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Long circulating liposomes: Past, present and future
Biotechnology Advances, Vol. 14, No. 2, pp. 151-175, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0734-9750/96 $32.00 + .00 ELSEVIER
PH S0734-9750(96)00007-9
LONG CIRCULATING LIPOSOMES: PAST, PRESENT AND FUTURE J I H A N M. J. M A R J A N and T H E R E S A M. A L L E N
Department of Pharmacology, University of Alberta, Edmonton, Alberta T6G 2H7
* To whom correspondence should be addressed: Department of Pharmacology, University of Alberta, Edmonton, AB T6G 2H7 Tel:
(403) 492-5710
Fax:
(403) 492-8078
email: [email protected]
151
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J.M.J. M A R J A N and T.M. ALLEN
Liposomes were first described by the British scientist Alec Bangham and colleagues in 1965 (1). Upon hydration, lipid films spontaneously formed closed concentric spherical structures that encapsulated part of the liquid medium in their interior, The primary constituent of a liposome is phospholipid, molecules consisting of hydrophillic "heads" attached to long non-polar hydrophobic "tails" (Figure 1A). The hydrophillic head typically consists of a phosphate group, hence phospholipid, whereas the hydrophobic tail is made of two long hydrocarbon chains.
This
amphipathic structure of phospholipids leads to their aggregation into ordered structures that sequester the hydrophobic tails away from contact with water (Figure 1A). Since their conception,
both industry and academia
have had high
expectations for practical applications of these small spherical structures.
These
expectations included their use as simple model membrane systems as well as a possible role as carriers for drugs. Liposomes were attractive as drug carriers as they were easily biodegradable and had the potential to be targeted to diseased cells....a 'magic bullet'! They quickly achieved their potential as model membranes for a variety of applications.
However, the route to the development of liposomal
drug carriers has been arduous, long and in many cases not fruitful.
Yet the
liposomes' appeal as targetable vehicles for site-selective delivery of therapeutics has not been lost.
This review will briefly outline the evolution of new liposomal
formulations, with long circulating times, from their conception, to their current approval for clinical use in humans, to future applications presently in the development stage.
LONG CIRCULATING LIPOSOMES
153
FIGURE 1. Schematic illustration of various liposome structures (not to scale). A. Classical liposome (CL) demonstrating how the hydrophillic "heads" and hydrophobic "tails" of the phospholipids aggregate into spherical sealed structures. CL attract plasma proteins (opsonins) to the liposome surface which contribute to the rapid removal of CL from circulation by the cells of the MPS. B. Long circulating liposomes (SL) with entrapped drug molecules, demonstrating how the hydrophillic polyethylene glycol (PEG) surface polymers repel plasma opsonins. C. Targeted long circulating liposomes where an antibody (or other ligand) is coupled to the terminus of the PEG polymer to form sterically stabilized immunoliposomes (SIL).
A, CLASSICAL LIPOSOME [CL]
Hydrophilllc
Entrapped in aqueous
y
drug interior
f Opsonins
~ Phospholipid
~. Cholesterol
B.LONG-CIRCULATING LIPOSOME[SL]
C. LONG-CIRCULATING IMMUNOLIPOSOME (SIL]
OpsOnlns are repel~d by water~yer at IkOosomesurface
~Y
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J.M.J. MARIAN and T.M. ALLEN
THE PAST Nowadays, liposomes are commonly classified into two categories: classical and long-circulating. The term classical liposomes (CL) has been used for liposomes that have dose-dependent pharmacokinetics
and short circulation
half-lives,
particularly after i.v. administration of low doses. They are rapidly cleared to the mononuclear phagocytic system (MPS). This was problematic in the early attempts to create drug delivery systems for certain applications, e.g. liposomal anti-cancer drugs. As a result, research focused on the formulation of liposomes with extended circulation times. With the inclusion of ganglioside GM1 (monosialoganglioside), or hydrogenated phosphatidylinositol, in the phospholipid bilayer, the circulation times of liposomes increased (2,3) and these long-circulating formulations were shown to have increased tumour uptake (3). Their success at hiding from the cells of the MPS resulted in the term Stealth m liposomes being bestowed on them (4).
There are a number of
reviews describing their dose-independent pharmacokinetics, biodistribution and the mechanism of action of these first-generation long-circulating liposomes (5,6). However, monosialogangliosides, prepared by extraction of natural resources (bovine brain), or by synthesis, make therapeutic application of GMl-containing liposomes expensive both financially and ethically, since purification from brain carries with it the potential risk of a slow virus contamination of GM 1. The second generation of long-circulating formulations contained
1 Stealth R is a trademark of SEQUUS Pharmaceuticals, Inc. Menlo Park, CA.
lipid
LONG CIRCULATING LIPOSOMES
derivatives of the polymer polyethylene glycol (PEG), e.g.
155
PEG-distearoyl
phosphatidylethanolamine (PEG-DSPE), with the hydrophillic polymer stabilizing the bilayer surface (Figure 1B).
These compositions have been termed sterically
stabilized liposomes (SL). SL, like GMl-containing liposomes, also possessed long circulation half-lives, dose-independent pharmacokinetics and were more successful at avoiding the cells of the MPS compared to their classical counterparts. There are several publications on the properties of SL (7,8,9,10,11,12).
SL are presently
preferred for therapeutic applications compared to liposomes containing GM1 for reasons of lower cost and increased safety.
Several products containing PEG have
already received U.S. Food and Drug Administration approval, including the enzyme PEG-adenosine deaminase (13) and SL containing entrapped doxorubicin (DoxilR2). The mechanism behind the extended circulation times and reduced MPS uptake seen after surface modification of liposomes is becoming well established. It is thought that the PEG polymer provides a hydrophillic steric barrier that reduces the interaction of plasma components (opsonins) with the liposome surface (14,15) (Figure 1B). As a result, the rate and extent of removal of the liposomes into the MPS is decreased, resulting in longer circulation times (for a review see 16). The hydrophillic barrier is dependent on the molecular weight of PEG, with the optimum molecular weight being around 2000 Da (17).
For lower molecular weight PEG
(<1000 Da) the circulation times decrease and the clearance to the MPS increases
(18).
2 Doxil R is a trademark of SEQUUS Pharmaceuticals, Inc., Menlo Park, CA.
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J.M.J. MARJAN and T.M. ALLEN
Current research is looking for better and newer polymers other than PEG, and a number of polymers have shown promise in this area (19,20), but to date none has surpassed PEG in conferring Stealth R properties to the liposomes. a) Important characteristics of long circulatin.q liposomes for in vivo applications There were a number of technical advances which had to be realized before SL could enter into the clinic. These include: i)
Uniform Size Distribution
For many applications, e.g. solid tumors, SL have to be able to move (extravasate) from the vasculature into the target tissues and, in order for this to be accomplished, a population of liposomes of a uniform, small size appears to be critical. One of the commonly used methods to achieve a uniform population size is through extrusion (21). Pressure is used to pass liposomes through polycarbonate filters of specific size. The most useful size range appears to be between 50- 200 nm in diameter (22), and for tumour uptake, liposomes of around 60-100 nm in diameter are preferred (23 and 29). ii)
Efficient Remote Drug Loading and Controlled Leakage
For reasons of economy, drugs such as the anti-cancer drug doxorubicin need to be loaded efficiently into liposomes. Once at the target site, the drugs must be released at rates which will result in drug levels within the therapeutic range. In other words, liposomes must not only protect the drug from premature release, metabolism and degradation while in the circulation, but also release the drug in a controlled manner at the target site (for a review see 24). Methods to efficiently load weak acids and bases such as the anti-cancer drugs doxorubicin, daunorubicin and
LONG CIRCULATING LIPOSOMES
157
vincristine have been developed. These methods rely on pH or chemical gradients across the lipid bilayer to load the drug into the liposome interior (25,26,27). Close to 100% of drug can be driven from the liposome exterior into the liposome interior by these techniques, and rates of leakage of the drugs in the circulation are very slow when these remote loading techniques are employed. iii)
Extended Shelf Life and Ease of Handling
In order for liposomes to be useful in the clinic they should have a long shelf life and, for maximum ease of use, they should come in a liquid or lyophilized form with the drug already loaded. These criteria are now realizable. For example, Sliposomal doxorubicin (Doxil ") can be stored for up to two years as an injectable liquid with little or no degradation of lipid or drug and minimal release of the entrapped contents. THE PRESENT There have been, to date, two major types of applications explored for the SL: i) circulating carriers for sustained release of drugs and ii) passive targeting to diseased tissues. This section will discuss briefly each of these uses. Sustained release systems are attractive for applications in which the drug of choice has a short half-life in the body.
Encapsulation of the drug will prevent
degradation and will lead to favourable alterations in the pharmacokinetics and the biodistribution of the entrapped drug. SL will passively localize in solid tumors or other sites of disease in higher levels than C-liposomes, which are cleared to the MPS too quickly to permit much localization in tumors.
SL localize in regions of the body which have "leaky
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capillaries" allowing extravasation of the liposomes into the diseased site (28,29). For example, as tumors grow, they recruit blood vessels in order to receive supplies of oxygen and nutrients (a process called angiogenesis). These growing capillaries have increased permeability compared to normal vasculature, allowing small liposomes to "leak out" (Figure 2).
These observations have applications in
chemotherapy of solid tumors, tumor imaging, and in treatment of infections where leaky capillaries can also occur. This section will discuss some of the applications of SL in cancer chemotherapy and refers the reader to a number of reviews in the area of tumor imaging and treatment of infections. Animal studies
Sustained release of anti-cancer drugs The first application of SL in vivo, in animals, was as a sustained release system for 1-r~-D-arabinofuranosylcytosine (ara-C) (30). Ara-C is an anti-cancer drug, used in the treatment of leukemia, with a half-life in vivo in mice or humans of approximately 20 min (31,32). Because the drug is only effective in killing cancer cells when the cells are exposed to the drug over a long period of time, ara-C is given as a slow i.v. infusion in the treatment of acute leukemia in humans.
Our
laboratory was the first to test the hypothesis that long circulating liposomes would mimic this slow infusion and act as a drug "cache" releasing ara-C in a sustained manner. When tested in mice bearing L1210 murine leukemia this hypothesis was shown to be true. Our experiments demonstrated that SL were more therapeutically effective than free drug infusions in treating mouse leukemia (33).
These
experiments also suggested that SL might prove useful as sustained delivery systems
L O N G C I R C U L A T I N G LIPOSOMES
159
FIGURE 2. Schematic illustration of passive targeting of SL via leaky vasculature of solid tumors (not to scale). During the process of angiogenesis, capillaries are recruited into growing tumors. These capillaries have higher permeability than normal vasculature allowing liposomes to percolate out into the surrounding tumor. Gradual release of drug from the liposomes localized in the tumor leads to cell kill,
PJ V,
i
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J.M.J. MARJAN and T.M. ALLEN
for many classes of rapidly degraded drugs including the new therapeutic peptides. Similar results were observed when vincristine was entrapped in SL. Here, treatment of mouse P388 leukemia using the S-liposomal drug was more efficacious than administration of the free drug alone. Passive targeting of anti-cancer drugs to tumors Vincristine is an anti-cancer drug primarily used in the clinic in the treatment of adult carcinomas and lymphomas. Passive targeting of S-liposomal vincristine was observed in both colon cancer (34) and murine mammary carcinoma (35) implanted subcutaneously in mice.
Vincristine entrapped in SL caused substantial tumor
regression and therapeutic benefit over the free drug given at the same total dose. Doxorubicin, another anti-tumor drug used widely in treating human solid tumors, when entrapped in SL has also been shown to have increased therapeutic efficacy in the treatment of a variety solid tumors in animals compared to the free drug alone, including colon cancer, mammary cancer and lymphoma (36-42). Passive targeting of antibiotics to areas of infection One of the common characteristics of an infected tissue or damaged endothelium is the increased capillary permeability. This characteristic may be used to our advantage by encapsulating agents to fight the infection within SL. Studies conducted by Bakker-Woudenberg et al. (43) have shown increased localization of SL in a rat lung model infected with Klebsiella pneumonia.
The efficacy, in the
treatment of the infected rat lungs with two clinically relevant antibiotics, gentamicin and ceftazidime entrapped in SL, appeared to be greater than the free drug alone.
LONG CIRCULATING LIPOSOMES
161
Gene therapy and tumour imaging Long circulating liposomes in the area gene therapy and imaging are currently attracting much attention. Although this review does not touch on these topics, they are of growing importance. To date, liposomal tumour imaging agents have yet to compete successfully in the imaging market, although SL may improve the outlook for these applications. In the area of gene therapy, although liposomes are attractive vectors for mediating transient DNA expression in cells in vitro, considerable basic research needs to be done before long circulating formulations of liposomal DNA with high transfection efficiencies will be achieved. There have been a number of recent review articles published both in the area of liposomes as DNA delivery systems (44) and liposomes in the area of imaging (45,46). Human studies
Passive targeting of doxorubicin to tumors The dose-limiting side effects of doxorubicin in human cancer chemotherapy include acute cardiomyopathy and myelosuppression. As previously mentioned, the efficacy of doxorubicin entrapped in SL has been demonstrated in solid tumors in animals.
Efficacy has also been demonstrated in human clinical trials in the
treatment of refractory Kaposi's sarcoma in patients infected with the HIV virus (47). In multi-centre trials, where Doxil R (doxorubicin entrapped in SL) was evaluated as a treatment for refractory Kaposi's sarcoma, both cutaneous and visceral lesions in the patients regressed during the course of treatment with a complete plus partial response rate of over 70% (47). No cardiotoxicity was observed in the patients and myelosupression was comparable to that seen for the free drug. The dose-limiting
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toxicities for Doxil Rwere stomatitis and hand and foot syndrome both of which would resolve upon dose reduction. Recently this product has been approved for use by the U.S. Food and Drug Administration in the treatment of refractory Kaposi's sarcoma. Doxil R is also currently in clinical trials for advanced breast, ovarian, and head and neck cancer. Daunorubicin, another anticancer drug, has been encapsulated in liposomes (DaunoXome R3) and has recently also been approved by the FDA as a first-line therapy for Kaposi's sarcoma in HIV-infected patients.
In DaunoXome R, the
liposomes contain the solid phase lipid distearoylphosphatidylcholine (DSPC) and this, combined with their small size, also results in prolonged circulation times, although the clearance rate in humans is somewhat shorter than that of Doxil R (48). Unlike S-liposomal doxorubicin, these liposomes are reported to possess dosedependent pharmacokinetics (48). THE FUTURE With SL in clinical trials or in the clinic, considerable recent effort in liposome research has been directed at developing a third generation of liposomes, i.e. one in which there is site-specific targeting of the SL by attaching "homing devices" such as monoclonal antibodies or receptor-specific ligands to the liposome surface (Figure 1C). A number of coupling methods have been developed for the attachment of antibodies or ligands to liposomes (reviewed in reference 49). Our laboratory has investigated a number of cancer models in mice to test
3 DaunoXome R is a trademark of NeXstar Inc., San Dimas, CA
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163
whether selective toxicity can be achieved in vivo using antibody- or ligand-targeted SL.
The
models
have
included
routine
squamous
lung
carcinoma
(a
pseudometastatic model), human ovarian cancer (a solid tumor model) and human B cell lymphoma (a hematological model).
Targeted SL (SIL) appear to have
increased efficacy over non-targeted liposomes or free drug in the treatment of human proliferative disorders of B or T-cell lineages grown as xenograft models in SCID mice (50). Increased therapeutic efficacy of targeted SL over non-targeted SL was possible in the treatment of solid tumors early (i.e. at the micrometastatic stage), but not late, in their development in a squamous lung carcinoma model in mice (50). On the other hand, non-targeted liposomes, e.g. SL- doxorubicin, appeared to be more effective than SIL-doxorubicin in treating human ovarian tumors, grown in SCID mice, in a more advanced stage of development (50) In order to explain the differences in efficacy between targeted and nontargeted SL in the treatment of hematological cancers versus early solid tumors versus more advanced solid tumors, we suggest the following explanation.
As has
been mentioned above, non-targeted SI_ can percolate through leaky capillaries of tumors which are undergoing the process of angiogenesis. A solid tumor will contain relatively large numbers of cells by the time it begins to become deficient in oxygen and nutrients and starts to send out signals recruiting capillary growth into the tumor. When metastatic cells from solid tumors are migrating in blood and lymph, or exist as micrometatases, they consist of individual cells or small groups of cells and lack the capillary network which allows the passive targeting of non-ligand bearing liposomes (SL) to the tumor. Therefore SL appear to be less effective against very
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early solid tumors, although they have good efficacy against more advanced solid tumors undergoing angiogenesis. Even if the SL cannot reach all tumor cells, once they have localized in the tumor drug released from the liposomes will diffuse further into the tumor resulting in an additional cytotoxic response.
Targeted liposomes
(SIL), on the other hand, will have relatively unimpeded access to, and will therefore be able to bind to, most of the cells in very small tumors, and to individual metastatic cells released from primary tumors (Figure 3A).
By the same argument, SIL are
effective in hematological malignancies, as they have easy access to the diseased cells within the circulation, and their ability to bind to these cells gives them an advantage over non-targeted (i.e. non-binding) liposomes. However when SIL reach an advanced solid tumour, they will bind to the first target cells they recognize. This prevents them from percolating further into the tumor to kill target cells deep in the tumor mass (Figure 3B). This phenomenon has been referred to as the "binding site barrier" (51). Once targeted liposomes have arrived at the desired target there are a number of possible scenarios.
Internalization of the drug-liposome package into the
endocytotic apparatus of the cell can occur if the targeting moiety is against an internalizing epitope on the cell surface.
If the drug is capable of surviving the
endocytotic environment, it would then be released into the cell cytoplasm once the carrier is destroyed by lysosomal enzymes (Figure 4A), Alternatively, binding of the targeted liposomes to a non-internalizing epitope will result in a sustained release of the liposomal contents in locally high concentrations at the cell surface. The released drug will enter the cell either by passive diffusion or by a transport system (Figure
LONG CIRCULATING LIPOSOMES
165
FIGURE 3. Schematic illustration of the binding of SIL to tumor cells (not to scale). A, Binding of SIL to micrometastases (or indivdual tumor cells). SIL bind to epitopes at the surface of the cancer cell. A small number of cells allows for targeting and drug delivery to all of the cells. B. Binding of SIL to advanced solid tumors. The SIL bind to epitopes on the first cancer cells they encounter, making it difficult for the SIL to penetrate further into the interior of the tumor mass (binding site barrier). Drug released from the liposomes does not reach effective concentrations in the inner portion of the tumor mass.
A. BINDING OF IMMUNOLIPOSOMES TO MICROMETASTASES
.~
cln.~-fllledtargeted tmmunc~igx)some[SILl
.'.
released drug molecules
•
surface eloitope on cancer cells
B. BINDINO-.-~ITEBARRIER FOR I M M U N O L I ~ M E S ~ D TO MORE ADVANCED SOLID TUMORS
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J.M.J. MARJAN and T.M. ALLEN
FIGURE 4. Schematic illustrations of some possible mechanisms for the cytotoxic effects of SIL (not to scale). A. Binding of SIL to an internalizing epitope on the surface of the cell triggers the internalization of the drug-liposome package. Cytotoxicity is achieved when the internalized liposome is degraded within the endocytotic apparatus of the cell and the drug reaches the cell cytoplasm and/or nucleus. B. Binding of SIL to a non-internalizing epitope on the surface of the cell. Drug will leak from liposomes bound to the cell surface possibly achieving locally high drug concentrations. The cytotoxic effect is dependent on the rate of leakage of the drug from the liposome and the rate at which the drug enters the cell either by passive diffusion, or by active transport. Diffusion may, however, carry the drug away from the cell surface faster than it can enter the cell. C. Bystander effect. Drug released from liposomes floating free in the tumor or attached via a non-internalizing epitope at the surface of a nearby cancer cell has the ability to be cytotoxic to a cancer cell in close proximity (bystander) that bears nonrecognized epitopes at the cell surface.
SOME MECHANISMS OF ACTION OF IMMUNOLIPOSOMES
Internalizing epilope
Q
endc< Non-internalizing epitope Non*recognized epitooe
diffusion
X .
!
LONG CIRCULATING LIPOSOMES
167
4B). The amount of drug entering the cell will have an inverse relationship to the rate of diffusion of the released drug away from the liposome surface.
Another
mechanism for cell kill in solid tumors is the "bystander effect" in which cells lacking the target epitope may be killed by drug released at the surface of neighbouring cells having bound liposomes (52) (Figure 4C). Another proposed mechanism of action is the "target-cell dragging" concept (53) whereby the targeted liposomes binds to its target cell in the bloodstream and is flagged by phagocytic cells due to the size of the complex or the presence of antibodies.
Phagocytic cells may engulf this complex and the drug and/or the
macrophage could kill the target cell. This concept resembles a long recognized pathway in the immune system to dispose of circulating antigens.
However, the
phagocytic cell may also be killed by the released drug and the consequences of this are not well understood. The successful achievement of targeting in animal models in our laboratory (36,50,54) has given us some insights into the requirements that are needed for this therapy to reach clinical development.
The ideal targeted carrier would have to
include at least four characteristics: i)
the ability to retain long circulating half-life despite the presence of antibodies or other targeting ligands.
ii)
coupling methodologies must be efficient, simple, rapid and compatible for use in humans.
iii)
the presence of the antibody should not interfere with drug loading and release mechanisms.
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iv)
SIL's should retain their target recognition in vivo and be able to bind in sufficient numbers in order to release the drug and achieve therapeutic efficacy superior to the free drug alone or the liposomal drug in the absence of antibody.
With the careful choice of drug, antibody, coupling method, and tumor model, e.g. hematological versus solid tumor, these criteria appear to be achievable. Before antibody-targeted long circulating liposomes reach the clinic, additional problems need to be addressed, including the potential immunogenicity of murine antibodies presented on liposomes. Can we get away from this problem by coupling "humanized antibodies" to liposomes? Will antibody fragments be less immunogenic than whole antibodies? How can the "binding-site barrier" be overcome? Rodent (monoclonal antibodies) have been "humanized" by linking rodent variable regions and human constant regions (chimeric antibodies) (55). "Humanizing" an antibody reduces
its immunogenicity; however a recent publication describing the clinical
application of a "humanized" antibody demonstrated that there is a partial immune response that is mounted by the patient (56). On the other hand, in a recent clinical trial, a humanized rat therapeutic antibody directed against mature human leukocytes proved clinically effective in destroying a large tumour mass in two patients (57). CONCLUSIONS This review has briefly described the journey of long circulating liposomes from their inception to their present applications, with some discussion of future directions for their development. The frequently asked question is why liposomes have not had more of a clinical impact? Liposomes, like most new technologies (including gene
LONG CIRCULATING LIPOSOMES
169
therapy at present) have been the victims of exaggerated claims in the absence of the basic research to support these claims. Once the basic research into formulation, stability, pharmacokinetics, etc. was undertaken, liposome formulations could be designed which married our understanding of liposome properties with current concepts of disease, e.g. tumor cell biology, leading to rational clinical applications. With five liposomal formulations now in the clinic, and many more in clinical trials, the wisdom of taking this approach is evident. We look forward to a bright future for this emerging technology.
REFERENCES
1)
Bangham, A.D., Standish, M.M., Watkins, J.C.
Diffusion of univalent ions
across the lamellae of swollen phospholipids. J. Mol. Biol. 13:328-252, 1965.
2)
Allen, T.M. and Chonn, A. Large unilamellar liposomes with low uptake into the reticuloendothelial system. FEBS Lett., 223:42-46, 1987.
3)
Gabizon, A. and Papahadjopoulos, D. Liposome formulations with prolonged circulation time in blood and enhanced uptake by tumors. Proc. Natl. Acad. Sci. USA, 85:6949-4953, 1988.
4)
Allen, T.M.
Stealth liposomes, Avoiding reticuloendothelial uptake, in
Liposomes in the Therapy of Infectious Diseases and Cancer, New Series, Vol. 89, Eds. G. Lopez-Berestein and I. Fidler, Alan R.Liss Inc, New York, 1989, pp. 405-415.
5)
Allen, T.M and Papahadjopoulos,
D.
Sterically stabilized ("Stealth")
liposomes: Pharmacokinetics and therapeutic advantages in: Liposome Technology, 2rid Edition, Vol. 111, Ed. G. Gregoriadis, CRC Press, Boca Raton, Florida, 1993 pp. 59-72.
6)
Mumtaz, S., Ghosh, P.C. and Bachhawat, B.K. Design of liposomes for circumventing the reticuloendothelial cells. Glycobiol., 1:505-510, 1991.
170
J,M.J. MARJAN and T.M. A L L E N
7)
Blume G. and Cevc, G. Molecular mechanism of the lipid vesicle longevity in vivo. Biochim. Biophys. Acta, 1029:91-97, 1990.
8)
Chonn, A., Semple, S.C. and Cullis, P.R. Association of blood proteins with large unilamellar liposomes in vivo, Relation to circulation lifetimes. J. Biol. Chem., 267:18759-18765, 1992.
9)
Gabizon, A. and Papahadjopoulos, D.
The role of surface charge and
hydrophillic groups on liposome clearance in vivo. Biochim. Biophys. Acta, 1103:94-100, 1992. 10)
Lasic, D.D., Martin, F.J., Gabizon, A., Huang, S.K. and Papahadjopoulos, D. Sterically stabilized liposomes, A hypothesis on the molecular origin of the extended circulation times. Biochim. Biophys. Acta, 1070:187-192, 1991.
11)
Needham, D., Kristova, K., Mclntosh, T.J., Dewhirst, M., Wu, N. and Lasic D.D. Polymer-grafted liposomes, Physical basis for the "Stealth" property. J. Liposome Res., 2:411-430, 1992.
12)
Woodle, M.C. and Lasic D.D.
Sterically stabilized liposomes.
Biochim.
Biophys. Acta, 1113:171-199, 1992. 13) 14)
Pool, R. "Hairy" enzymes stay in the blood. Science, 248:305, 1990. Torchilin, V.P, Omelyanenko, V.G., Papisov, M.I., Bogdanov, A.A., Trubetskoy, V.S., Herron, J.N., Gentry, C.A. Poly(ethylene glycol) on the liposome surface: on the mechanism of polymer-coated liposome longevity. Biochim. Biophys. Acta, 1195:11-20, 1995.
15)
Needham, D., Mclntosh, T.J., Lasic, D.D.
Repulsive interactions and
mechanical stability of polymer-grafted lipid membranes. Biochim. Biophys. Acta, 1108:40-48, 1992. 16)
Gabizon, A., Dagan, A., Goren, D., Barenholz, Y. and Fuks, A.
Liposomes
as in vivo carriers of adriamycin, Reduced cardiac uptake and preserved antitumour activity in mice. Cancer Res., 42:4734-4739, 1982.
LONG CIRCULATING L1POSOMES
17)
171
Maruyama, K., Yuda, T., Okamoto, A., Ishikura, C., Kojima, S., Iwatsuru, M. Effect of molecular weight in amphipathic polyethylene glycol on prolonging the circulation time of large unilamellar liposomes. Chem. Pharm. Bull, 39:16201622, 1991.
18)
Woodle, M.C.
Surface modified liposomes: assessment and stability and
prolonged circulation. Chem. Phys. Lipids, 64:249-262, 1993. 19)
Zalipsky, S., Hansen, C.B., Oaks, J.M. and Allen, T.M. Evaluation of blood clearance and biodistribution of poly(2-oxazoline)-grafted
liposomes.
J.
Pharm. Sci., (in press). 20)
Torchilin, V.P., Shtilman, M.I., Trubetskoy, V.S., Whiteman, K., Milstein, A.M. Amphiphilic vinyl polymers effectively prolong liposome circulation time in vivo. Biochim. Biophys. Acta., 1195:181-184, 1994.
21)
Olson, F., Hunt, C.A., Szoka, F.C., Vail, W.J., Papahadjopoulos,
D.
Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. Biochim. Biophys. Acta, 557:9-23, 1979. 22)
Litzinger, D.C., Buiting, AM.J, van Rooijen, N., Huang, L. Effect of liposome size on the circulation time and intraorgan distribution of amphipathic poly(ethylene glycol)-containing liposomes. Biochim. Biophys. Acta, 1190:99107, 1994.
23)
Papahadjopoulos, D., Allen, T.M., Gabizon, A., Mayhew, E., Matthay, K., Huang, S.K., Lee, K.D., Woodle, M.C., Lasic, D.D., Redemann, C., Martin, F.J. Sterically stabilized liposomes: Improvements in pharmacokinetics
and
antitumor therapeutic efficacy. Proc. Natl. Acad. Sci. USA, 88:11460-11464, 1991. 24)
Allen, T.M., Hansen, C.B., de Menezes, D.E.L. Pharmacokinetics of longcirculating liposomes. Adv. Drug. Deliv. Rev., 16:267-285, 1995.
25)
Mayer, L.D., Tai, L.C.L., Bally, M.B., Mitilenes, G.N., Ginsberg, R.S., Cullis, P.R. Characterization of liposomal systems containing doxorubicin entrapped in response to pH gradients. Biochim.Biophys.Acta 1025:143-151, 1990.
172
J.M.J. M A R J A N and T.M. A L L E N
26)
Mayer, L.D., Bally, M.B., Cullis, P.R.
Uptake of adriamycin into large
unilamellar vesicles in response to a pH gradient.
Biochim.Biophys.Acta
857:123-126, 1986. 27)
Haran, G., Cohen, R., Bar, L.K., Barenholz, Y. Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases. Biochim.Biophys.Acta. 1151:201-215, 1993.
28)
Dvorak, H.F., Nagy, J.A., Dvorak, J.T., Dvorak, A.M.
Identification and
characterization of the blood vessels of solid tumors that are leaky to circulating macromolecules Am. J. Pathol., 133:95-109, 1988. 29)
Wu, N.Z., Da, D., Rudoll, T.L., Needham, D., Dewhirst, M.W.
Increased
microvascular permeability contributes to preferential accumulation of Stealth liposomes in tumor tissue. Cancer Res., 53:3765- 3770, 1993. 30)
Allen, T.M. and Mehra T.
Recent advances in sustained release of
antineoplastic drugs using liposomes which avoid uptake in to the reticuloendothelial system. Proc. Western Pharmacol. Soc., 32:111-114, 1989. 31)
Baguley, B.C. and Falkenhaug, E.M. Plasma half-life of cytosine arabinoside (NSC-63878) in patients treated for acute myeloblastic leukaemia. Cancer Chemother. Rep., 55:291-298 (1971)
32)
Borsa, J., Whitmore, G.R., Valeriote, F.A., Collines, D. and Bruce, W.R. Studies on the persistence of methotrexate, cytosine arabinoside and leucovorin in the serum of mice. J. Nat. Cancer Inst., 42:235-242, 1969.
33)
Allen, T.M., Mehra, T., Hansen, C., Chin, Y-C.
Stealth liposomes: an
improved sustained release system for 1-1~-D-arabinofuranosylcytosine. Cancer Res., 52:2431-2439, 1992. 34)
Allen, T.M., Newman, M.S., Woodle, M.C., Mayhew, E., Uster, P.S. Pharmacokinetics and antitumor activity of vincristine encapsulated in sterically stabilized liposomes. Int. J. Cancer, 62:199-204, 1995
35)
Vaage, J., Donovan, A., Mayhew, E., Uster, P., Woodle, M. Therapy of mouse mammary carcinomas with vincristine and doxorubicin encapsulated in sterically stabilized liposomes. Int. J. Cancer, 54:959-964, 1993.
LONG CIRCULATING LIPOSOMES
36)
173
Ahmad, I., Longenecker, M., Samuel, J. and Allen, T.M. Antibody-targeted delivery of doxorubicin entrapped in sterically stabilized liposomes can eradicate lung cancer in mice. Cancer Res., 53:1484-1488, 1993.
37)
Gabizon, A.A. Selective tumor localization and improved therapeutic index of anthracyclines encapsulated in long-circulating liposomes.
Cancer Res.,
52:891-896, 1992. 38)
Jain, R.K.
Transport of molecules across tumor vasculature.
Cancer
Metastasis Rev., 6:559-593, 1987. 39)
Mayhew, E.G., Lasic, D., Babbar, S., and Martin, F.J. Pharmacokinetics and antitumor activity of epirubicin encapsulated in long-circulating liposomes incorporating a polyethylene glycol-derivatized phospholipid. Int. J. Cancer, 51:302-309, 1992.
40)
Papahadjopoulos, D., Allen, T.M., Gabizon, A., Mayhew, E., Matthay, K., Huang, S.L., Lee, K-D, Woodle, M.C., Lasic, D.D., Redemann, C. and Martin, F.J. Sterically stabilized liposomes, Improvements in pharmacokinetics and antitumor therapeutic efficacy. Proc. Natl. Acad. Sci. USA, 88:11460-11464, 1991.
41)
Vaage, J., Mayhew, E., Lasic, D.D. and Martin, F.J. Therapy of primary and metastatic mouse mammary carcinomas with doxorubicin encapsulated in long circulating liposomes. Int. J. Cancer, 51:942-948, 1992.
42)
Williams, S.S., Alosco, T.R., Mayhew, E., Lasic, D.D., Martin, F.J., and Bankert, R.B.
Arrest of human lung tumor xenograft grown in severe
combined immunodeficient mice using doxorubicin encapsulated in sterically stabilized liposomes. Cancer Res., 53:3964-3967, 1993. 43)
Bakker-Woudenberg, I.A.J.M., Storm, G., Woodle, M.C.
Liposomes in the
treatment of infections. J. Drug Targeting, 2:363-371, 1994. 44)
Miller, N., Vile, R. Targeted vectors for gene therapy. FASEB J., 9:190-199, 1995.
174
J,M.J. M A R I A N and T.M. A L L E N
45)
Weissleder, R., Bogdanov, A., Neuwlt, E.A., Papisov, M. Long circulating iron oxides for magnetic resonance imaging. Adv. Drug Deliv. Rev., 16:3231-334, 1995.
46)
Bogdanov, A.Jr., Weissleder, IR., Brady, T.
Long circulating blood pool
imaging agents. Adv. Drug Deliv. Rev., 16:335-348, 1995. 47)
Northfelt, D.W., Martin, F.J., Kaplan, L.D., Russell, J., Anderson, M., Lang, J., Volberding, P.A. Pharmacokinetics, tumor localization and safety of Doxil (liposomal Doxorubicin) in AIDS patients with Kaposi's sarcoma. Proc. Am. Soc. Clin. Oncol., 12:51, 1993.
48)
Gill, P.S., Espina, B.M., Muggia, F., Cabriales, S., Tulpule, A., Esplin, J.A., Liebman, H.A., Forssen, E., Ross, M.E., Levine, A.M. Phase 1/11clinical and pharmacokinetic evaluation of liposomal daunorubicin. J. Clin. Oncol., 13:9961003, 1995.
49)
Hansen, C.B., Kao, G.Y., Moase, E.H., Zalipsky, S., Allen, T.M. Attachment of antibodies to sterically stabilized liposomes: evaluation, comparison and optimization of coupling procedures. Biochim. Biophys, Acta, 1239:133-144, 1995.
50)
Allen,
T.M.,
Ahmad,
I.,
Lopes
de
Menezes,
D.E.,
Moase,
E.H.
Immunoliposome-mediated targeting of anticancer drugs in vivo. Biochem. Society Trans., 23:1073-1079,1995. 51)
Weinstein, J.N. and van Osdol, W.
Early intervention in cancer using
monoclonal antibodies and other biological ligands:micropharmacology and the "Binding Site Barrier". Cancer Res., 52:2747-2751, 1992. 52)
Allen, T.M. Long-circulating (sterically stabilized) liposomes for targeted drug delivery. Trends in Pharmacological Sciences, 15:215-220, 1994.
53)
Vingerhoeds, M.H., Storm, G., Crommelin, D.J.A. Immunoliposomes in vivo. Immunomethods, 4:259-272, 1994.
54)
ALlen, T.M., Agrawal, A.K., Ahmad, I., Hansen, C.B. and Zalipsky, S. Antibody-mediated targeting of long-circulating (Stealth R) liposomes. Liposome IRes., 4:1-25, 1994.
J.
LONG CIRCULATING LIPOSOMES
55)
175
Neuberger, M.S., Williams, G.T., Mitchell, E.B., Jouhal, S.S., Flanagan, J.G., Rabbitts, T.H.
A hapten-specific chimeric IgE antibody with human
physiological effector function. Nature, 314:268-270, 1985. 56)
LoBuglio, A.F., Wheeler, R.H., Trang, J., Haynes, A., Rogers, K., Harvey, E.B., Sun, L., Ghrayeb, J., Khazaeli, M.B. Mouse/human chimeric monoclonal antibody in man: kinetics and immune response. Proc. Natl. Acad. Sci. USA, 86:4220-4224, 1989.
57)
Hale, G., Dyer, M.J., Clark, M.R., Phillips, J.M., Marcus, R., Reichmann, L., Winter, G., Waldmann, H. Remission induction in non-Hodgkin lymphoma with reshaped human monoclonal antibody CAMPATH-1H. Lancet, ii(8625):13941399, 1988.
PH S0734-9750(96)00007-9
LONG CIRCULATING LIPOSOMES: PAST, PRESENT AND FUTURE J I H A N M. J. M A R J A N and T H E R E S A M. A L L E N
Department of Pharmacology, University of Alberta, Edmonton, Alberta T6G 2H7
* To whom correspondence should be addressed: Department of Pharmacology, University of Alberta, Edmonton, AB T6G 2H7 Tel:
(403) 492-5710
Fax:
(403) 492-8078
email: [email protected]
151
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J.M.J. M A R J A N and T.M. ALLEN
Liposomes were first described by the British scientist Alec Bangham and colleagues in 1965 (1). Upon hydration, lipid films spontaneously formed closed concentric spherical structures that encapsulated part of the liquid medium in their interior, The primary constituent of a liposome is phospholipid, molecules consisting of hydrophillic "heads" attached to long non-polar hydrophobic "tails" (Figure 1A). The hydrophillic head typically consists of a phosphate group, hence phospholipid, whereas the hydrophobic tail is made of two long hydrocarbon chains.
This
amphipathic structure of phospholipids leads to their aggregation into ordered structures that sequester the hydrophobic tails away from contact with water (Figure 1A). Since their conception,
both industry and academia
have had high
expectations for practical applications of these small spherical structures.
These
expectations included their use as simple model membrane systems as well as a possible role as carriers for drugs. Liposomes were attractive as drug carriers as they were easily biodegradable and had the potential to be targeted to diseased cells....a 'magic bullet'! They quickly achieved their potential as model membranes for a variety of applications.
However, the route to the development of liposomal
drug carriers has been arduous, long and in many cases not fruitful.
Yet the
liposomes' appeal as targetable vehicles for site-selective delivery of therapeutics has not been lost.
This review will briefly outline the evolution of new liposomal
formulations, with long circulating times, from their conception, to their current approval for clinical use in humans, to future applications presently in the development stage.
LONG CIRCULATING LIPOSOMES
153
FIGURE 1. Schematic illustration of various liposome structures (not to scale). A. Classical liposome (CL) demonstrating how the hydrophillic "heads" and hydrophobic "tails" of the phospholipids aggregate into spherical sealed structures. CL attract plasma proteins (opsonins) to the liposome surface which contribute to the rapid removal of CL from circulation by the cells of the MPS. B. Long circulating liposomes (SL) with entrapped drug molecules, demonstrating how the hydrophillic polyethylene glycol (PEG) surface polymers repel plasma opsonins. C. Targeted long circulating liposomes where an antibody (or other ligand) is coupled to the terminus of the PEG polymer to form sterically stabilized immunoliposomes (SIL).
A, CLASSICAL LIPOSOME [CL]
Hydrophilllc
Entrapped in aqueous
y
drug interior
f Opsonins
~ Phospholipid
~. Cholesterol
B.LONG-CIRCULATING LIPOSOME[SL]
C. LONG-CIRCULATING IMMUNOLIPOSOME (SIL]
OpsOnlns are repel~d by water~yer at IkOosomesurface
~Y
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J.M.J. MARIAN and T.M. ALLEN
THE PAST Nowadays, liposomes are commonly classified into two categories: classical and long-circulating. The term classical liposomes (CL) has been used for liposomes that have dose-dependent pharmacokinetics
and short circulation
half-lives,
particularly after i.v. administration of low doses. They are rapidly cleared to the mononuclear phagocytic system (MPS). This was problematic in the early attempts to create drug delivery systems for certain applications, e.g. liposomal anti-cancer drugs. As a result, research focused on the formulation of liposomes with extended circulation times. With the inclusion of ganglioside GM1 (monosialoganglioside), or hydrogenated phosphatidylinositol, in the phospholipid bilayer, the circulation times of liposomes increased (2,3) and these long-circulating formulations were shown to have increased tumour uptake (3). Their success at hiding from the cells of the MPS resulted in the term Stealth m liposomes being bestowed on them (4).
There are a number of
reviews describing their dose-independent pharmacokinetics, biodistribution and the mechanism of action of these first-generation long-circulating liposomes (5,6). However, monosialogangliosides, prepared by extraction of natural resources (bovine brain), or by synthesis, make therapeutic application of GMl-containing liposomes expensive both financially and ethically, since purification from brain carries with it the potential risk of a slow virus contamination of GM 1. The second generation of long-circulating formulations contained
1 Stealth R is a trademark of SEQUUS Pharmaceuticals, Inc. Menlo Park, CA.
lipid
LONG CIRCULATING LIPOSOMES
derivatives of the polymer polyethylene glycol (PEG), e.g.
155
PEG-distearoyl
phosphatidylethanolamine (PEG-DSPE), with the hydrophillic polymer stabilizing the bilayer surface (Figure 1B).
These compositions have been termed sterically
stabilized liposomes (SL). SL, like GMl-containing liposomes, also possessed long circulation half-lives, dose-independent pharmacokinetics and were more successful at avoiding the cells of the MPS compared to their classical counterparts. There are several publications on the properties of SL (7,8,9,10,11,12).
SL are presently
preferred for therapeutic applications compared to liposomes containing GM1 for reasons of lower cost and increased safety.
Several products containing PEG have
already received U.S. Food and Drug Administration approval, including the enzyme PEG-adenosine deaminase (13) and SL containing entrapped doxorubicin (DoxilR2). The mechanism behind the extended circulation times and reduced MPS uptake seen after surface modification of liposomes is becoming well established. It is thought that the PEG polymer provides a hydrophillic steric barrier that reduces the interaction of plasma components (opsonins) with the liposome surface (14,15) (Figure 1B). As a result, the rate and extent of removal of the liposomes into the MPS is decreased, resulting in longer circulation times (for a review see 16). The hydrophillic barrier is dependent on the molecular weight of PEG, with the optimum molecular weight being around 2000 Da (17).
For lower molecular weight PEG
(<1000 Da) the circulation times decrease and the clearance to the MPS increases
(18).
2 Doxil R is a trademark of SEQUUS Pharmaceuticals, Inc., Menlo Park, CA.
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J.M.J. MARJAN and T.M. ALLEN
Current research is looking for better and newer polymers other than PEG, and a number of polymers have shown promise in this area (19,20), but to date none has surpassed PEG in conferring Stealth R properties to the liposomes. a) Important characteristics of long circulatin.q liposomes for in vivo applications There were a number of technical advances which had to be realized before SL could enter into the clinic. These include: i)
Uniform Size Distribution
For many applications, e.g. solid tumors, SL have to be able to move (extravasate) from the vasculature into the target tissues and, in order for this to be accomplished, a population of liposomes of a uniform, small size appears to be critical. One of the commonly used methods to achieve a uniform population size is through extrusion (21). Pressure is used to pass liposomes through polycarbonate filters of specific size. The most useful size range appears to be between 50- 200 nm in diameter (22), and for tumour uptake, liposomes of around 60-100 nm in diameter are preferred (23 and 29). ii)
Efficient Remote Drug Loading and Controlled Leakage
For reasons of economy, drugs such as the anti-cancer drug doxorubicin need to be loaded efficiently into liposomes. Once at the target site, the drugs must be released at rates which will result in drug levels within the therapeutic range. In other words, liposomes must not only protect the drug from premature release, metabolism and degradation while in the circulation, but also release the drug in a controlled manner at the target site (for a review see 24). Methods to efficiently load weak acids and bases such as the anti-cancer drugs doxorubicin, daunorubicin and
LONG CIRCULATING LIPOSOMES
157
vincristine have been developed. These methods rely on pH or chemical gradients across the lipid bilayer to load the drug into the liposome interior (25,26,27). Close to 100% of drug can be driven from the liposome exterior into the liposome interior by these techniques, and rates of leakage of the drugs in the circulation are very slow when these remote loading techniques are employed. iii)
Extended Shelf Life and Ease of Handling
In order for liposomes to be useful in the clinic they should have a long shelf life and, for maximum ease of use, they should come in a liquid or lyophilized form with the drug already loaded. These criteria are now realizable. For example, Sliposomal doxorubicin (Doxil ") can be stored for up to two years as an injectable liquid with little or no degradation of lipid or drug and minimal release of the entrapped contents. THE PRESENT There have been, to date, two major types of applications explored for the SL: i) circulating carriers for sustained release of drugs and ii) passive targeting to diseased tissues. This section will discuss briefly each of these uses. Sustained release systems are attractive for applications in which the drug of choice has a short half-life in the body.
Encapsulation of the drug will prevent
degradation and will lead to favourable alterations in the pharmacokinetics and the biodistribution of the entrapped drug. SL will passively localize in solid tumors or other sites of disease in higher levels than C-liposomes, which are cleared to the MPS too quickly to permit much localization in tumors.
SL localize in regions of the body which have "leaky
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capillaries" allowing extravasation of the liposomes into the diseased site (28,29). For example, as tumors grow, they recruit blood vessels in order to receive supplies of oxygen and nutrients (a process called angiogenesis). These growing capillaries have increased permeability compared to normal vasculature, allowing small liposomes to "leak out" (Figure 2).
These observations have applications in
chemotherapy of solid tumors, tumor imaging, and in treatment of infections where leaky capillaries can also occur. This section will discuss some of the applications of SL in cancer chemotherapy and refers the reader to a number of reviews in the area of tumor imaging and treatment of infections. Animal studies
Sustained release of anti-cancer drugs The first application of SL in vivo, in animals, was as a sustained release system for 1-r~-D-arabinofuranosylcytosine (ara-C) (30). Ara-C is an anti-cancer drug, used in the treatment of leukemia, with a half-life in vivo in mice or humans of approximately 20 min (31,32). Because the drug is only effective in killing cancer cells when the cells are exposed to the drug over a long period of time, ara-C is given as a slow i.v. infusion in the treatment of acute leukemia in humans.
Our
laboratory was the first to test the hypothesis that long circulating liposomes would mimic this slow infusion and act as a drug "cache" releasing ara-C in a sustained manner. When tested in mice bearing L1210 murine leukemia this hypothesis was shown to be true. Our experiments demonstrated that SL were more therapeutically effective than free drug infusions in treating mouse leukemia (33).
These
experiments also suggested that SL might prove useful as sustained delivery systems
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159
FIGURE 2. Schematic illustration of passive targeting of SL via leaky vasculature of solid tumors (not to scale). During the process of angiogenesis, capillaries are recruited into growing tumors. These capillaries have higher permeability than normal vasculature allowing liposomes to percolate out into the surrounding tumor. Gradual release of drug from the liposomes localized in the tumor leads to cell kill,
PJ V,
i
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for many classes of rapidly degraded drugs including the new therapeutic peptides. Similar results were observed when vincristine was entrapped in SL. Here, treatment of mouse P388 leukemia using the S-liposomal drug was more efficacious than administration of the free drug alone. Passive targeting of anti-cancer drugs to tumors Vincristine is an anti-cancer drug primarily used in the clinic in the treatment of adult carcinomas and lymphomas. Passive targeting of S-liposomal vincristine was observed in both colon cancer (34) and murine mammary carcinoma (35) implanted subcutaneously in mice.
Vincristine entrapped in SL caused substantial tumor
regression and therapeutic benefit over the free drug given at the same total dose. Doxorubicin, another anti-tumor drug used widely in treating human solid tumors, when entrapped in SL has also been shown to have increased therapeutic efficacy in the treatment of a variety solid tumors in animals compared to the free drug alone, including colon cancer, mammary cancer and lymphoma (36-42). Passive targeting of antibiotics to areas of infection One of the common characteristics of an infected tissue or damaged endothelium is the increased capillary permeability. This characteristic may be used to our advantage by encapsulating agents to fight the infection within SL. Studies conducted by Bakker-Woudenberg et al. (43) have shown increased localization of SL in a rat lung model infected with Klebsiella pneumonia.
The efficacy, in the
treatment of the infected rat lungs with two clinically relevant antibiotics, gentamicin and ceftazidime entrapped in SL, appeared to be greater than the free drug alone.
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161
Gene therapy and tumour imaging Long circulating liposomes in the area gene therapy and imaging are currently attracting much attention. Although this review does not touch on these topics, they are of growing importance. To date, liposomal tumour imaging agents have yet to compete successfully in the imaging market, although SL may improve the outlook for these applications. In the area of gene therapy, although liposomes are attractive vectors for mediating transient DNA expression in cells in vitro, considerable basic research needs to be done before long circulating formulations of liposomal DNA with high transfection efficiencies will be achieved. There have been a number of recent review articles published both in the area of liposomes as DNA delivery systems (44) and liposomes in the area of imaging (45,46). Human studies
Passive targeting of doxorubicin to tumors The dose-limiting side effects of doxorubicin in human cancer chemotherapy include acute cardiomyopathy and myelosuppression. As previously mentioned, the efficacy of doxorubicin entrapped in SL has been demonstrated in solid tumors in animals.
Efficacy has also been demonstrated in human clinical trials in the
treatment of refractory Kaposi's sarcoma in patients infected with the HIV virus (47). In multi-centre trials, where Doxil R (doxorubicin entrapped in SL) was evaluated as a treatment for refractory Kaposi's sarcoma, both cutaneous and visceral lesions in the patients regressed during the course of treatment with a complete plus partial response rate of over 70% (47). No cardiotoxicity was observed in the patients and myelosupression was comparable to that seen for the free drug. The dose-limiting
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toxicities for Doxil Rwere stomatitis and hand and foot syndrome both of which would resolve upon dose reduction. Recently this product has been approved for use by the U.S. Food and Drug Administration in the treatment of refractory Kaposi's sarcoma. Doxil R is also currently in clinical trials for advanced breast, ovarian, and head and neck cancer. Daunorubicin, another anticancer drug, has been encapsulated in liposomes (DaunoXome R3) and has recently also been approved by the FDA as a first-line therapy for Kaposi's sarcoma in HIV-infected patients.
In DaunoXome R, the
liposomes contain the solid phase lipid distearoylphosphatidylcholine (DSPC) and this, combined with their small size, also results in prolonged circulation times, although the clearance rate in humans is somewhat shorter than that of Doxil R (48). Unlike S-liposomal doxorubicin, these liposomes are reported to possess dosedependent pharmacokinetics (48). THE FUTURE With SL in clinical trials or in the clinic, considerable recent effort in liposome research has been directed at developing a third generation of liposomes, i.e. one in which there is site-specific targeting of the SL by attaching "homing devices" such as monoclonal antibodies or receptor-specific ligands to the liposome surface (Figure 1C). A number of coupling methods have been developed for the attachment of antibodies or ligands to liposomes (reviewed in reference 49). Our laboratory has investigated a number of cancer models in mice to test
3 DaunoXome R is a trademark of NeXstar Inc., San Dimas, CA
LONG CIRCULATING LIPOSOMES
163
whether selective toxicity can be achieved in vivo using antibody- or ligand-targeted SL.
The
models
have
included
routine
squamous
lung
carcinoma
(a
pseudometastatic model), human ovarian cancer (a solid tumor model) and human B cell lymphoma (a hematological model).
Targeted SL (SIL) appear to have
increased efficacy over non-targeted liposomes or free drug in the treatment of human proliferative disorders of B or T-cell lineages grown as xenograft models in SCID mice (50). Increased therapeutic efficacy of targeted SL over non-targeted SL was possible in the treatment of solid tumors early (i.e. at the micrometastatic stage), but not late, in their development in a squamous lung carcinoma model in mice (50). On the other hand, non-targeted liposomes, e.g. SL- doxorubicin, appeared to be more effective than SIL-doxorubicin in treating human ovarian tumors, grown in SCID mice, in a more advanced stage of development (50) In order to explain the differences in efficacy between targeted and nontargeted SL in the treatment of hematological cancers versus early solid tumors versus more advanced solid tumors, we suggest the following explanation.
As has
been mentioned above, non-targeted SI_ can percolate through leaky capillaries of tumors which are undergoing the process of angiogenesis. A solid tumor will contain relatively large numbers of cells by the time it begins to become deficient in oxygen and nutrients and starts to send out signals recruiting capillary growth into the tumor. When metastatic cells from solid tumors are migrating in blood and lymph, or exist as micrometatases, they consist of individual cells or small groups of cells and lack the capillary network which allows the passive targeting of non-ligand bearing liposomes (SL) to the tumor. Therefore SL appear to be less effective against very
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early solid tumors, although they have good efficacy against more advanced solid tumors undergoing angiogenesis. Even if the SL cannot reach all tumor cells, once they have localized in the tumor drug released from the liposomes will diffuse further into the tumor resulting in an additional cytotoxic response.
Targeted liposomes
(SIL), on the other hand, will have relatively unimpeded access to, and will therefore be able to bind to, most of the cells in very small tumors, and to individual metastatic cells released from primary tumors (Figure 3A).
By the same argument, SIL are
effective in hematological malignancies, as they have easy access to the diseased cells within the circulation, and their ability to bind to these cells gives them an advantage over non-targeted (i.e. non-binding) liposomes. However when SIL reach an advanced solid tumour, they will bind to the first target cells they recognize. This prevents them from percolating further into the tumor to kill target cells deep in the tumor mass (Figure 3B). This phenomenon has been referred to as the "binding site barrier" (51). Once targeted liposomes have arrived at the desired target there are a number of possible scenarios.
Internalization of the drug-liposome package into the
endocytotic apparatus of the cell can occur if the targeting moiety is against an internalizing epitope on the cell surface.
If the drug is capable of surviving the
endocytotic environment, it would then be released into the cell cytoplasm once the carrier is destroyed by lysosomal enzymes (Figure 4A), Alternatively, binding of the targeted liposomes to a non-internalizing epitope will result in a sustained release of the liposomal contents in locally high concentrations at the cell surface. The released drug will enter the cell either by passive diffusion or by a transport system (Figure
LONG CIRCULATING LIPOSOMES
165
FIGURE 3. Schematic illustration of the binding of SIL to tumor cells (not to scale). A, Binding of SIL to micrometastases (or indivdual tumor cells). SIL bind to epitopes at the surface of the cancer cell. A small number of cells allows for targeting and drug delivery to all of the cells. B. Binding of SIL to advanced solid tumors. The SIL bind to epitopes on the first cancer cells they encounter, making it difficult for the SIL to penetrate further into the interior of the tumor mass (binding site barrier). Drug released from the liposomes does not reach effective concentrations in the inner portion of the tumor mass.
A. BINDING OF IMMUNOLIPOSOMES TO MICROMETASTASES
.~
cln.~-fllledtargeted tmmunc~igx)some[SILl
.'.
released drug molecules
•
surface eloitope on cancer cells
B. BINDINO-.-~ITEBARRIER FOR I M M U N O L I ~ M E S ~ D TO MORE ADVANCED SOLID TUMORS
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J.M.J. MARJAN and T.M. ALLEN
FIGURE 4. Schematic illustrations of some possible mechanisms for the cytotoxic effects of SIL (not to scale). A. Binding of SIL to an internalizing epitope on the surface of the cell triggers the internalization of the drug-liposome package. Cytotoxicity is achieved when the internalized liposome is degraded within the endocytotic apparatus of the cell and the drug reaches the cell cytoplasm and/or nucleus. B. Binding of SIL to a non-internalizing epitope on the surface of the cell. Drug will leak from liposomes bound to the cell surface possibly achieving locally high drug concentrations. The cytotoxic effect is dependent on the rate of leakage of the drug from the liposome and the rate at which the drug enters the cell either by passive diffusion, or by active transport. Diffusion may, however, carry the drug away from the cell surface faster than it can enter the cell. C. Bystander effect. Drug released from liposomes floating free in the tumor or attached via a non-internalizing epitope at the surface of a nearby cancer cell has the ability to be cytotoxic to a cancer cell in close proximity (bystander) that bears nonrecognized epitopes at the cell surface.
SOME MECHANISMS OF ACTION OF IMMUNOLIPOSOMES
Internalizing epilope
Q
endc< Non-internalizing epitope Non*recognized epitooe
diffusion
X .
!
LONG CIRCULATING LIPOSOMES
167
4B). The amount of drug entering the cell will have an inverse relationship to the rate of diffusion of the released drug away from the liposome surface.
Another
mechanism for cell kill in solid tumors is the "bystander effect" in which cells lacking the target epitope may be killed by drug released at the surface of neighbouring cells having bound liposomes (52) (Figure 4C). Another proposed mechanism of action is the "target-cell dragging" concept (53) whereby the targeted liposomes binds to its target cell in the bloodstream and is flagged by phagocytic cells due to the size of the complex or the presence of antibodies.
Phagocytic cells may engulf this complex and the drug and/or the
macrophage could kill the target cell. This concept resembles a long recognized pathway in the immune system to dispose of circulating antigens.
However, the
phagocytic cell may also be killed by the released drug and the consequences of this are not well understood. The successful achievement of targeting in animal models in our laboratory (36,50,54) has given us some insights into the requirements that are needed for this therapy to reach clinical development.
The ideal targeted carrier would have to
include at least four characteristics: i)
the ability to retain long circulating half-life despite the presence of antibodies or other targeting ligands.
ii)
coupling methodologies must be efficient, simple, rapid and compatible for use in humans.
iii)
the presence of the antibody should not interfere with drug loading and release mechanisms.
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J.M.J. MARJAN and T.M. ALLEN
iv)
SIL's should retain their target recognition in vivo and be able to bind in sufficient numbers in order to release the drug and achieve therapeutic efficacy superior to the free drug alone or the liposomal drug in the absence of antibody.
With the careful choice of drug, antibody, coupling method, and tumor model, e.g. hematological versus solid tumor, these criteria appear to be achievable. Before antibody-targeted long circulating liposomes reach the clinic, additional problems need to be addressed, including the potential immunogenicity of murine antibodies presented on liposomes. Can we get away from this problem by coupling "humanized antibodies" to liposomes? Will antibody fragments be less immunogenic than whole antibodies? How can the "binding-site barrier" be overcome? Rodent (monoclonal antibodies) have been "humanized" by linking rodent variable regions and human constant regions (chimeric antibodies) (55). "Humanizing" an antibody reduces
its immunogenicity; however a recent publication describing the clinical
application of a "humanized" antibody demonstrated that there is a partial immune response that is mounted by the patient (56). On the other hand, in a recent clinical trial, a humanized rat therapeutic antibody directed against mature human leukocytes proved clinically effective in destroying a large tumour mass in two patients (57). CONCLUSIONS This review has briefly described the journey of long circulating liposomes from their inception to their present applications, with some discussion of future directions for their development. The frequently asked question is why liposomes have not had more of a clinical impact? Liposomes, like most new technologies (including gene
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therapy at present) have been the victims of exaggerated claims in the absence of the basic research to support these claims. Once the basic research into formulation, stability, pharmacokinetics, etc. was undertaken, liposome formulations could be designed which married our understanding of liposome properties with current concepts of disease, e.g. tumor cell biology, leading to rational clinical applications. With five liposomal formulations now in the clinic, and many more in clinical trials, the wisdom of taking this approach is evident. We look forward to a bright future for this emerging technology.
REFERENCES
1)
Bangham, A.D., Standish, M.M., Watkins, J.C.
Diffusion of univalent ions
across the lamellae of swollen phospholipids. J. Mol. Biol. 13:328-252, 1965.
2)
Allen, T.M. and Chonn, A. Large unilamellar liposomes with low uptake into the reticuloendothelial system. FEBS Lett., 223:42-46, 1987.
3)
Gabizon, A. and Papahadjopoulos, D. Liposome formulations with prolonged circulation time in blood and enhanced uptake by tumors. Proc. Natl. Acad. Sci. USA, 85:6949-4953, 1988.
4)
Allen, T.M.
Stealth liposomes, Avoiding reticuloendothelial uptake, in
Liposomes in the Therapy of Infectious Diseases and Cancer, New Series, Vol. 89, Eds. G. Lopez-Berestein and I. Fidler, Alan R.Liss Inc, New York, 1989, pp. 405-415.
5)
Allen, T.M and Papahadjopoulos,
D.
Sterically stabilized ("Stealth")
liposomes: Pharmacokinetics and therapeutic advantages in: Liposome Technology, 2rid Edition, Vol. 111, Ed. G. Gregoriadis, CRC Press, Boca Raton, Florida, 1993 pp. 59-72.
6)
Mumtaz, S., Ghosh, P.C. and Bachhawat, B.K. Design of liposomes for circumventing the reticuloendothelial cells. Glycobiol., 1:505-510, 1991.
170
J,M.J. MARJAN and T.M. A L L E N
7)
Blume G. and Cevc, G. Molecular mechanism of the lipid vesicle longevity in vivo. Biochim. Biophys. Acta, 1029:91-97, 1990.
8)
Chonn, A., Semple, S.C. and Cullis, P.R. Association of blood proteins with large unilamellar liposomes in vivo, Relation to circulation lifetimes. J. Biol. Chem., 267:18759-18765, 1992.
9)
Gabizon, A. and Papahadjopoulos, D.
The role of surface charge and
hydrophillic groups on liposome clearance in vivo. Biochim. Biophys. Acta, 1103:94-100, 1992. 10)
Lasic, D.D., Martin, F.J., Gabizon, A., Huang, S.K. and Papahadjopoulos, D. Sterically stabilized liposomes, A hypothesis on the molecular origin of the extended circulation times. Biochim. Biophys. Acta, 1070:187-192, 1991.
11)
Needham, D., Kristova, K., Mclntosh, T.J., Dewhirst, M., Wu, N. and Lasic D.D. Polymer-grafted liposomes, Physical basis for the "Stealth" property. J. Liposome Res., 2:411-430, 1992.
12)
Woodle, M.C. and Lasic D.D.
Sterically stabilized liposomes.
Biochim.
Biophys. Acta, 1113:171-199, 1992. 13) 14)
Pool, R. "Hairy" enzymes stay in the blood. Science, 248:305, 1990. Torchilin, V.P, Omelyanenko, V.G., Papisov, M.I., Bogdanov, A.A., Trubetskoy, V.S., Herron, J.N., Gentry, C.A. Poly(ethylene glycol) on the liposome surface: on the mechanism of polymer-coated liposome longevity. Biochim. Biophys. Acta, 1195:11-20, 1995.
15)
Needham, D., Mclntosh, T.J., Lasic, D.D.
Repulsive interactions and
mechanical stability of polymer-grafted lipid membranes. Biochim. Biophys. Acta, 1108:40-48, 1992. 16)
Gabizon, A., Dagan, A., Goren, D., Barenholz, Y. and Fuks, A.
Liposomes
as in vivo carriers of adriamycin, Reduced cardiac uptake and preserved antitumour activity in mice. Cancer Res., 42:4734-4739, 1982.
LONG CIRCULATING L1POSOMES
17)
171
Maruyama, K., Yuda, T., Okamoto, A., Ishikura, C., Kojima, S., Iwatsuru, M. Effect of molecular weight in amphipathic polyethylene glycol on prolonging the circulation time of large unilamellar liposomes. Chem. Pharm. Bull, 39:16201622, 1991.
18)
Woodle, M.C.
Surface modified liposomes: assessment and stability and
prolonged circulation. Chem. Phys. Lipids, 64:249-262, 1993. 19)
Zalipsky, S., Hansen, C.B., Oaks, J.M. and Allen, T.M. Evaluation of blood clearance and biodistribution of poly(2-oxazoline)-grafted
liposomes.
J.
Pharm. Sci., (in press). 20)
Torchilin, V.P., Shtilman, M.I., Trubetskoy, V.S., Whiteman, K., Milstein, A.M. Amphiphilic vinyl polymers effectively prolong liposome circulation time in vivo. Biochim. Biophys. Acta., 1195:181-184, 1994.
21)
Olson, F., Hunt, C.A., Szoka, F.C., Vail, W.J., Papahadjopoulos,
D.
Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. Biochim. Biophys. Acta, 557:9-23, 1979. 22)
Litzinger, D.C., Buiting, AM.J, van Rooijen, N., Huang, L. Effect of liposome size on the circulation time and intraorgan distribution of amphipathic poly(ethylene glycol)-containing liposomes. Biochim. Biophys. Acta, 1190:99107, 1994.
23)
Papahadjopoulos, D., Allen, T.M., Gabizon, A., Mayhew, E., Matthay, K., Huang, S.K., Lee, K.D., Woodle, M.C., Lasic, D.D., Redemann, C., Martin, F.J. Sterically stabilized liposomes: Improvements in pharmacokinetics
and
antitumor therapeutic efficacy. Proc. Natl. Acad. Sci. USA, 88:11460-11464, 1991. 24)
Allen, T.M., Hansen, C.B., de Menezes, D.E.L. Pharmacokinetics of longcirculating liposomes. Adv. Drug. Deliv. Rev., 16:267-285, 1995.
25)
Mayer, L.D., Tai, L.C.L., Bally, M.B., Mitilenes, G.N., Ginsberg, R.S., Cullis, P.R. Characterization of liposomal systems containing doxorubicin entrapped in response to pH gradients. Biochim.Biophys.Acta 1025:143-151, 1990.
172
J.M.J. M A R J A N and T.M. A L L E N
26)
Mayer, L.D., Bally, M.B., Cullis, P.R.
Uptake of adriamycin into large
unilamellar vesicles in response to a pH gradient.
Biochim.Biophys.Acta
857:123-126, 1986. 27)
Haran, G., Cohen, R., Bar, L.K., Barenholz, Y. Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases. Biochim.Biophys.Acta. 1151:201-215, 1993.
28)
Dvorak, H.F., Nagy, J.A., Dvorak, J.T., Dvorak, A.M.
Identification and
characterization of the blood vessels of solid tumors that are leaky to circulating macromolecules Am. J. Pathol., 133:95-109, 1988. 29)
Wu, N.Z., Da, D., Rudoll, T.L., Needham, D., Dewhirst, M.W.
Increased
microvascular permeability contributes to preferential accumulation of Stealth liposomes in tumor tissue. Cancer Res., 53:3765- 3770, 1993. 30)
Allen, T.M. and Mehra T.
Recent advances in sustained release of
antineoplastic drugs using liposomes which avoid uptake in to the reticuloendothelial system. Proc. Western Pharmacol. Soc., 32:111-114, 1989. 31)
Baguley, B.C. and Falkenhaug, E.M. Plasma half-life of cytosine arabinoside (NSC-63878) in patients treated for acute myeloblastic leukaemia. Cancer Chemother. Rep., 55:291-298 (1971)
32)
Borsa, J., Whitmore, G.R., Valeriote, F.A., Collines, D. and Bruce, W.R. Studies on the persistence of methotrexate, cytosine arabinoside and leucovorin in the serum of mice. J. Nat. Cancer Inst., 42:235-242, 1969.
33)
Allen, T.M., Mehra, T., Hansen, C., Chin, Y-C.
Stealth liposomes: an
improved sustained release system for 1-1~-D-arabinofuranosylcytosine. Cancer Res., 52:2431-2439, 1992. 34)
Allen, T.M., Newman, M.S., Woodle, M.C., Mayhew, E., Uster, P.S. Pharmacokinetics and antitumor activity of vincristine encapsulated in sterically stabilized liposomes. Int. J. Cancer, 62:199-204, 1995
35)
Vaage, J., Donovan, A., Mayhew, E., Uster, P., Woodle, M. Therapy of mouse mammary carcinomas with vincristine and doxorubicin encapsulated in sterically stabilized liposomes. Int. J. Cancer, 54:959-964, 1993.
LONG CIRCULATING LIPOSOMES
36)
173
Ahmad, I., Longenecker, M., Samuel, J. and Allen, T.M. Antibody-targeted delivery of doxorubicin entrapped in sterically stabilized liposomes can eradicate lung cancer in mice. Cancer Res., 53:1484-1488, 1993.
37)
Gabizon, A.A. Selective tumor localization and improved therapeutic index of anthracyclines encapsulated in long-circulating liposomes.
Cancer Res.,
52:891-896, 1992. 38)
Jain, R.K.
Transport of molecules across tumor vasculature.
Cancer
Metastasis Rev., 6:559-593, 1987. 39)
Mayhew, E.G., Lasic, D., Babbar, S., and Martin, F.J. Pharmacokinetics and antitumor activity of epirubicin encapsulated in long-circulating liposomes incorporating a polyethylene glycol-derivatized phospholipid. Int. J. Cancer, 51:302-309, 1992.
40)
Papahadjopoulos, D., Allen, T.M., Gabizon, A., Mayhew, E., Matthay, K., Huang, S.L., Lee, K-D, Woodle, M.C., Lasic, D.D., Redemann, C. and Martin, F.J. Sterically stabilized liposomes, Improvements in pharmacokinetics and antitumor therapeutic efficacy. Proc. Natl. Acad. Sci. USA, 88:11460-11464, 1991.
41)
Vaage, J., Mayhew, E., Lasic, D.D. and Martin, F.J. Therapy of primary and metastatic mouse mammary carcinomas with doxorubicin encapsulated in long circulating liposomes. Int. J. Cancer, 51:942-948, 1992.
42)
Williams, S.S., Alosco, T.R., Mayhew, E., Lasic, D.D., Martin, F.J., and Bankert, R.B.
Arrest of human lung tumor xenograft grown in severe
combined immunodeficient mice using doxorubicin encapsulated in sterically stabilized liposomes. Cancer Res., 53:3964-3967, 1993. 43)
Bakker-Woudenberg, I.A.J.M., Storm, G., Woodle, M.C.
Liposomes in the
treatment of infections. J. Drug Targeting, 2:363-371, 1994. 44)
Miller, N., Vile, R. Targeted vectors for gene therapy. FASEB J., 9:190-199, 1995.
174
J,M.J. M A R I A N and T.M. A L L E N
45)
Weissleder, R., Bogdanov, A., Neuwlt, E.A., Papisov, M. Long circulating iron oxides for magnetic resonance imaging. Adv. Drug Deliv. Rev., 16:3231-334, 1995.
46)
Bogdanov, A.Jr., Weissleder, IR., Brady, T.
Long circulating blood pool
imaging agents. Adv. Drug Deliv. Rev., 16:335-348, 1995. 47)
Northfelt, D.W., Martin, F.J., Kaplan, L.D., Russell, J., Anderson, M., Lang, J., Volberding, P.A. Pharmacokinetics, tumor localization and safety of Doxil (liposomal Doxorubicin) in AIDS patients with Kaposi's sarcoma. Proc. Am. Soc. Clin. Oncol., 12:51, 1993.
48)
Gill, P.S., Espina, B.M., Muggia, F., Cabriales, S., Tulpule, A., Esplin, J.A., Liebman, H.A., Forssen, E., Ross, M.E., Levine, A.M. Phase 1/11clinical and pharmacokinetic evaluation of liposomal daunorubicin. J. Clin. Oncol., 13:9961003, 1995.
49)
Hansen, C.B., Kao, G.Y., Moase, E.H., Zalipsky, S., Allen, T.M. Attachment of antibodies to sterically stabilized liposomes: evaluation, comparison and optimization of coupling procedures. Biochim. Biophys, Acta, 1239:133-144, 1995.
50)
Allen,
T.M.,
Ahmad,
I.,
Lopes
de
Menezes,
D.E.,
Moase,
E.H.
Immunoliposome-mediated targeting of anticancer drugs in vivo. Biochem. Society Trans., 23:1073-1079,1995. 51)
Weinstein, J.N. and van Osdol, W.
Early intervention in cancer using
monoclonal antibodies and other biological ligands:micropharmacology and the "Binding Site Barrier". Cancer Res., 52:2747-2751, 1992. 52)
Allen, T.M. Long-circulating (sterically stabilized) liposomes for targeted drug delivery. Trends in Pharmacological Sciences, 15:215-220, 1994.
53)
Vingerhoeds, M.H., Storm, G., Crommelin, D.J.A. Immunoliposomes in vivo. Immunomethods, 4:259-272, 1994.
54)
ALlen, T.M., Agrawal, A.K., Ahmad, I., Hansen, C.B. and Zalipsky, S. Antibody-mediated targeting of long-circulating (Stealth R) liposomes. Liposome IRes., 4:1-25, 1994.
J.
LONG CIRCULATING LIPOSOMES
55)
175
Neuberger, M.S., Williams, G.T., Mitchell, E.B., Jouhal, S.S., Flanagan, J.G., Rabbitts, T.H.
A hapten-specific chimeric IgE antibody with human
physiological effector function. Nature, 314:268-270, 1985. 56)
LoBuglio, A.F., Wheeler, R.H., Trang, J., Haynes, A., Rogers, K., Harvey, E.B., Sun, L., Ghrayeb, J., Khazaeli, M.B. Mouse/human chimeric monoclonal antibody in man: kinetics and immune response. Proc. Natl. Acad. Sci. USA, 86:4220-4224, 1989.
57)
Hale, G., Dyer, M.J., Clark, M.R., Phillips, J.M., Marcus, R., Reichmann, L., Winter, G., Waldmann, H. Remission induction in non-Hodgkin lymphoma with reshaped human monoclonal antibody CAMPATH-1H. Lancet, ii(8625):13941399, 1988.