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UBC Theses and Dissertations
Systemic administration of liposomal encapsulated [beta]- galactosidase: a model to investigate the development of therapeutic protein drug Mok, Wilson Wing Ki
Abstract
With advances in recombinant protein technology, a growing number of therapeutic protein products have become available for clinical applications. But their wide use is limited by poor biodistribution, limited circulation time and the generation of immune responses to the drug by the patient. The specific aim of the work described in this thesis is to characterize the pharmacokinetic behaviour of an immunogenic protein encapsulated in a liposomal delivery system. Liposomes have been demonstrated to increase the therapeutic index of drugs by ameliorating toxicity and enhancing biodistribution to sites of disease. The therapeutic index of a broad spectrum of 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 of 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 of P-gal antibodies and the pharmacokinetics of both protein and lipid vehicle were monitored following multiple, weekly injections. An 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. An 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 PEG 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 Chapter 3. Normal mice were subjected to five weekly injections of encapsulated P-gal in vesicles ± PEG. The results show that both preparations elicit P-gal antibodies at the same rate and to the same level during the course of administration. Despite this, the rate of clearance for (3-gal-containing, PEG-coated liposomes is increased dramatically by the second injection compared to the naked vesicles. After the full course of five weekly injections, both types of 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 PEG-coated vesicles are recognized more readily than naked vesicles. Possible reasons for the pharmacokinetic differences observed are discussed in Chapter 4. These include the physical characteristics of the vesicles and the exposure of protein epitopes at the surface of the vesicles, as well as the nature of the immune response and the possibility that antibodies are raised against the PEG anchor.
Item Metadata
Title |
Systemic administration of liposomal encapsulated [beta]- galactosidase: a model to investigate the development of therapeutic protein drug
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
1997
|
Description |
With advances in recombinant protein technology, a growing number of
therapeutic protein products have become available for clinical applications. But their
wide use is limited by poor biodistribution, limited circulation time and the generation of
immune responses to the drug by the patient. The specific aim of the work described in
this thesis is to characterize the pharmacokinetic behaviour of an immunogenic protein
encapsulated in a liposomal delivery system.
Liposomes have been demonstrated to increase the therapeutic index of drugs by
ameliorating toxicity and enhancing biodistribution to sites of disease. The therapeutic
index of a broad spectrum of 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 of 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 of P-gal antibodies
and the pharmacokinetics of both protein and lipid vehicle were monitored following
multiple, weekly injections. An 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. An
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 PEG 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 Chapter 3. Normal mice were subjected to five weekly injections of
encapsulated P-gal in vesicles ± PEG. The results show that both preparations elicit P-gal
antibodies at the same rate and to the same level during the course of administration.
Despite this, the rate of clearance for (3-gal-containing, PEG-coated liposomes is
increased dramatically by the second injection compared to the naked vesicles. After the
full course of five weekly injections, both types of 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 PEG-coated
vesicles are recognized more readily than naked vesicles. Possible reasons for the
pharmacokinetic differences observed are discussed in Chapter 4. These include the
physical characteristics of the vesicles and the exposure of protein epitopes at the surface
of the vesicles, as well as the nature of the immune response and the possibility that
antibodies are raised against the PEG anchor.
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Extent |
6905628 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2009-04-30
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0088519
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
1998-05
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Item Media
Item Citations and Data
Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.