverse phase evaporation [Szoka and Papahadojopoulos, 1978] or by detergent dialysis [Kagawa and Packer, 1971; M i m m s et al., 1981]. Both o f these techniques, however, have the disadvantage o f being dependent on lipid composition, exhibit inconsistent reproducibility, and are complicated by the need to remove residual organic solvents or detergents from the final products. M o r e recently, L U V have been produced from M L V by the extrusion technique, where M L V are forced through polycarbonate filters o f  5  defined pore sizes under medium pressure (< 6000 kPa) [Hope et al., 1985]. Using extrusion, L U V composed from a wide variety o f lipid species can be readily made at high concentration i n the absence o f contaminating detergents or organic solvents [Hope et al., 1985; Nayar et al., 1989]. Light scattering measurement shows that L U V produced by the extrusion method are uniform i n size (Figure 1.2). Experiments reported i n this thesis mostly employ L U V produced by extrusion through filters with a pore size o f 200 nm.  6  Figure 1.2  Freeze fracture electron micrographs of LUV produced by extrusion  Vesicles were prepared from M L V at a concentration of 50 mg/ml by extrusion through two stacked polycarbonate filters of various sizes. The bar represents 200 nm.  7  1.3  Properties of liposomes influencing their circulation lifetime in vivo  The use o f liposomes as Ehrlich's "magic bullet" for targeting toxic drugs to sites of disease was first challenged by empirical findings that early liposomes were unstable in blood [Gregoriadis and Ryman, 1972]. In the past twenty years, significant advances have been made i n understanding the factors involved i n the clearance o f liposomes from the circulation. Circulating blood proteins (opsonins) identify liposomes as foreign and mark them for clearance by the R E organs and elements o f the host defense system known as the M P S (See section 1.3.2.2). Macrophages, which line blood vessels in organs such as liver and spleen, recognize opsonized foreign particles and remove them by receptor mediated phagocytosis.  L i p i d composition is one o f the most important factors contributing to the stability and clearance o f liposomes from the blood. It affects vesicle permeability, surface charge, and interaction with plasma protein [Allen, 1988; A l l e n et al., 1990; Gabizon and Papahadjopoulos, 1988] (Figure 1.3). The two major lipid components used to make the vesicles described here are phospholipids and cholesterol.  8  F i g u r e 1.3  B i o p h y s i c a l properties of liposomes that influence stability and  clearance in vivo. Surface charge Surface charge attracts serum proteins to the vesicle surface which enhances clearance and can cause hematological toxicity through complement activation, for example.  Bilayer fluidity Acyl chain order and cholesterol influence the ability of opsonins and other serum proteins to interact with the bilayer.  Vesicle size Vesicle size influences circulation lifetime as well as extravastation into disease sites.  9  1.3.1  Chemistry and physics of lipids  1.3.1.1 Phospholipid  The importance o f phospholipids to living organisms is underscored by the nearly complete lack o f genetic defects in the metabolism o f these lipids i n humans. Presumably, any such defects are lethal at early stages o f development and therefore are never observed. A l l phospholipids are composed o f various combinations o f polar (hydrophilic) headgroups coupled to apolar (hydrophobic) tails v i a a glycerol-3phosphate backbone (Figure 1.4). The hydroxyls on carbons 1 and 2 are usually acylated with fatty acids, and i n most phospholipids the fatty acid substituent at carbon-1 is saturated, while the one at carbon-2 is unsaturated. The physical properties o f the lipid bilayer are dictated by the combination o f headgroup and acyl chain (Table 1.1). The acyl chain length and degree o f saturation govern the temperature o f the gel (rigid) to liquid-crystalline (disordered and fluid) phase transition for the lipid bilayers. In general, longer acyl chains and higher degrees o f saturation w i l l give rise to a higher phase transition temperature (T ). Above the T , the acyl chains are less ordered or more "fluid" c  c  in nature (liquid-crystalline phase). Long, saturated acyl chains form extensive van der Waals interactions with each other i n the bilayer thus limiting their motion. However, ew-double bonds, present i n unsaturated phospholipid acyl chains, produce kinks, which impede nearest neighbour interactions and increase motion. In general, biological bilayers are i n the fluid state at physiological temperatures and fluid model membranes  10  tend to be more permeable than membranes in the gel state [Papahadjopoulos et al., 1973; Bittman and Blau, 1972].  Figure 1.4  General structure of a phospholipid showing commonly occurring headgroups and fatty acid moieties.  A-  x  I  Headgroup  Choline (Phosphatidylcholine) Ethanolamine (Phosphatidylethanolamine)  o '—0"  O— I)  • Glycerol backbone  I CH —CH—CH 2  I I  0  0  1  I  o=c  2  Neutral phospholipids  -4-  Structures  CH CH N (CH ) +  2  2  3  3  -CH CH N H3 +  2  2  Negative phospholipids Serine -CH CH-N+H (Phosphatidylserine) I 2  3  coo-  Glycerol (Phosphatidylglycerol)  -CH CH(OH)CH OH 2  Inositol (Phosphatidylinositol)  c=o  2  H  on  Saturated Fatty Acids Laurie Myristic Palmitic Stearic Arachidic  CH (CH )j COOH 2  3  0  CH (CH ) C00H 2  3  3  2  14  CH (CH ), C00H 2  3  6  CH (CH ) C00H 2  3  Lignocenc  12  CH (CH ) COOH  lg  CH (CH ), COOH 3  2  2  Unsaturated Fatty Acids L Acyl Chain  Palmitoleic Oleic Linolcic Linolenic Arachidonic  CH (CH ) CI«:II(CH2) COOII 3  2  5  7  ra (CH ) CH=CH(CH ) cooii 3  C L  2  7  2  7  I (CH ) CH=CIICI I CI i=cii(ci I ) COOH 3  2 5  2  2  7  CH CH CH=CHCH CH<HCII CH-CII(CII ) COOH 3  2  2  2  2 7  CH (CH ) (CH<HCH ) CH=CH(CH ) COOH 3  2  4  2  3  2  3  11  Table 1.1  Transition temperature (Tc) of various combinations of acyl chain length, degree of saturation, and headgroup moiety. The highlighted phospholipid, POPC was used in this thesis.  Lipid species (acyl chain # dilauroyl PC dimyristoyl PC dipalmitoyl PC distearoyl PC stearoyl, oleoyl PC stearoyl, linoleoyl PC stearoyl, linolenoyl PC stearoyl, arachidonyl PC dioleoyl PC palmitoyl, oleoyl PC dipalmitoyl P A dipalmitoyl P E dipalmitoyl PS dipalmitoyl P G  1 (12:0, (14:0, (16:0, (18:0, (18:0, (18:0, (18:0, (18:0, (18:1, (10:0. (16:0, (16:0, (16:0, (16:0,  2) 12 0) 14 0) 16 0) 18 0) 18 1) 18 2) 18 3) 20 4) 18 1) 18 1) 16 0) 16 0) 16 0) 16 0)  Transition temperature (± 2 °C) -1 24 41 55 6 -16 -13 -13 -19 67 63 55 41  In an aqueous environment, phospholipids adopt a variety of structures. This is known as lipid polymorphism [Cullis et al., 1986] (Figure 1.5). When the cross sectional area of the hydrophobic head group is greater than that swept by the acyl tails, the phospholipid molecules can be considered to adopt a cone shape and thus will tend to pack into micelles, structures typically adopted by detergents. Unsaturated phosphatidylethanolamine (PE), does not form a bilayer when hydrated as the crosssectional area of the relatively small, neutral headgroup is less than that occupied by the acyl chains. The dynamic shape of unsaturated PE can be conceptualized as an inverted cone. These lipids tend to form the hexagonal H phase in aqueous medium [Cullis and n  de Kruijff, 1979], however, they will form bilayers when stabilized by bilayer forming lipids. These lipids behave as cylinders and therefore prefer to pack into a bilayer  12  configuration. A n example is phosphatidylcholine (PC), the most common phospholipid in eukaryotic plasma membranes. P C is a zwitterion composed o f a glycerol-phosphate ester with a choline headgroup and two acyl chains esterified to the sn-1 and sn-2 positions [Small, 1986]. In naturally occurring P C , the fatty acid at the sn-1 position is saturated, while the sn-2 acyl chain is usually unsaturated [Small, 1986]. The phospholipid used to form vesicles described here was l-palmitoyl-2-oleoyl phosphatidylcholine (POPC), a synthetic analogue o f one o f the most common, naturally occurring P C .  13  F i g u r e 1.5  L i p i d p o l y m o r p h i s m - organization of l i p i d molecules i n micelle, bilayer, and hexagonal phases.  SHAPE  STRUCTURE  14  1.3.1.2 Cholesterol  Cholesterol is the major neutral lipid component o f eukaryotic plasma membranes. It is an amphipathic molecule with a hydrophobic, rigid steroid nucleus, aliphatic side chain and 3-f3-hydroxyl group. In the bilayer, cholesterol is oriented such that the hydrophobic moieties lay next to the phospholipid acyl chains and the hydroxyl group is positioned close to the carbonyl ester bond linking the acyl chain to the glycerol backbone (Figure 1.6a). Cholesterol has a very specific interaction with phospholipids and increases the "order" in the acyl chains o f P C s that are i n the liquid-crystalline state but decreases the order o f P C s which are in the gel state [Demel and de Kruyff, 1976]. Furthermore, the incorporation o f cholesterol into membranes composed o f saturated P C progressively decreases the enthalpy o f the gel-liquid crystalline phase transition (Figure 1.6b). A t 30 m o l % o f cholesterol or higher, the transition can no longer be detected [Chapman, 1975]. This cholesterol-phospholipid interaction gives rise to the condensing effect measured using lipid monolayer techniques which have shown that the surface area occupied by a mixture o f liquid-crystalline P C and cholesterol is actually less than the sum o f the area o f the two components [Hyslop et al., 1990].  The inclusion o f cholesterol helps stabilize liposomes i n blood [Mayhew et al., 1979; Senior 1987; Patel et a l , 1983] by reducing the disruptive effects o f plasma proteins, which i n turn enhances solute retention i n the aqueous core [Papahadjopoulos et al., 1973; Inoue, 1974]. In culture, Kupffer cells (responsible for the majority o f liposomal clearance in vivo) exhibit decreased uptake and intracellular degradation o f  15  cholesterol-containing liposomes [Roerdink et al, 1989]. The addition o f cholesterol has also been shown to reduce the net transfer o f phospholipid from liposomes to high density lipoprotein (see detail discussion on section 1.3.2.1) [Kirby et al., 1980a, b; Gregoriadis and Davis, 1979].  Figure 1.6  The structure of cholesterol (a) and its effect on the structure of lipid bilayers (b).  If III  - '— ~ y  1.3.2  In vivo behavior of liposomes  The majority o f a dose o f liposomes administered intravenously is cleared from the circulation by the liver and spleen [Gregoriadis and Ryman, 1972; Gregoriadis, 1988]. The exact in vivo mechanisms responsible for this clearance and the recognition o f liposomes as foreign particles are still unsolved. However, two major factors contribute to liposome clearance from the blood: (1) interaction with opsonins and plasma  16  lipoproteins, and (2) uptake o f liposomes by cells o f the M P S primarily in the liver and spleen [Gregoriadis, 1988].  1.3.2.1 Interaction of liposomes with plasma protein  Liposome clearance from the blood is believed to be mediated by two different groups o f plasma proteins: opsonins and lipoproteins [Patel, 1992]. Opsonins are serum proteins that adsorb onto the surface o f a foreign particle, thereby rendering the particle more palatable to phagocytes. They promote phagocytosis primarily by forming a bridge between the particle and macrophage, but they are generally not involved i n the internalization or digestion o f particles. There are two types o f opsonins: specific and nonspecific. Specific opsonins, which include immunoglobulins and components o f the complement system, interact directly with receptors on macrophages, whereas nonspecific ones function by altering the surface properties o f the foreign particle or the phagocyte or both, thus rendering them more adhesive to phagocytes. It has been suggested that opsonins associate with the surface o f liposomes v i a nonspecific hydrophobic interactions [Gregoriadis, 1988]. The degree o f opsonization is affected by the surface charge and molecular packing o f the lipid bilayer which is dictated by phospholipid headgroups, vesicle size, acyl chain composition and cholesterol content [Scherphof et al., 1984]. Vesicles with loosely packed bilayers are more susceptible to opsonin adsorption [Scherphof et al, 1984; M o g h i m i and Patal, 1988a]. The surface charge o f vesicles has also been demonstrated to play a role in mediating the non-specific adsorption o f IgG and complement proteins, which mark vesicles for clearance v i a macrophages that exhibit specific receptors for IgG, the complement protein C 3 ,  17  fibronectin, and other extracellular matrix components [reviewed i n Patel, 1992]. The role o f scavenger receptors on macrophages i n mediating the uptake o f liposomes has also been described [Nishikawa et al., 1990]. The circulation lifetime o f liposomes has been determined to be inversely related to the affinity o f liver macrophages for the liposomes. Furthermore, it has been suggested that serum may contain organ-specific opsonins that selectively enhance liposome uptake by macrophages o f either the liver or spleen [Moghimi and Patel, 1988b]. Recent findings based on in situ liver perfusion assays demonstrated that hepatic uptake o f neutral or negatively charged liposomes in mice is an opsonin independent process and does not involve serum components [ L i u and L i u , 1996], whereas hepatic uptake o f liposomes i n rats appears to depend on serum opsonins [Liu et al., 1995].  In addition to opsonins, the interaction o f liposomes with other plasma proteins plays a role i n determining liposome clearance and permeability characteristics. In the absence o f cholesterol i n the membrane, S U V w i l l interact with plasma lipoproteins which leads to lipid exchange, dissolution o f the carrier, and premature release o f the encapsulated materials [Kirby et al., 1980a]. M o r e specifically, high-density lipoproteins ( H D L ) , HDL-associated apolipoprotein A p o A - 1 [Klausner et al., 1985], and complement proteins [Devine et al., 1994] can penetrate vesicle membranes and disrupt the permeability barrier. Some lipid compositions can activate the complement pathway inducing the formation o f membrane attack complexes, which lyse the liposomal membrane [Silverman et al., 1984; M a l i n s k i and Nelsestuen, 1989]. Recent findings indicate that liposomes activate complement in a dose-dependent manner, require the  18  inclusion o f phospholipid bearing a net negative or positive charge, and can activate both the classical and alternative pathways [for review, see Patel, 1992]. In addition, liposome size can also contribute to complement activation: for example, large L U V (400 nm) are found to be more effective in activating complement than small (50 nm) vesicles [Devine etal., 1994].  1.3.2.2 Interaction with the mononuclear phagocyte system (MPS)  Following i.v. injection, the majority o f liposomes are removed by fixed macrophages residing i n the liver and spleen, as well as i n the lung, lymph nodes, and bone marrow. Macrophages patrol the circulatory system in search o f unwanted particles. In the course o f their travels, macrophages ingest sick and dying cells, invading pathogens and anything else that appears to be foreign. Macrophages were originally classified as part o f the reticuloendothelial system ( R E S ) [Metchnikoff, 1963] which included reticular cells, endothelial cells, fibrocytes, histocytes, and monocytes. However, since this classification did not fulfill the criteria o f common morphology, function and origin o f the macrophage, a new classification, the mononuclear phagocyte system ( M P S ) , was adopted i n 1969. The M P S was described on the basis o f knowledge about the monocyte precursors in the bone marrow and about the monocyte-derived macrophages i n various locations under normal and pathological conditions [van Furth et al., 1971]. The M P S is composed o f monocytes, macrophages, and precursor cells (stem cells, monoblasts, and promocytes) in the bone marrow, as well as several other cell types found i n the circulation, tissue, and body cavities under normal and inflammation conditions (Table 1.2). It is now known that the uptake o f foreign particles by M P S is  19  dependent on the recognition o f membrane associated opsonins by specific receptors on the macrophage surface [reviewed by Coleman, 1986]. A s mentioned earlier, the size o f liposomes and the total amount o f bound protein on the liposome surface [Chonn et al., 1992] are factors which can dictate the rate o f liposome clearance by macrophages.  Table 1.2  Cells o f Mononuclear Phagocytic System ( M P S )  Tissue Bone marrow Blood Liver Lung  Cells Stem cells, monoblasts, promocytes, and monocytes Monocytes Kupffer cells Alveolar macrophages, monocytes in the pleural fluid  Spleen L y m p h nodes Thymus Lymphoid tissues Gastrointestinal tract Colostrum Endocrine organs  Free and fixed macrophages  Nervous system Serous cavities Connective tissue  Microglial cells . Peritoneal macrophages Histiocytes  1.3.3  Liposomes coated b y Poly(ethylene glycol) ( P E G )  In order to enhance the circulation lifetime o f liposomes and deflect their biodistribution away from the liver and spleen attempts have been made to inhibit opsonization. Certain biological precedents suggested that this might be possible. For  20  example, the basic structure o f red blood cell membranes is similar to that o f liposomes, and erythrocytes are able to circulate for several months before being removed by the M P S . Subsequent incorporation o f specific glycolipids such as monosialo-ganglioside ( G ) or hydrogenated soy phosphatidylinositol (HPI) have been shown to result in M1  prolonged circulation and reduced M P S uptake [Allen and Chonn, 1987; Gabizon and Papahadjopoulos, 1988; L i u and Huang 1990]. Furthermore, coating the surface o f liposomes with P E G has been shown to reduce uptake by Kupffer cells in the first few hours following i.v. administration.  P E G is a hydrophilic polymer that has previously been grafted onto proteins to prolong their lifetime i n the bloodstream. Based on monitoring the structural organization, interbilayer repulsion, as well as lipid bilayer elasticity, the addition o f P E G to liposome does not change the normal structure o f the bilayer interior [Needham et al., 1992]. It is proposed that P E G forms a polymeric "cloud" extended ~ 5.0 n m above the liposome surface [Torchilin 1994; Torchilin et al., 1995] which reduces the hydrophobic and electrostatic interaction o f lipoproteins and opsonins to the lipid surface (Figure 1.7). This phenomenon is known as surface steric stabilization and it decreases the rate at which liposomes are cleared from the circulation by macrophages [Parr et al., 1993].  Both the length o f the P E G polymer and its density at the lipid bilayer surface are important factors for maximizing the stabilizing effect. It has been reported that the optimal polymer size for extending circulation time is 2000 D a , at a density o f about 5 mole % o f the total liposome lipid [Klibanov et al., 1991; Woodle et al., 1992; Litzinger  21  and Huang, 1992]. The sterically stabilized vesicles used i n the work described in this thesis contained PEG-2000, which consist o f an average 45 ethylene glycol monomers per P E G chain, conjugated to a distearoylphosphatidylethanolamine ( D S P E ) lipid anchor via a succinate linker group. This molecule has been shown to provide a stable polymer coat for 24 hours in vivo [Parr et al., 1994].  22  Figure 1.7  PEG-coated vesicle and the chemical structure o f D S P E - P E G .  1.4 The P-galactosidase The p-gal employed in this work is derived from yeast Kluyveromyces fragilis. It is an evolutionary conserved protein: the prokaryotic P-gal and the eukaryotic P-gal are closely related i n both structure and sequence [Guiso et al., 1978; Poch et al., 1992]. The active form o f P-gal is a tetramer [Edwards et a l , 1988]. Each monomeric unit o f P-gal is composed o f 1025-aa polypeptide with M W o f 117,618 D a . Electron micrographs [Karlsson et al., 1964], Fourier-transform infrared spectroscopy [Arrondo et al., 1989], and X-ray diffraction [Jacobson and Matthews, 1992; Jacobson et al., 1994] have revealed that the tetrameric P-gal from E. coli is roughly 17.5 x 13.5 x 9.0 n m i n size and particles with three dimensions have been drawn to scale inside a 200 n m L U V to help provide some perspective (Figure 1.8). This protein is composed o f 35% a-helix, 40% Psheet (mainly parallel), 12% random coil, and 13% P-turn [Arrondo et al., 1989]. Each tetramer contains four active sites and the residues G l u 461, M e t 502, T y r 503 and G l u 537 are believed to be important for catalytic function, or reside near the active site [Jacobson et al., 1994]. The presence o f M g , and monovalent cations K and N a is 2 +  +  +  required for maximal P-gal activity [Wallenfels and W e i l , 1972, Huber et al., 1994]. However, the specific role o f these cations i n the mechanism o f action is not yet understood.  24  Figure 1.8  Ribbon representation of the P-galactosidase tetramer.  Ribbon representation o f the P-galactosidase tetramer showing the largest face o f the molecule. The constituent monomers form two different monomer-monomer contact that are referred to as the "activating" interface and the "long" interface. Contacts between red/green and blue/yellow dimers form the long interface. Contacts between the red/yellow and blue/green dimers form the activating interface. Formation o f the tetrameric particle results in two deep clefts that run across opposite faces o f the molecule and each contain two active sites (Adopted from Jacobson et al., 1994). •  . - ••  W  J7  S a  o o  The dimensions o f the tetrameric P-gal are shown in scale to the dimension o f a 200 n m vesicle. The number o f protein encapsulated inside the vesicle is estimated as outlined in table 2.1.  25  This protein was chosen for these studies for several reasons. (1) P-gal is a Common  enzyme reagent used in many biochemical, histochemical and analytical assays.  Consequently, commercial kits and substrates are readily available. (2) It is important for the objectives o f this study that an enzyme is employed because retention o f biological activity was o f interest not simply the presence o f protein per se. The activity o f P-gal is sensitive to protein structure and integrity which may be compromised by an immune response. (3) The P-gal enzyme employed here is available i n bulk as a pure protein used in the dairy industry. This is relevant because the process o f encapsulation used is inefficient, more than 85% o f the starting protein is discarded, so the work had to be conducted using a cost effective protein. Finally, (4) this large protein isolated from a microorganism was expected to be immunogenic.  1.5  Thesis objectives  The objective o f this thesis is to test the hypothesis that a biologically active protein, encapsulated i n a lipid based drug delivery system, can be administered intravenously and repeatedly to mice without loss o f activity or reduced bioavailability due to the immune response induced against it.  Specific objectives were: 1. Develop a well characterized formulation i n which P-gal was encapsulated i n a lipid carrier.  26  2. Develop an assay that could measure protein latency, and would work in plasma with sufficient sensitivity to detect < 10% o f the injected dose i n blood. 3. Measure the stability o f free protein and encapsulated protein i n buffer and plasma in vitro to confirm the feasibility o f conducting in vivo experiments. 4. Measure the pharmacokinetics o f free protein, encapsulated protein, empty liposome and liposomes containing encapsulated protein i n normal mice following a single injection. 5. Develop assays to measure antibody production i n mice immunized by subcutaneous injection o f protein. 6. Measure the pharmacokinetics o f free protein, encapsulated protein, empty liposomes and liposomes containing encapsulated protein (following a single injection) in mice that have been immunized against the delivered protein. 7. Measure the pharmacokinetics o f encapsulated protein following repeat multiple weekly injections.  27  CHAPTER 2 F o r m u l a t i o n o fl i p o s o m a l  2.1  (3-galactosidase  Introduction  Liposomes have been widely used as delivery vehicles for a variety o f conventional, low molecular weight, synthetic drugs. However, the molecular size and structural sensitivity o f large protein molecules presents formulation challenges. Fluctuations i n temperature and p H outside o f the normal physiological range, as well as the presence o f organic solvent and/or detergent can denature protein or alter protein conformation in such a way as to have adverse effects on the biological activity.  For  the purposes o f this study p-galactosidase was chosen as the biologically  active protein, and the reasons for this choice have been outlined i n section 1.4. The encapsulation procedure employed was the mildest possible i n order to minimize any loss in specific activity o f the protein. This involved passive hydration o f lipid mixtures i n protein solutions at room temperature to form M L V ,  followed by sequential extrusion  through filters with defined pore sizes to produce 200 nm diameter LUV.  The lipid  composition was chosen to form the most stable vesicle that could be produced at 37 °C. L o n g chain, saturated phospholipids form very stable liposomes (section 1.3.1.1), however, they must be formed at temperatures above their T . For example, vesicles c  composed o f D S P C must be hydrated and extruded at temperatures > 65 °C, even in the  28  presence of cholesterol [Nayar et al., 1989], and at this temperature P-gal is rapidly denatured. Therefore the lipid mixture P O P C / C h o l was chosen, which readily forms vesicles at room temperature. However, it was determined that P-gal was sufficiently , stable at 37 °C to allow extrusion at this temperature, thus speeding the formulation process. Polymer coated vesicles were produced by incorporating 5 m o l % D S P E - P E G 2000. This amount o f saturated lipid in the formulation slightly slowed the extrusion process but did not prevent vesicles from being formed.  2.2  2.2.1  Materials and methods  Materials  l-Palmitoyl-2-01eoyl-sn-Glycero-3-Phosphocholine ( P O P C ) was obtained from Northern Lipids Inc. (Vancouver, B C , Canada). Cholesterol (Choi), para-nitrophenyl-PD-galactoside ( P N P G ) , and Triton X - 1 0 0 (TX-100) were purchased from Sigma Chemical C o . (St. Louis, M O ) .  l,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-  [Poly(ethylene glycol)-2000] ( D S P E - P E G 2000) was a gift from Dr. Steven Ansell (Inex Pharmaceuticals Corp. Vancouver, B C , Canada) and [ H]-cholesterylhexadecylether ( H 3  3  C H E ) was purchased from Dupont Canada, p-galactosidase solution (Lactozym 3000L) was a gift from N o v o Nordisk Biochem North America, Inc. (Franklinton, N C ) . Modified Hepes buffer ( H B S ) optimized for P-gal activity assay consists o f 0 . 1 M Hepes, 50 m M K C 1 , 2 m M M g C l « 6 H 0 (pH 6.5) was used for all preparations. 2  2  29  2.2.2  Measurement of P-galactosidase activity  The lactozym 3000L (commercial P-gal product) is supplied as protein i n glycerol solution. Before encapsulation and extrusion the glycerol was removed by overnight dialysis at 4 °C against H B S , typically 3 m l o f P-gal solution was dialyzed against 2 L o f H B S . This is because the presence o f glycerol made the protein solution too viscous to extrude. Protein concentration was determined by using the standard bicinchoninic acid ( B C A ) assay and the P-gal concentration post dialysis was 10.3 ± 0.2 mg'ml" (n=4). The 1  activity o f P-gal was measured by monitoring the cleavage o f the substrate P N P G (3.0 m l of P N P G at 0.5 mg'ml" i n H B S ) after 10 minutes incubation at room temperature. The 1  reaction was stopped by the addition o f 500 p i o f 1 M N a O H , which raised the p H to above 9.0. The absorbance was measured at 420 n m using a Perkin Elmer U V / V I S spectrometer - Lambda B i o .  2.2.3  Encapsulation of p-galactosidase in liposomes  Encapsulation o f P-gal was carried out by forming M L V i n the presence o f the protein solution followed by extrusion as described previously [Hope et al., 1985]. Briefly, total lipid composed o f P O P C : C h o l (55:45, mole ratio), or P O P C : C h o l : D S P E P E G 2000 (55:40:5, mole ratio) was dissolved i n C H C 1 at a concentration o f 100 mg«ml" 3  1  and mixed with radiolabeled lipid marker H - C H E (20 p C i ) . B u l k C H C 1 was removed 3  3  under a stream o f nitrogen gas, then trace solvent was removed b y exposing the lipid films to high vacuum overnight.  30  The dried lipid film (100 mg) was rehydrated by the addition o f one m l o f dialyzed P-gal solution, and M L V were formed during vigorous vortexing. M L V were first extruded 10 times through two stacked polycarbonate filters with a pore size o f 400 nm (Nuclepore, Pleasanton, C A ) . These were then replaced by two stacked filters with 200 n m pore size and the vesicles extruded for a further 10 passes. Extrusion was conducted at 37°C using a thermal regulated extruder (Lipex Biomembranes, Vancouver, B C , Canada). This process produced a homogeneous population o f vesicles with a mean diameter o f 200 ± 40 nm (n = 10). Liposome sizes were determined by Quasi-Elastic Light Scattering ( Q E L S ) (Nicomp Particle Sizing System M o d e l 370, Santa Barbara, C A ) . Encapsulation efficiency was measured by using gel filtration (Sepharose C L - 4 B ) . Typically, 1.0 m l o f L U V solution was loaded on a 1.5 x 11 cm Sepharose C L - 4 B column equilibrated i n H B S . The column was eluted with H B S at a flow rate o f 0.5 ml'min" and 1  600 p i per fraction were collected. Aliquots o f each fraction were assayed for lipid content by liquid scintillation counting ( L S C ) and P-gal activity by P N P G assay.  2.2.4  L a t e n c y of P-gal and  in vitro  stability study  To determine the activity o f P-gal encapsulated within the liposomes the lipid vesicles must first be disrupted to release the encapsulated protein, because the vesicle membrane is impermeable to the enzyme substrate. Latent enzyme activity was measured by conducting the P N P G assay in the presence and absence o f 1% T X - 1 0 0 (v/v), a detergent solution capable o f dissolving these vesicles.  31  % Latency = P ' (  g a l  activity + TX100) - (p-gal activity - TXIOO)  x  j  0 0  (p-gal activity+ TX100)  Liposomal P-gal fractions collected from the gel filtration column, determined to be greater than 95% latent, were pooled and used for the in vitro stability and pharmacokinetic studies. The amount of protein encapsulated was determined from P-gal standard curves constructed with known amounts o f protein i n the presence and absence o f detergent. The standards were prepared from a 1:5000 P-gal dilution i n H B S with or without 1% T X - 1 0 0 (v/v). Then, 0 to 400 p i o f diluted enzyme was assayed by P N P G assay as outlined on section 2.2.3.  The in vitro stability o f free P-gal and encapsulated P-gal was determined by incubating samples i n 50% diluted fresh mouse (BDF-1 strain) plasma at 37°C for various lengths o f time. In the presence o f plasma, P-gal activity was determined as described previously using the P N P G substrate solution containing 1% T X - 1 0 0 (v/v). Light scattering due to intact liposomes and plasma components was corrected by carrying out reactions in H B S containing detergent.  2.3  Results  32  2.3.1  Assay for measuring p-galactosidase activity  The activity o f P-gal was determined using an assay based on the substrate P N P G [Hill and Huber, 1971; Huber et al., 1979]. A l l spectroscopic measurements were made 10 minutes after the addition o f the stopping solution (NaOH). A linear relationship between absorbance at 420 n m and P-gal concentration was measured up to 0.8 p g o f protein (Figure 2.1). Furthermore, the absorbance was not affected by the presence o f Triton X - I 0 0 .  Standard curve for P-gal activity  Figure 2.1  1.2  •  withTX-100 without TX-100 Linear (without TX100) Linear (with TX-100)  0  0.2  0.4  0.6  0.8  ng P-gai (3-gal was reacted with P N P G substrate in the presence or absence of 1% (v/v) Triton X-100. Lines represent linear regression by least squares fit with correlation coefficient > 0.96.  2.3.2  Characterization of encapsulated P-gal  33  Despite the fact that glycerol was removed from the p-gal solution, the protein activity throughout the formulation process remained stable with no change i n the protein specific activity detected (Figure 2.2). It is common practice to employ 100 n m diameter L U V for in vivo applications (Hope et al., 1986). However, the 200 n m P-gal L U V could not be further extruded through a 100 nm pore size, presumably the presence o f this large molecular weight protein inhibited the vesicle deformation required in order for the 200 nm L U V to extrude through the smaller pore size. The lipid free protein solution was found to pass through easily.  Figure 2.3 shows a gel filtration profile for a typical 200 nm L U V preparation. Separation o f encapsulated and unencapsulated protein is good, despite the fact that the Pgal protein has a molecular weight o f - 500,000 D a . Vesicles that elute i n fractions 12 14 were collected for latency analysis. The latency o f both P O P C / C h o l and P O P C / C h o l / D S P E - P E G P-gal L U V (Figure 2.4a and b respectively) were routinely measured at > 95%. The external activity is most likely due to small amounts o f contamination from the free protein peak. This is supported by the observation that passing the vesicles down a second column produced a preparation with a latency > 99%, with the detection o f unencapsulated protein at the limits o f the assay procedure (Figure 2.4c). In addition, when the liposomal P-gal was briefly subjected to low p H treatment (pH 4.0) with H S 0 , vesicles with > 99 % latency were achieved with no loss i n the 2  4  entrapped protein activity (Figure 2.4d). The encapsulation efficiency (what proportion o f the initial protein has been encapsulated) was ~ 18-20% for both formulations, which  34  is consistent with a vesicle trapped volume o f approximately 2 pl/umole of total lipid, expected for the size o f vesicles used here.  Figure2.2  Comparison of protein specific activity before and after extrusion.  1.4 1.2 =  1.3  1.2  1  o Q-  u>  -3-  °  0.8 0.6  (A  <  0.4 0.2 -\ 0 After extrusion  Before extrusion  Protein activity was assayed in the presence of TX-100, and expressed as protein activty (Abs 420)  per ug of protein.  Based on calculations which assume the L U V formulations consist of uniform spheres with a bilayer thickness o f 5 nm and a lipid surface area o f 0.6 nm /molecule 2  [Deamer and Bramhall, 1986], it is estimated that there are 341,540 lipid molecules per lipid vesicle o f 200 n m diameter [Harrigan, 1992]. Therefore, for a P O P C : C h o l (55:45) preparation, there are approximately 1.63 x 10 vesicles per mg o f total lipid (see table 12  2.1). A typical formulation has 10 pg o f P-gal encapsulated inside the liposomes per mg of total lipid (figure 2.4a for example), this corresponds to encapsulating 8 P-gal tetramers per lipid vesicle. To put this number i n perspective, the internal volume o f a 200 nm vesicle is 3.58 x 10" m , and each P-gal tetramer is roughly 17.5 x 13.5 x 9.0 nm 21  3  35  in size and occupies a volume o f 2.12 x 10~ m (section 1.4). Consequently, the 24  3  maximum number o f P-gal molecules that could be packed inside a 200 nm L U V is -1700.  F i g u r e 2.3  Separation o f liposomal P-gal from free protein  Fraction #  Typical elution profile of liposomal P-gal (POPC:Chol + P-gal) expressed as DPM (lipid carrier) and Abs 420 (P-gal activity) assayed as lOul per fraction. Similar elution profile was obtained for the PEG-coated liposomal P-gal vesicles.  36  F i g u r e 2.4  Latency measurement  Figure 2.4b  Figure 2.4a.  12  14  10.87  12.62 10  10 J sncy  tency  -  -  o>  CM  6  in  to -  2  A  0.57  I  "  0.23  I  w i t h o u t TX-100  with TX-100  w i t h o u t TX-100  Figure 2.4c.  w i t h TX-100  Figure 2.4d.  14  X 12.94  X [T] Without TX-100  10  [Without TX-100  11  • With TX100  • With TX100 6-1  After 1 p  After 2 passages  Before acid  After acid  treatment  treatment  Latency measurement of the (a) 200 nm POPC:Chol (55:45) liposomal P-gal, and (b) POPC:Chol:DSPEPEG (55:40:5) liposomal P-gal. (c) Attempts to isolate vesicles with > 99 % latency was achieved by passing the PEG-coated liposomal P-gal through the sizing column twice, as well as by (d) exposing the pooled PEG-coated liposomal P-gal to pH 4.0 for two minutes. (n=3, ±SD).  37  Calculation of the number of P-gal molecules encapsulated inside POPCrChol (55:45) liposomes.  Table 2.1  200 341540 1.76 x 10  Vesicle diameter (nm) Total lipid molecules per vesicle Vesicles per Vesicles per Vesicles per pg P-gal per  u m o l phospholipid mg phospholipid m g total lipid mg total lipid  Outside o f liposome Encapsulated inside liposome Outside o f liposome  2.31 x 10 1.63 x 10 0.5 10 0.4  Encapsulated inside liposome  8  1  2  3  Number o f P-gal molecule per lipid vesicle  12  12  12  4  M W of phospholipid = 760 g*mor' M W o f P O P C : C h o l (55:45) liposome = 592 g.mol" See figure 2.4a M W o f p-gal = 470,472 g.mol"  1  1  2.3.3  In vitro stability of P-gal and liposomal P-gal in buffer and plasma  Prior to conducting the planned extensive studies in vivo it was important to validate the P-gal assay i n the presence o f serum proteins and measure the stability o f both free and encapsulated protein in mouse plasma at 37 °C. F r o m the encapsulation . characteristics described above, it is possible to generate L U V that contain approximately 10 ug o f P-gal per mg o f lipid. After a single pass through a gel filtration column the total lipid concentration becomes diluted to approximately 20 m g « m f ' . The maximum injectable dose (bolus) v i a the tail vein o f a typical 20 g mouse is on the order o f 200 p i , therefore the maximum amount o f L U V that could be administered for the in vivo studies would be 4 mg o f total lipid, equivalent to 40 pg o f P-gal enzyme. A 20 g mouse contains 2 m l of blood that yields about 1 m l of plasma. In other words, immediately after i.v. administration o f a 4 mg dose o f lipid, and before any clearance from the  38  circulation, a 100 u l aliquot o f plasma would be expected to contain ~ 400 ug o f lipid. In order to adequately follow the kinetics o f L U V and protein clearance from the blood it is desirable to be able to detect a circulating lipid dose that is greater than or equal to ~ 5% o f the initial dose (i.e. 20 ug o f lipid in a 100 p i aliquot o f serum equivalent to 0.2 ug o f P-gal protein). The graphs shown i n figure 2.5 a and b show that there is excellent linearity i n the presence o f serum over the required range and that the level o f sensitivity is such that the limit o f detection is ~ 5 % o f the initial dose.  Detection o f P-gal was dependent on the addition o f T X - 1 0 0 , indicating that the P-gal activity being measured was encapsulated i n liposomes and not accessible to the substrate even in the presence o f plasma. A problem with assaying i n plasma is the background scattering from lipoproteins when detergent is not present. Consequently, all samples, including blanks, were run i n the presence o f detergent and the reaction controlled by the presence or absence o f the P N P G substrate (Fig. 2.5b). Samples from experiments with liposomal protein administered i.v. (chapter 3) were therefore assayed in the presence o f T X - 1 0 0 and i n the presence or absence o f P N P G . Absorbance measured in the absence o f P N P G was taken as background scattering and subtracted from the absorbance i n the presence o f P N P G to obtain the true absorbance value arising from P-gal activity.  39  F i g u r e 2.5  P N P G assay validation Figure 2.5a. 2  0  -1  ,  ,  ,  ,  ,  1  0  20  40  60  80  100  120  ng lipid added  Figure 2.5b. 3  -i  •  •  with P N P G  m  without P N P G Linear (with P N P G ) Linear (without P N P G )  0  50  100  150  ug lipid added  Samples were analyzed in (a) the presence or absence of 1% Trition X-100, and (b) the presence or absence of the substrate PNPG with Trition X-100.  40  Specific activity for free protein, liposomal P-gal and PEG-coated liposomal P-gal incubated i n buffer and fresh plasma at 37°C are shown in figure 2.6. Free protein loses 60% o f its activity over 8 hours when incubated in a 50 % dilution of mouse plasma. However, only a 20 % reduction in activity of the encapsulated P-gal is observed over the same period. Since both the free protein and the encapsulated P-gal exhibit the same 20% reduction in activity over 8 hours when incubated in buffer, it is likely that this loss is due to thermal destabilization o f the protein at 37°C. It is not known why the loss o f activity for free enzyme is greater in plasma than i n buffer but it may be the result of protease degradation.  Figure 2.6  In vitro stability of P-gal, liposomal P-gal, and PEG-coated liposomal P-gal.  "b-gal in buffer 'b-gal in plasma "liposomal b-gal in buffer "liposomal b-gal in plasma "PEG-coated liposomal fagal in plasma  2  4  6  8  10  Time (hours)  Samples were incubated in buffer or in 5 0 % dilution of fresh mouse plasma at 37°C. At the indicated times, p-gal activity was determined by P N P G assay as outlined in section 2.2.3. Data represent the average from two different experments.  41  2.4  Summary  Two p-gal formulations, composed o f P O P C : C h o l and P O P C : C h o l : D S P E - P E G 2000, were prepared and characterized. Protein was trapped by hydrating lipid films in a protein solution at 37°C to form M L V . M L V were then sized by extrusion through polycarbonate filters with a pore size o f 200 nm to form a homogeneous population o f L U V with diameters o f 200 ± 40 nm. Unencapsulated P-gal was removed by gel filtration. A n assay was developed to measure enzyme activity i n the presence o f detergent and mouse serum. It exhibits a linear response over the concentration range required to conduct the in vivo experiments. Furthermore, the assay is sensitive enough to detect as little as 5% o f the projected i.v. dose which was set as one o f the criteria to be achieved before proceeding into animals.  Both formulations can be made routinely with > 95% latency after a single pass through a gel filtration column. External enzyme activity appears to arise from unencapsulated protein, which is either free i n solution, or weakly associated with the vesicle surface. This external activity can be removed by a second gel filtration step or eliminated by briefly exposing the vesicles to low p H , which inactivates P-gal, without any loss o f encapsulated activity.  Finally, both formulations appear to be quite stable i n serum at 37°C over eight hours. Approximately 20-30% o f the encapsulated proteins specific activity is lost during the incubation which is most likely due to the temperature, because a similar loss is  42  measured for the protein suspended i n buffer alone. The free protein i n serum loses 60% o f its activity over the same incubation period. The data indicate that both formulations can be made reproducibly and their properties are suitable for the in vivo phase o f this project.  4 3  CHAPTER 3  P h a r m a c o k i n e t i c s o f L i p o s o m a l ( 3 - G a l a c t o s i d a s e i n N o r m a l a n d I m m u n i z e d M i c e 3.1  Introduction The rationale for encapsulating therapeutic proteins i n lipid carriers is to enhance  their therapeutic index. This is accomplished by (1) and biodistribution to sites o f disease, (2)  increasing their circulation lifetime  reducing their toxicity by gradual exposure o f  biological activity, in contrast to a bolus injection o f unencapsulated protein and (3) minimizing any loss of biological potency by protecting the protein from denaturation i n blood and tissue. However, it is not known whether these positive attributes o f encapsulation are outweighed by the enhanced immune response expected due to the increase i n delivery o f therapeutic protein to antigen presenting cells such as macrophages and dendritic cells.  This chapter addresses the in vivo pharmacokinetic characteristics o f free protein, empty vesicles and vesicles containing P-gal i n normal mice, i n mice that have been preimmunized against P-gal, as well as i n normal mice receiving repeated injections.  44  3.2  3.2.1  Materials and methods  Materials  Unless otherwise specified, all lipids and chemicals used i n this chapter were the same as outlined i n section 2.2.1.  3.2.2  Measurement of anti P-gal antibodies in mouse serum  Antibody against P-gal i n plasma was determined using an indirect, two-step E L I S A . The following reagents: SuperBlock™ Blocking buffer i n P B S , Tween 20, horseradish peroxidase conjugated I m m u n o P u r e ® G o a t anti-mouse I g G (F ), and 1-Step™ c  Turbo T M B - E L I S A solution were purchased from Pierce (Rockford, II). A l l other reagents were o f analytical grade. Plasma samples from individual mice were assayed in triplicate as follows. Briefly, E L I S A plates (Becton Dickinson Labware, L i n c o l n Park, NJ) were coated overnight at 4°C with 40 pg^ml" o f P-gal i n carbonate-bicarbonate 1  buffer (0.015 M N a C 0 , 0.035 M N a F £ C 0 , 0.003 M N a N , p H 9.6). Plates were washed 2  3  3  3  three times with 200 p i PBS-Tween 20 containing 0.1% (w/w) B S A , followed by blocking with 200 p i SuperBlock™ blocking buffer. Mouse plasma to be assayed was diluted from 10 to 78,000 x i n SuperBlock™ Blocking Buffer with 0.05% (v/v) Tween 20 and applied to the plates as the primary A b . Samples were incubated at room temperature for 1 hour, after which a 100 p i aliquot o f the horseradish peroxidase conjugated goat anti-mouse I g G (diluted 1:1000) was used. After incubation for 2 hours at room  45  temperature, plates were washed and 125 pi of 1-Step™ Turbo T M B substrate was added for 15 minutes. The reaction was stopped by the addition of 125 pi of 1 M H S 0 . Plates 2  4  were read in a Dynatech 5000 ELISA plate reader at 450 nm. Controls consisted of plasma samples obtained from untreated mice.  F i g u r e 3.1  Measurement of anti P-gal I g G by indirect two-step E L I S A . P r i m a r y antibody reacts with b o u n d antigen and a labeled secondary antibody reacts with the p r i m a r y antibody. substrate Reaction stopped by addition of 1M H SO . colored precipitate  HRP-labelled goat antimouse IgG  mouse plasma  P-gal protein  46  3.2.3  Immunization of mice against P-galactosidase  A variation o f the method described by Tardi et al. [1997] was employed to immunize mice against P-gal protein. These authors showed that PEG-coated liposomes acted as potent adjuvants i n generating a humoral response against ovalbumin when mixed with the protein and administered intraperitoneally to mice. In addition, Tardi et al. (personal communication) demonstrated that repeat administration o f liposomal ovalbumin subcutaneously was even more effective at eliciting an antibody response. Therefore, the PEG-coated vesicle formulation o f P-gal described i n chapter 2 was used as the vaccination material and was administered subcutaneously. A 100 p i aliquot i n H B S containing 0.96 m g o f total lipid ( P O P C : C h o l : D S P E - P E G 2000, 55:40:5 mole ratio) and 11.7 p g o f encapsulated P-gal was injected under the skin i n the abdominal area o f B D F - 1 mice. Injections were given at intervals o f 9 day for a total o f 3 injections.  To investigate the therapeutic effect upon repeated administration o f identical formulations, two groups o f mice were established to compare P E G free and PEG-coated preparations with P-gal encapsulated. Identical dosage was used for each i.v. injection. A t one hour following administration, the blood was analyzed to determine the percentage o f initial dose remains in the circulation, as well as for monitoring the generation o f P-gal antibodies. This was repeated for a total o f five weekly injections.  47  3.2.4  Pharmacokinetic studies  The pharmacokinetic studies were carried out in female B D F - 1 mice (Harlan Sprague Dawley, Prattville, A l ) . M i c e were 8 weeks old at the beginning o f the experiment and weighed approximately 20 g. A total lipid dose o f 60 mg»Kg"' body weight was administered for this series o f experiments. The liposomal formulations were diluted i n H B S , such that the required dose could be administered i n approximately a 200 p i injection volume via the tail vein. The P-gal content o f the liposomal preparation was determined using the latency assay described, so that the same dose o f free P-gal could be administered to the experimental group o f mice receiving protein only. A t various times following i.v. administration o f the samples a group o f 4 mice per time point were given an i.p. injection o f a ketamine/xylazine cocktail (160/4 mg*Kg ) for general anesthesia. _1  B l o o d was collected by cardiac puncture and transferred to Microtainer® tubes containing E D T A as anticoagulant. The blood was centrifuged at 500 g for 10 minutes and aliquots of the plasma were removed for scintillation counting to determine lipid concentrations and P N P G assay for P-gal activity.  3.3  3.3.1  Results  Pharmacokinetics of free and encapsulated P-galactosidase in normal mice  In normal (non-immunized) mice free P-gal, tracked by measuring enzyme activity in the serum, is cleared from the circulation at approximately the same rate as Pgal encapsulated i n PEG-free vesicles (Figure 3.2a). In contrast, the effect o f P E G -  48  coating on opsonization and clearance o f P-gal containing vesicles is striking. A t 3 hours 70 % o f the injected dose o f P-gal is still i n the blood for the PEG-coated formulation compared to < 10 % for both the naked vesicle formulation and free protein. L i p i d clearance profiles are shown in Figure 3.2b, and are almost identical to the protein -activity curves (Figure 3.2a). In other words, the specific activity o f encapsulated P-gal per mole o f lipid does not change in the blood during the 3 hour time course for either formulation. Furthermore, empty P O P C : C h o l vesicles exhibit the same clearance profile as P O P C : C h o l vesicles loaded with P-gal protein, indicating that the two preparations are treated similarly by the bodies particle clearance mechanisms. The rate o f vesicle clearance is consistent with the lipid dose and vesicle diameter.  49  Figure 3.2  Pharmacokinetics of free and encapsulated p-galactosidase in normal mice. Figure 3.2a. 120  T  0  1  2  3  4  Time post i.v. administration (hours)  Figure 3.2b. 120 -|  0  1  2  3  4  Time post i.v. administration (hours)  In vivo clearance kinetics of (a) the encapsulated protein, and (b) the lipid carrier in normal mice. Blood was collected at various time points and the amount of (3-gal and lipid carrier remaining in the circulation was determined by PNPG assay, and by LSC, respectively. The result is plotted as an average obtained from four animals. (n=4, ± S.D.)  50  Figure 3.3  Generation o f anti P-gal IgG upon repeat s.c. injection.  o  <  Day 9  D a y 18  D a y 27  The level of anti p-gal IgG induced after three s.c. inoculations with the PEG-coated liposomal p-gal was monitored by ELISA as outlined in section 3.2.2. Measurement was taken from 0.025 dilution of mouse plasma. (n=4, ±SD)  3.3.2  Pharmacokinetics of free and encapsulated P-galactosidase in immunized mice  In the previous section it was shown that i n normal mice there is not a large difference between the clearance o f free P-gal and P-gal encapsulated i n P O P C : C h o l vesicles. However, in mice carrying antibodies against P-gal the free protein is expected to immediately form antibody-antigen complexes that are rapidly cleared from the circulation, whereas encapsulated P-gal w i l l be protected and therefore the circulation time should be the same as that measured in normal animals.  1  51  To test this hypothesis mice were immunized against P-gal protein as described i n section 3.2.3. The generation o f anti P-gal antibodies was monitored by E L I S A (Figure 3.3), and after the third subcutaneous injection (day 27) a strong humoral response could be detected. A s expected, the effect o f immunization on clearance o f free protein from the circulation is pronounced (Figure 3.4). A t the earliest time point that can be measured (10 minutes) only 10% o f the injected dose o f free P-gal is detected i n the blood o f the immunized group o f mice. This level drops to the background limits o f the P N P G assay (< 5% o f the injected dose) for the remainder o f the 3 hour time course. However, when the same dose o f protein is administered encapsulated in P O P C : C h o l vesicles there is no apparent effect on vesicle or protein clearance rates as the kinetics are almost identical to those observed i n normal animals (Figure 3.5a and b). The same behaviour is not observed for PEG-coated, P-gal containing vesicles. Surprisingly, this formulation is cleared as rapidly as free protein when administered at the same dose to immunized animals (Figure 3.6a and b). For this experiment P-gal levels i n the blood were not measured as early as 10 minutes, but at 30 minutes both P-gal activity and lipid concentration were < 10 % o f the injected dose compared to 90 % i n the non-immunized group.  52  Figure 3.4  A comparison of the circulation life-time of free P-gal in immunized and non-immunized mice.  120  100*  ~*"~ irrmriaad ncrHrrrruiaad  Q5  1  1.5  2  25  35  lime post i.v. arJiiristnaticn (hcurs)  Mice were immunized by repeat s.c. administration with PEG-coated liposomal (3-gal. Plasma was collected, and P-gal activity was determined by PNPG assay. (n=4, ±SD).  53  Clearance kinetics o f liposomal P-gal.  F i g u r e 3.5  Figure 3.5a. 120  -i  100 •  0 -I 0  , 1  ,  ,  2  1 4  3  Time post i.v. administration (hours)  Figure 3.5b. 120 -,  -•— immunized -•— non-immunized  0.5  1  1.5  2  2.5  3  3.5  Time post i.v. administration (hours)  Comparison of the clearance kinetics of liposomal p-gal (POPC:Chol + P-gal) in immunized and nonimmunized mice. The clearance kinetic was monitored by following the rate of clearance of (a) the lipid carrier, and (b) the protein activity. (n=4, ±SD).  54  Clearance kinetics o f PEG-coated liposomal P-gal.  F i g u r e 3.6  Figure 3.6a. 120  -•—immunized -•—non-immunized  1  2  3  Time post i.v. administration (hours)  Figure 3.6b.  120  —  100  in  o  "D  •o O +.  •free  80  protein  u  a  t;  60 • e n c a p s u l a t e d in PEG 40  coated  liposomes  1  20  0  1  2  3  Time post i.v. administration (hours)  (a) Comparison of the clearance kinetics of PEG-coated liposomal P-gal (POPC:Chol:DSPE-PEG + P-gal) in immunized and non-immunized mice was monitored by following the serum concentration of the lipid carrier, (b) In immunized mice, both free protein and protein encapsulated in the PEG-coated liposomes exhibit similar rapid clearance profiles (n=4, ±SD).  55  This result indicates that the PEG-coated vesicles are recognized by a component o f the immune system i n the immunized animals whereas the naked P O P C : C h o l vesicles are not. The physical characterizations indicate that both formulations are similar with respect to size, protein encapsulation and latency (Figure 2.4). One important point to note is that for the purposes o f this study mice were immunized using (3-gal encapsulated in PEG-coated vesicles. Therefore it is possible that antibodies may also have been raised against the PEG-coating on the vesicle surface. This is unlikely as P E G has been widely used to mask the immunogenicity o f proteins and is considered non-immunogenic (Ueno et al., 1996; recent review by Duncan and Spreafico, 1994). However, in order to test whether this was the case, two groups o f mice were "immunized" with either empty P O P C : C h o l or PEG-coated P O P C : C h o l vesicles, following the same procedure as outlined in section 3.2.4. for immunization against (3-gal. Vesicle clearance was then measured and compared to non-immunized animals. The data show no effect from immunization with lipid only on the clearance kinetics o f either PEG-coated P O P C : C h o l or P O P C : C h o l vesicles (Figure 3.7).  56  Figure 3.7  Immunization with protein free vesicles has no effect on the pharmacokinetics of vesicle clearance.  120  - • - P O P C i C h o l (normal)  - • -  POPC:Chol:DSPE-PEG 2000 (normal)  - P O P C : C h o l (immunized)  - * -  POPC:Chol:DSPE-PEG 2000 (immunized)  0 0  2  3  5  4  Time post i.v. administration (hours) Mice were immunized by three repeat s.c. injection with either empty POPC:Chol or P E G coated POPC:Chol vesicles given at 9 day intervals as oulined in section 3.2.3. Following immunization the kinetics of vesicle clearance was measured and compared to a normal, non-immunized experimental group. Plasma was m l l p r t f v l  3.3.3  a t tV»p. i n H i r a t p H t i m p c  anA  l i n i H r n n r p n t r f l t i n n c  H ^ t e r m i r w l  K \ /T S P  1  (n=A  -k^TYl  Pharmacokinetics and humoral immune response to encapsulated (3galactosidase following repeated weekly administration  The experiments described above were conducted i n mice pre-immunized against (3-gal and show that both free protein and protein encapsulated i n PEG-coated liposomes are sensitive to the presence o f antibodies which cause them to be cleared rapidly from the circulation compared to their circulation T  1 / 2  in normal mice. Interestingly, naked  P O P C : C h o l vesicles protect P-gal, and the clearance kinetics for protein i n this formulation are the same i n both normal and immunized mice. In the experiment  57  described below, the effect o f repeated injection on vesicle and P-gal clearance is monitored.  Two experimental groups were established to compare PEG-free and PEG-coated preparations, each containing enough mice to allow the analysis o f 5 animals per time point. A l l the animals received the same dose o f encapsulated P-gal. One hour following the first administration five mice from each group were sacrificed and the blood analyzed for radioactivity to determine the % o f initial dose still i n the circulation and a sample was also analyzed for the presence o f anti P-gal antibodies. This was repeated for a total of five weekly injections. After the first injection the level o f lipid in the blood was within the normal range previously measured for the two formulations. Approximately 50% o f the injected dose for P O P C : C h o l p-gal and 90% for the PEG-coated vesicles (Figure 3.8). However, after the second i.v. administration there was a dramatic drop in the circulating concentration o f PEG-coated vesicles at one hour, from 90% to 20% o f the injected dose. In contrast, the % injected dose remaining for the uncoated, P O P C : C h o l Pgal formulation was unchanged at 50%. The effect o f the immune response on P E G coated vesicle clearance does not change significantly over the next three weekly injections. However, the % dose remaining at one hour for the uncoated formulation gradually decreases between weeks two and three, so that at week five both preparations appear to be cleared at similar rates with only - 2 0 % o f the initial dose i n the blood (Figure 3.8).  58  Interestingly there does not appear to be a direct correlation between the extent of an IgG humoral response to the repeat injections and the rate of particle clearance from the blood. The most dramatic change in clearance occurs for the second administration of PEG-coated vesicles when the antibody response can only just be detected. Moreover, the antibody response to both vesicle formulations of P-gal is identical but the effect on vesicle clearance, particularly the second injection, is very different.  Figure 3.8  Correlation between liposomal P-gal clearance kinetics and the generation of anti P-gal IgG following weekly i.v. administration.  Time (weeks)  A l l measurements were made at 1 hour post administration. T h e level o f A b was determined b y E L I S A ( A b s 450) as discussed in section 3.2.2, and the clearance kinetic was assessed b y determining lipid concentration b y L S C  and expressing the data as % o f injected dose. (n=5,  ±SD).  59  3.3.4  Phenotypic response of mice to P-galactosidase formulations  The work described in this thesis is designed to test the hypothesis that immunogenic proteins can be repeatedly administered in an encapsulated form without causing acute toxicity resulting from the formation o f antibody-antigen complexes i n plasma. The experiments described so far have quantified both the I g G humoral response and clearance o f protein and carrier from the circulation. However, i f an animal suffers an acute immune reaction to material being injected this can usually be observed through the animals' behaviour. In this section the gross phenotypical responses o f the mice during, and for the first hour following, each administration are summarized.  Table 3.1  Phenotypic response following bolus i.v. administration to mice preimmunized against P-gal (section 3.2.2).  Free P-gal  POPC/Chol P-gal  PEG-coated POPC/Chol P-gal  # React/Total # Animals Severity o f Reaction Pre-immunized:  0/16 0  0/16 0  0/16 0  # React/Total # Animals Severity o f Reaction  3/16 1  2/16 2  6/12 3  Experimental Group Non-immunized:  Scale of severity: 0 No observable effects from injection. 1 Animals motionless but recovery within minutes. 2 Some convulsions immediately upon injection and recovery takes several minutes. 3 Severe response resulting in convulsions, lack of balance, respiratory difficulty and recovery 0.5-2 hours.  60  Table 3.2  Phenotypic response following five weekly bolus i.v. administrations to n o r m a l mice.  T i m e of Injection (weeks)  Free P-gal  POPC/Chol P-gal  PEG-coated POPC/Chol  3-gal Weekl  # React/Total # Animals Severity of Reaction  0/43 0  0/40 0  0/43 0  3/33 2  3/33 2  27/33 3  1/29 1  1/29 2  10/28 2  1/23 1  0/23 0  0/21 0  2/18 1  1/18 1  0/16 0  W e e k 2:  # React/Total # Animals Severity of Reaction W e e k 3:  # React/Total # Animals Severity of Reaction W e e k 4:  # React/Total # Animals Severity of Reaction W e e k 5:  # React/Total # Animals Severity of Reaction  Scale of severity: 0 No observable effects from injection. 1 Animals motionless but recovery within minutes. 2 Some convulsions immediately upon injection and recovery takes several minutes. 3 Severe response resulting in convulsions, lack of balance, respiratory difficulty and recovery 0.5-2 hours. :  It is interesting to note (table 3.1) that 2 out of 16 animals in the pre-immunized group receiving P-gal encapsulated in POPC:Chol exhibited responses upon injection despite the fact that there was no effect seen upon the clearance kinetics of vesicles from the plasma. This is in contrast to the free protein, which produced a similar phenotypic reaction (3 out 16 mice at grade 1) but the rate of protein clearance also increased  61  markedly. The rate o f clearance was also enhanced for the PEG-coated vesicles, but i n this group 50% o f the animals experienced a severe reaction to the administration.  In the multiple injection experiment (table 3.2) there was very little response seen for the free protein or P O P C : C h o l groups. Whereas, the second and third injections given to the group receiving PEG-coated vesicles induced a severe immune reaction. But by the 4th and 5th week the animals appeared to tolerate the injection even though the vesicles were still cleared rapidly from the circulation.  3.4  Summary  In this chapter the key objectives o f the thesis were completed. It was demonstrated that encapsulating an antigenic protein inside a lipid vesicle can protect the protein from humoral antibodies but that this protection may be limited and vesicle dependent. The blood clearance profiles o f two P-gal formulations, P O P C : C h o l and PEG-coated P O P C : C h o l , were measured in normal and immunized mice. In normal mice the two vesicle preparations behaved as expected. The 200 nm L U V vehicle was cleared from the circulation with a half-life o f approximately 45 minutes, which was similar to that measured for free protein at the same protein dose. The presence o f a P E G coat slowed clearance o f the lipid carrier and its protein payload substantially, increasing the circulation half-life more than 5-fold. However, in the presence o f P-gal antibodies P E G coated vesicles were rapidly cleared from the blood with kinetics similar to the free protein which exhibited a half-life < 10 minutes. This contrasts to p-gal encapsulated in P O P C : C h o l vesicles, for which both lipid and protein circulation times remain unchanged  62  from that measured i n normal mice. Furthermore, the PEG-coated formulation was more toxic upon injection into immunized animals than either free protein or protein encapsulated i n PEG-free vesicles.  Paradoxically, PEG-coated vesicles were included i n this study because it was assumed they would represent the most effective vehicle for systemic delivery o f antigenic proteins. The effect observed in immunized mice, however, was also seen in normal mice receiving weekly injections. B y only the second injection the amount o f the administered dose remaining i n the blood at one hour dropped from 90% to 20% for PEG-coated vesicles, but remained unchanged for PEG-free vesicles. B y the 5th injection both formulations were cleared at similar rates. Moreover, the PEG-coated formulation o f P-gal was toxic to more than 80% o f the animals following the 2nd injection. These data indicate that the effect o f an immune response against an antigenic protein encapsulated i n a lipid carrier is complex and may limit the application o f particulate carrier systems for antigenic drugs.  63  CHAPTER 4  DISCUSSION  4.1 Enzyme replacement and protein therapeutics Many diseases are caused by genetic defects i n which a specific protein, such as an enzyme, is either missing or inactive. Advances i n sequencing the human genome mean that many o f the genes responsible for producing these proteins have been or w i l l shortly be identified. This knowledge, combined with the ability to produce pharmaceutical grade proteins on a large scale from genetically engineered microorganisms or cultured animal cells, means that protein therapy to manage these diseases is possible. Enzyme replacement strategies are an illustration o f how large, biologically active proteins can be useful in controlling a disease. One example is i n severe combined immune deficiency (SCID) a disorder caused by a missing or defective enzyme in the metabolic pathway responsible for the catabolism o f purines. The enzyme is adenosine deaminase ( A D A ) , and ADA-deficient SCID arises because the accumulation o f a toxic metabolite (deoxyadenosine monophosphate and deoxyadenosine triphosphate) i n T-cells disables the immune system [Blaese et al., 1995]. Lipid storage disorders are also caused by single enzyme deletions. Gaucher's disease is the most prevalent and is caused by an excessive accumulation o f glucocerebroside in organs and tissues. Patients lack the enzyme glucocerebrosidase, which normally catalyzes the  64  cleavage o f glucose from glucocerebroside. In the absence o f this enzyme, glucocerebroside accumulates in macrophages, especially those located in liver and bone marrow. These cells are responsible for the turnover and reutilization o f membrane lipids from senescent red blood cells, which are rich in glycolipids [Brady & Barton, 1996; Grabowski et al., 1995].  Both diseases are currently managed quite effectively by regular, systemic administration o f purified enzymes. In the case o f ADA-deficient S C I D , the therapeutic enzyme is covalently linked to P E G , which slows opsonization and enhances the circulation lifetime o f the protein [recent review by Burnham, 1994] by the same steric hindrance mechanism described earlier for liposomes. In Gaucher's disease the rationale is to target glucocerebrosidase to macrophages rather than develop a drug which exhibits extended circulation characteristics. Therefore, the enzyme's carbohydrate groups are modified by sequential treatment with exoglycosidases to expose mannose groups. Mannose is recognized by a lectin associated with the surface o f macrophages. In both o f these examples the enzyme drags are the only therapy available for patients suffering from a fatal disease, they do not represent a cure but a means o f managing their disorder. Consequently, the immune side effects resulting from long-term applications are tolerated. The most common toxicity results when patients develop antibodies against the therapeutic protein. In addition to the increased rate o f clearance and resulting decrease in the drug's effectiveness, antigen-antibody immune complexes can cause kidney, joint and blood vessel damage over time.  65  4.2  Formation of antigen-antibody complexes and vesicle clearance  Encapsulation o f enzymes i n lipid vesicles or liposomes represents a potential means o f administering long-term protein therapy whilst avoiding chronic toxicity associated with the formation o f antigen-antibody complexes once patients become seropositive to the drug. However, paradoxically, the lipid vehicle is known to behave as an adjuvant and enhance the immune response against encapsulated antigen [Shek et a l , 1986; A l v i n g , 1995]. The purpose o f this study was to determine whether this presented a practical problem with respect to delivering biologically active protein to a target site or not.  The assumption is that any increase in the rate o f clearance o f vesicles or protein from the circulation results from the formation o f immune complexes with circulating antibodies, which increase particle size by multivalent cross-linking, enhance macrophage uptake through F c receptors and accelerate opsinization through complement activation. The data indicate that lipid encapsulation prevents the formation o f P-gal antigen-antibody immune complexes i n pre-immunized animals, as the rate o f clearance for encapsulated p-gal is almost identical to that measured i n normal mice. It is reasonable to assume that the lipid membrane represents an impermeable barrier to the antibodies. This is i n contrast to free protein, the majority of which is cleared immediately. Surprisingly, the rate o f clearance o f PEG-coated p-gal vesicles also increases dramatically. One explanation could be that there is sufficient P-gal adhered to the surface of PEG-coated vesicles to form immune complexes i n the serum. However, there is little evidence to support this. The small amount o f unencapsulated protein that  66  co-elutes with the vesicle fraction from the gel filtration column does not appear to be bound to the vesicle surface as it is readily removed by additional filtration steps. Moreover, the same amount o f unencapsulated "contaminating" P-gal is administered with both types o f vesicle, uncoated and PEG-coated, yet only the clearance o f P E G coated vesicles is affected. Furthermore, the P E G coat would be expected to decrease protein binding to the vesicle surface, which is why it is included i n the composition. It is interesting to note that the PEG-coated formulation was used to immunize the mice. Perhaps a humoral immune response was raised against the P E G as well as the encapsulated P-gal. However, the data shown i n figure 3.7 demonstrate that empty vesicles (lipid only, no P-gal) are not strongly immunogenic. Perhaps the presence o f protein acts as an adjuvant, enhancing the humoral response to a degree that anti-PEG antibodies are generated (see section 4.4 for further discussion).  4.3  Repeated administration and vesicle clearance  A critical aspect o f enzyme replacement therapy is that treatment manages the disease but does not cure it. Therefore, protein drug must be administered regularly throughout the lifetime o f the patient. Repeat administration o f P-gal i n uncoated and PEG-coated vesicles revealed that eventually a serum reaction against both drug delivery vehicles was elicited. The data, however, support the first observation i n the preimmunized animals, that PEG-coated vesicles are either more immunogenic or more sensitive to the mounting immune response. Despite the fact that by the second administration there is barely sufficient anti-P-gal antibody to detect (figure 3.8), there is a dramatic serum reaction against PEG-coated vesicles, which causes their rapid  67  clearance from the circulation and severely shocks the animals upon injection. Uncoated vesicles, on the other hand, raise the same level o f p-gal antibody but a serum effect is not detected, similar to what is seen i n pre-immunized animals. But there is a progressive increase i n the recognition o f the uncoated lipid system, so that a gradual increase i n vesicle clearance occurs over the course o f treatment.  4.4  On the difference between uncoated and PEG-coated vesicles  W h y are PEG-coated, p-gal containing vesicles apparently more sensitive to immune complex formation than the same formulation without P E G ? Similar P E G effects have been reported by Phillips et al. [Phillips et al., 1994; Phillips et al., 1996; Phillips & Dahman, 1995]. The difference between Phillips' data and that reported i n this thesis is that he used immunoliposomes, where antibodies are attached to the outside of the lipid carrier to target encapsulated drug to specific cells. Such systems are immunogenic, particularly i f the vesicle associated I g G is from a different species from that o f the host, however, the adjuvant activity o f the liposome is sufficiently potent to generate antibodies against autologous IgG as well [Phillips et al., 1994]. P E G coating has been used in an attempt to limit the immune response against immunoliposomes [Phillips et a l , 1994; Phillips et a l , 1996; Phillips & Dahman, 1995]. However, the presence o f this polymer has been shown to not only give rise to a powerful humoral immune response to protein antigens associated with the carrier but also to enhance the generation o f antibodies to the liposomal phospholipids and the linkers used to attach ligands to their phospholipid anchors [Phillips et al., 1994]. These data suggest that PEG-coated vesicles are better adjuvants than naked vesicles when they carry a protein  68  payload. This may be because these vesicles are targeted to more powerful antigen presenting cells, such as dendritic cells, compared to P E G free vesicles. The latter are removed mostly by Kupfer cells in the liver and spleen based macrophages, whereas PEG-coated vesicles avoid the liver and can access distal tissue sites such as the skin [Gabizon et al., 1997] an organ rich in Langerhans cells, one o f the most potent antigen presenting cells known. Different intracellular processing o f the P-gal antigen delivered to antigen presenting cells may also be an important factor [Harding et al 1991; Rao et al., 1995].  The hypothesis that PEG-coated vesicles raise a humoral response against the surface polymer or polymer linker is readily testable. A group o f mice w i l l be immunized by weekly injection of PEG-coated P-gal vesicles then challenged with the same vesicles without P-gal. If "vesicle" antibodies are present the lipid should be cleared rapidly from the blood. I f the clearance o f empty vesicles is not affected then the observations reported i n this thesis are likely due to P-gal epitopes exposed at the vesicle surface. The same experiment w i l l be conducted using naked vesicles. In this instance, i f empty P E G free vesicles are cleared more quickly from the immunized animals it may indicate the presence o f anti-phospholipid antibodies.  Finally, the key objective o f this work was to test the hypothesis that: A  biologically active protein, encapsulated in a lipid based drug delivery system, can be administered intravenously and repeatedly to mice without loss of activity or reduced bioavailability due to the immune response induced against it. The data  69  indicate that bioavailability may be severely reduced following repeat injections due to an increase i n the rate o f clearance o f the carrier.  70  BIBLIOGRAPHY Arrondo, J. L . R., M u g a , A . , Castresana, J., Bernabeu, C , Goni, F. M . (1989) A n infrared spectroscopic study o f P-galactosidase structure in aqueous solutions. F E B S Lett 252, 118-120. A l l e n , T . M . (1988) Stealth liposomes: avoiding reticuloendothelial uptake, i n Liposomes in the Therapy o f Infectious Diseases and Cancer, 89, U C L A Symp. on Molecular and Cellular Biology, Lopez-Berestein, G . and Fidler, I., Eds., A l a n R . Liss, N e w Y o r k , 405. A l l e n , T . 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