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BACKGROUND OF THE INVENTION
The present invention is directed to liposomes. More particularly, the
present invention is directed to the covalent and non-covalent coupling of
liposomes to proteins for purposes of in vivo targeting, or for use in
diagnostics.
Liposomes are completely closed structures composed of lipid bilayer
membranes containing an encapsulated aqueous volume. Liposomes may contain
many concentric lipid bilayers separated by aqueous phase (multilamellar
vesicles or MLVs), or may be composed of a single membrane bilayer
(unilamellar vesicles).
Liposome preparation has typically been achieved by the process of Bangham
et.al., (1965 J. Mol. Biol., 13: 238-252) whereby lipids suspended in
organic solvent are evaporated under reduced pressure to a dry film in a
reaction vessel. An appropriate amount of aqueous phase is then added to
the vessel and the mixture agitated, then allowed to stand, essentially
undisturbed for a time sufficient for the multilamellar vesicles to form.
The aqueous phase entrapped within the liposomes may comprise bioactive
agents including, but not limited to, drugs, hormones, proteins, dyes,
vitamins, or imaging agents.
The current state of the art is such that liposomes may be reproducibly
prepared using a number of techniques. Liposomes resulting from some of
these techniques are small unilamellar vesicles (SUVs) Papahadjopoulos and
Miller, Biochem. Biophys. Acta, 135, p. 624-638 (1967), reverse-phase
evaporation vesicles (REV) U.S. Pat. No. 4,235,871 issued Nov. 25, 1980,
stable plurilamellar vesicles (SPLV) U.S. Pat. No. 4,522,803 issued June
11, 1985, and large unilamellar vesicles produced by an extrusion
technique as described in copending U.S. patent application Ser. No.
788,017, filed Oct. 16, 1985, Cullis et.al., entitled "Extrusion Technique
for Producing Unilamellar Vesicles", relevant portions of which are
incorporated herein by reference.
One of the primary uses for liposomes is as carriers for a variety of
materials, such as, drugs, cosmetics, diagnostic reagents, bioactive
compounds, and the like. Such systems may be designed for both diagnostics
and in vivo uses. In this regard, the ability to produce an
antibody-directed vesicle would be a distinct advantage over similar
undirected systems (Gregoriadis, G., Trends Pharmacol Sci, 4, p. 304-307,
1983). Useful applications would be in the selective targeting of
cytotoxic compounds entrapped in vesicles to circulating tumor cells
(Wolff et.al., Biochim Biophys. Acta, 802, p. 259-273 1984), or
applications of these immunoglobulin-associated vesicles in the
development of diagnostic assays. As is well known in the art, liposomes
may be covalently coupled to proteins, antibodies and immunoglobins. Heath
et.al. (Biochim. Biophys. Acta., 640, p. 66-81, 1981), describe the
covalent attachment of immunoglobulins to liposomes containing
glycosphingolipid. Leserman et. al. (Liposome Technology, III, 1984, CRC
Press, Inc., CA., p. 29-40; Nature, 288, p. 602-604, 1980) and Martin et.
al., (J. Biol. Chem., 257, p. 286-288, 1982) have described procedures
whereby thiolated IgG or protein A is covalently attached to lipid
vesicles, and thiolated antibodies and Fab' fragments are attached to
liposomes, respectively. These protocols and various modifications (Martin
et.al, Biochemistry, 20, p. 4229-4238, 1981; and Goundalkar et.al., J.
Pharm. Pharmacol. 36, p. 465-466, 1984) represent the most versatile
approaches to coupling. Avidin-coupled and avidin and biotinyl-coupled
phospholid liposomes containing actinomycin D have successfully targeted
tumor cells expressing ganglio-N-triosylceramide (Urdal et al., J. Biol.
Chem., 255, p. 10509-10516, 1980). Huang et al. (Biochim. Biophys. Acta.,
716, p. 140-150, 1982) demonstrate the binding of mouse monoclonal
antibody to the major histocompatibility antigen H-2 (K), or goat antibody
to the major glycoprotein of Molony Leukemia Virus, to palmitic acid.
These fatty acid modified IgGs were incorporated into liposomes, and the
binding of these liposomes to cells expressing the proper antigens
characterized. Other in vitro efforts to specific binding of liposomes
coated with specific immunoglobins have been performed (Sharkey et al.,
Fed. Proc., 38, p. 1089, 1979). In still other coupling studies, Rahman
et. al. found that tissue uptake of liposomes could be altered by
attachment of glycolipids to the liposomes (J. Cell Biol., 83, p. 268a,
1979).
One aspect of the present invention is to couple biotinylated proteins such
as immunoglobulins and antibodies to liposomes with covalently-attached
streptavidin. Methods for this coupling are herein provided. The nature of
this covalent attachment between streptavidin and the liposomes is a
chemical bonding between the streptavidin, and derivatized
phosphatidylethanolamine incorporated in the liposome bilayer. In a second
aspect of the invention, Applicants provide a two-step method for the
non-covalent coupling of these biotinylated proteins to
biotinylated-phosphatidylethanolamine (PE)-containing liposomes through
the same streptavidin linker. This non-covalent attachment of streptavidin
and liposomes occurs through a specific association between four specific
biotin binding sites on streptavidin, and the biotin. These
antibody-liposome complexes bind specifically to target cells as directed
by the coupled antibody. Such liposomes may be made to contain bioactive
agents such as drugs.
In accordance with a primary use for liposomes, the entrapment of
antineoplastic agents inside liposomal bilayers has resulted in more
efficacious therapy as compared to direct administration of the drug.
(Forsben et al., Cancer Res., 43, p. 546, 1983; and Gabizon et al., Cancer
Res., 42, p. 4734, 1982). A problem with the encapsulation of
antineoplastic drugs is in the fact that many of these drugs have been
found to be rapidly released from liposomes after encapsulation. This is
an especially undesirable effect, in view of the fact that toxicity of
these agents can be significantly reduced through liposome encapsulation
as compared to direct administration. See, for example, Forssen et al.
Cancer Res. 43, 546 (1983) and Rahman et al. Cancer Res., 42, 1817 (1982).
Clearly, a method whereby drug could be loaded into preformed liposomes
would be advantageous. To achieve this object, the invention, in
accordance with one of its aspects, provides a method for loading
liposomes with ionizable antineoplastic agents wherein a transmembrane
potential is created across the walls of the liposomes and the
antineoplastic agent is loaded into the liposomes by means of the
transmembrane potential. See also U.S. patent application Ser. No.
749,161, Bally et al. entitled "Encapsulation of Antineoplastic Agents in
Liposomes", filed June 26, 1985, relevant portions of which are
incorporated herein by reference.
In accordance with these needs, a liposome composition is presented which
describes the use of protein-coupled liposomes which may be stored stably
for an indefinite period, in a dehydrated state, with loading of the
liposomes on an "as needed" basis.
SUMMARY OF THE INVENTION
We have prepared a liposome composition whereby the glycoprotein
streptavidin is coupled to liposomes for purposes of liposome targeting.
The streptavidin may in turn couple biotinated proteins such as
Immunoglobulin G or monoclonal antibodies and be loaded with a variety of
bioactive agents, depending on use. Such agents may be the antineoplastic
agents such as daunorubicin, doxorubicin, and vinblastine.
The liposomes are preferably prepared in such a way as to create a
transmembrane potential across their lamellae in response to a
concentration gradient. This concentration gradient may be created by
either Na.sup.+ /K.sup.+ potential or pH (H.sup.+). The difference in
internal versus external potential is the mechanism which drives the
loading of the liposomes with ionizable bioactive agents; delayed loading
of preformed liposomes will occur in response to the transmembrane
potential. These liposomes may be dehydrated in the presence of one or
more protecting sugars such as the disaccharides trehalose and sucrose,
stored in their dehydrated condition, and subsequently rehydrated with
retention of the ion gradient and associated ability to accumulate the
bioactive agent. Such bioactive agents may be those used as in vivo
pharmaceutical preparations, such as antineoplastic agents including
doxorubicin. These preparations may be administered to a subject for
treatment of disease. Alternatively, the coupled liposome preparations may
be used in diagnostic assays. Methods are provided for the preparation of
liposomes either covalently or non-covalently coupled to streptavidin,
which in turn are complexed with biotinylated proteins such as IgG or
monoclonal antibodies. In the case of non-covalent binding of liposomes to
streptavidin, the liposomes comprise biotinylated
Phosphatidylethanolamine. Such liposomes are incubated with, for example,
about 10-fold molar excess streptavidin to biotinylated
phosphatidylethanolamine (PE), to complete the coupling reaction. The
liposomes may be large unilamellar vesicles, and may also comprise egg
Phosphatidylcholine (EPC).
In preparations containing EPC and biotinylated PE, the latter is in an
about 0.1 to 0.5% mole ratio with the EPC, preferably an about 0.1% mole
ratio.
Compositions of protein-streptavidin-biotinylated PE liposomes wherein the
protein is a monoclonal antibody or Immunoglobulin G are claimed. These
liposomes may also comprise a bioactive agent. They may be used in vivo as
a pharmaceutical preparation in a subject or alternatively in in vitro
diagnostic assays by contacting a sample with the composition.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a graph showing coupling of anti-rat erythrocyte IgG to EPC
LUVETs (labeled with .sup.3 H-DPPC) containing PDP-PE (closed triangle)
and MPB-PE (closed circle) as a function of cholesterol content.
FIG. 2 is a graph characterizing the covalent coupling reaction of IgG to
vesicles with regards to time course, of (A), MPB-PE concentration (B) and
IgG concentration (C).
FIG. 3 is a graph showing the influence of reaction pH on covalent coupling
of anti-human erythrocyte IgG (closed circle) and streptavidin (closed
triangle) to EPC/Chol (50:45) vesicles containing 5 mol % MPB-PE.
FIG. 4 is a graph showing the efficiency of covalent coupling of anti-human
erythrocyte IgG to vesicles of variable size.
FIG. 5 is a graph showing the elution profile for biotinated anti-human
erythrocyte IgG to vesicles which have covalently coupled streptavidin.
FIG. 6 is a graph showing the accumulation of adriamycin into
covalently-coupled streptavidin-vesicles ("avisomes") which were prepared
with a transmembrane pH gradient. Vesicles composed of EPC (closed circle)
or EPC/Chol (closed triangle).
FIG. 7 is a graph showing the non-covalent coupling of streptavidin to LUVs
containing biotinylated phosphatidylethanolamine.
DETAILED DESCRIPTION OF THE INVENTION
As described above, the present invention describes a liposome composition
that results from the coupling of the liposomes to streptavidin. In
addition, the composition can be dehydrated and rehydrated. The liposome
portions can be loaded with a chosen bioactive agent by potential
difference of ions across the bilayer membranes during the rehydration
step or subsequently thereto. Alternatively, the bioactive agent may be
added to the liposomes prior to dehydration. The streptavidin-coupled
liposomes can be coupled to proteins such as Immunoglobulin G or
monoclonal antibodies which have been biotinated by coupling to biotin.
Quite surprising is the observed stability of the
streptavidin-liposome-biotinated protein complex which makes streptavidin
an attractive coupler between the liposomes and the targeting proteins.
The proteins bound to the liposomes aid in targeting the liposomes and
their contents to a specific site in the body.
In one embodiment of the present invention, liposomes are formed using the
LUVET apparatus described in copending U.S. patent application entitled
"Extrusion Technique for Producing Unilamellar Vesicles", Ser. No.
622,690, filed June 20, 1984, relevant portions of which are incorporated
herein by reference, and covalently coupled to streptavidin using a
modified technique of Leserman et al., (Liposome Technology, III, 1984,
CRC Press, Inc., N.Y., p. 29-40). Liposomes are formed with a
transmembrane potential i.e. Na.sup.+ /K.sup.+ gradient or H.sup.+
potential difference across the bilayers, see copending U.S. patent
application, Ser. No. 749,161, Bally et al., entitled "Encapsulation of
Antineoplastic Agents in Liposomes", filed June 26, 1985, relevant
portions of which are incorporated herein by reference; this potential
difference effected by the ionic concentrations of the internal versus the
external media of the liposome. Liposomes are then dehydrated either in
the presence or absence of sugars such as trehalose, and may be stored in
this state for indefinite periods of time; see copending U.S. patent
application, Ser. No. 759,419, Janoff et al., entitled "Dehydrated
Liposomes," filed July 26, 1985, relevant portions of which are
incorporated herein by reference.
In another embodiment of the present invention, biotinylated proteins are
non-covalently coupled to biotinylated PE-containing liposomes via
streptavidin. The non-covalent binding of the streptavidin to the
liposomes, the first step, involves incorporation of biotin-PE in the
liposomes, followed by a second step of binding the streptavidin to the
biotinylated protein. The proteins are prepared for this binding by the
use of fluorescent derivatizing reagents such as the fluorescent amine
reagent fluorescein-isothiocyanate (FITC).
There are four biotin binding sites on the streptavidin, which makes
liposomes containing biotin aggregate with streptavidin in an excess of
biotinylated PE. Thus, the amount of biotinylated PE to incorporate into
the liposomes was titrated in order to prevent this aggregation, while
maximizing the streptavidin coupling. Values for biotinylated PE may range
from about 0.05 to 0.5 mole % (with respect to total lipid in the liposome
preparation); if the amount of biotin is increased further than about
0.5%, complete aggregation and precipitation of liposomes is observed on
addition of streptavidin. This aggregation phenomenon may be exploited in
the use of these systems in an aggregation-type diagnostic assay.
The biotinylated antibody is then attached to the streptavidin coated
liposome. These liposomes effectively targeted specifically to their
target cells with little non-specific binding.
The liposomes used in the present invention can have a variety of
compositions and internal contents, and can be in the form of
multilamellar, unilamellar, or other types of liposomes, or more
generally, lipid-containing particles, now known or later developed. For
example, the lipid-containing particles can be in the form of steroidal
liposomes, Ser. No. 599,691, now abandoned, alpha-tocopherol containing
liposomes, Ser. No. 786,740, now abandoned, stable plurilamellar liposomes
(SPLVs), U.S. Pat. No. 4,522,803, issued June 11, 1985, monophasic
vesicles (MPVs), U.S. Pat. No. 4,588,578, issued May 13, 1986, or lipid
matrix carriers (LMC), U.S. Pat. No. 4,610,868, issued Sept. 9, 1986, the
pertinent portions of which are incorporated herein by reference. Within
the class of liposomes that may be used in the present invention is a
preferred subclass of liposomes characterized in having solute
distribution substantially equal to the solute distribution environment in
which prepared. This subclass may be defined as stable plurilamellar
vesicles (SPLV), monophasic vesicles (MPVs), and frozen and thawed
multilamellar vesicles (FATMLVs) as described in "Solute Distributions and
Trapping Efficiencies Observed in Freeze-Thawed Multilamellar Vesicles"
Mayer et al. Biochimica et Biophysica Acta 817:1983-196 (1985). It is
believed that the particular stability of the SPLV type liposomes arises
from the low energy state attendant to solute equilibrium.
Alternatively, techniques used for producing large unilamellar liposomes
(LUVs), such as, reverse-phase evaporation, infusion procedures, and
detergent dilution, can be used to produce the liposomes. A review of
these and other methods for producing liposomes can be found in the text
Liposomes, Marc J. Ostro, ed., Marcel Dekker, Inc., New York, 1983,
Chapter 1, the pertinent portions of which are incorporated herein by
reference.
Compounds which are bioactive agents can be entrapped within the liposomes
of the present invention. Such compounds include but are not limited to
antibacterial compounds such as gentamycin, antiviral agents such as
rifampacin, antifungal compounds such as amphotericin B, anti-parasitic
compounds such as antimony derivatives, tumoricidal compounds such as
adriamycin, anti-metabolites, peptides, proteins such as albumin, toxins
such as diptheriatoxin, enzymes such as catalase, polypeptides such as
cyclosporin A, hormones such as estrogen, hormone antagonists,
neurotransmitters such as acetylcholine, neurotransmitter antagonists,
glycoproteins such as hyaluronic acid, lipoproteins such as
alpha-lipoprotein, immunoglobulins such as IgG, immunomodulators such as
interferon or interleuken, vasodilators, dyes such as Arsenazo III,
radiolabels such as .sup.14 C, radio-opaque compounds such as .sup.90 Te,
fluorescent compounds such as carboxy fluorscein, receptor binding
molecules such as estrogen receptor protein, anti-inflammatories such as
indomethacin, antigalucoma agents such as pilocarpine, mydriatic
compounds, local anesthetics such as lidocaine, narcotics such as codeine,
vitamins such as alpha-tocopherol, nucleic acids such as thymine,
polynucleotides such as RNA polymers, psychoactive or anxiolytic agents
such as diazepam, mono- di- and polysaccharides, etc. A few of the many
specific compounds that can be entrapped are pilocarpine, a polypeptide
growth hormone such as human growth hormone, bovin growth hormone and
porcine growth hormone, indomethacin, diazepam, alpha-tocopherol itself
and tylosin. Antifungal compounds include miconazole, terconazole,
econazole, isoconazole, tioconazole, bifonazole, clotrimazole,
ketoconazole, butaconazole, itraconazole, oxiconazole, fenticonazole,
nystatin, naftifine, amphotericin B, zinoconazole and ciclopirox olamine,
preferably miconazole or terconazole, The entrapment of two or more
compounds simultaneously may be especially desirable where such compounds
produce complementary or synergistic effects. The amounts of drugs
administered in liposomes will generally be the same as with the free
drug; however, the frequency of dosing may be reduced.
The liposomes of the present invention are preferably dehydrated using
standard freeze-drying equipment or equivalent apparatus, and, if desired,
the liposomes and their surrounding medium can be frozen in liquid
nitrogen before being dehydrated. Alternatively, the liposomes can also be
dehydrated without prior freezing, by simply being placed under reduced
pressure. Dehydration with prior freezing requires the presence of one or
more protective sugars in the preparation. A variety of sugars can be
used, including such sugars as trehalose, maltose, sucrose, glucose,
lactose, and dextran. In general, disaccharide sugars have been found to
work better than monsaccharide sugars, with the disaccharide sugars
trehalose and sucrose being most effective.
The one or more sugars are included as part of either the internal or
external media of the liposomes. Most preferably, the sugars are included
in both the internal and external media so that they can interact with
both the inside and outside surfaces of the liposomes' membranes.
Inclusion in the internal medium is accomplished by adding the sugar or
sugars to the solute which the liposomes are to encapsulate. Since in most
cases this solute also forms the bathing medium for the finished
liposomes, inclusion of the sugars in the solute also makes them part of
the external medium. Of course, if an external medium other than the
original solute is used, e.g., to create a transmembrane potential (see
below), the new external medium should also include one or more of the
protective sugars.
In the case of dehydration without prior freezing, if the liposomes being
dehydrated have multiple lipid layers and if the dehydration is carried
out to an end point where there is sufficient water left in the
preparation so that a substantial portion of the membranes retain their
integrity upon rehydration, the use of one or more protective sugars may
be omitted. It has been found preferable if the preparation contains at
the end of the dehydration process at least about 2%, and most preferably
between about 2% and about 5%, of the original water present in the
preparation prior to dehydration.
Once the liposomes have been dehydrated, they can be stored for extended
periods of time until they are to be used. When the dehydrated liposomes
are to be used, rehydration is accomplished by simply adding an aqueous
solution, e.g., distilled water, to the liposomes and allowing them to
rehydrate.
As discussed above, in accordance with another of its aspects, the present
invention provides a method for loading liposomes with ionizable
antineoplastic agents wherein a transmembrane potential is created across
the bilayers of the liposomes and the antineoplastic agent is loaded into
the liposomes by means of the transmembrane potential. The transmembrane
potential is generated by creating a concentration gradient for one or
more charged species (e.g., Na.sup.+, K.sup.+ and/or H.sup.+) across the
liposome membranes. The concentration gradient is created by producing
liposomes having different internal and external media, i.e., internal and
external media having different concentrations of one or more charged
species.
Specifically, liposomes are prepared which encapsulate a first medium
having a first concentration of the one or more charged species. For a
typical liposome preparation technique (see discussion above), this first
medium will surround the liposomes as they are formed, and thus the
liposomes' original external medium will have the same composition as the
first medium. To create the concentration gradient, the original external
medium is replaced by a new external medium having a different
concentration of the one or more charged species. The replacement of the
external medium can be accomplished by various techniques, such as, by
passing the liposome preparation through a gel filtration column, e.g., a
Sephadex column, which has been equilibrated with the new medium, or by
centrifugation, dialysis, or related techniques.
In accordance with the invention, it has been found that this transmembrane
potential can be used to load ionizable antineoplastic agents into the
liposomes. Specifically, once liposomes having a concentration gradient
and thus a transmembrane potential of the appropriate orientation have
been prepared, the process of loading antineoplastic agents into the
liposomes reduces to the step of adding the agent to the external medium.
Once added, the transmembrane potential will automatically load the agent
into the liposomes.
The transmembrane potential loading method can be used with essentially any
antineoplastic agent which can exist in a charged state when dissolved in
an appropriate aqueous medium (e.g., organic compounds which include an
amino group which can be protonated). Preferably, the agent should be
relatively lipophilic so that it will partition into the liposome
membranes. Examples of some of the antineoplastic agents which can be
loaded into liposomes by this method include doxorubicin, mitomycin,
daunorubicin, streptozocin, vinblastine, vincristine, mechlorethamine
hydrochloride, melphalan, cyclophosphamide, triethylenethiophosphoramide,
carmustine, lomustine, semustine, hydroxyurea, thioguanine, decarbazine,
cisplatin, procarbazine, and pharmaceutically acceptable salts and
derivatives thereof.
In addition to loading a single antineoplastic agent, the method can be
used to load multiple antineoplastic agents, either simultaneously or
sequentially. Also, the liposomes into which the ionizable antineoplastic
agents are loaded can themselves be pre-loaded with other antineoplastic
agents or other drugs using conventional encapsulation techniques (e.g.,
by incorporating the drug in the buffer from which the liposomes are
made).
Turning now to the aspects of the invention relating to reducing the rate
of release of an ionizable antineoplastic agent or other ionizable
biologically-active agent (drug) from liposomes, it has been surprisingly
found that the rate of release can be markedly reduced by creating a
transmembrane potential across the liposome membranes which is oriented to
retain the agent in the liposomes. That is, for an agent which is
positively charged when ionized, a transmembrane potential is created
across the liposome membranes which has an inside potential which is
negative relative to the outside potential, while for an agent which is
negatively charged, the opposite orientation is used.
As with the transmembrane loading aspects of the invention, the
transmembrane potentials used to reduce the rate of drug release are
created by adjusting the concentrations on the inside and outside of the
liposomes of a charged species such as Na.sup.+, K.sup.+ and/or H.sup.+.
Indeed, if the liposomes have been loaded by means of a transmembrane
potential produced by such a concentration gradient, simply keeping the
liposomes in an external medium which will maintain the original
concentration gradient will produce the desired reduction in the rate of
release. Alternatively, if a transmembrane potential has not already been
created across the liposome membranes, e.g., if the liposomes have been
loaded using a conventional technique, the desired transmembrane potential
can be readily created by changing the composition of the external medium
using the exchange techniques described above.
Turning next to the aspects of the invention relating to the dehydration
protocols, two basic approaches are provided: (1) the liposomes can be
loaded with antineoplastic agents (e.g., using conventional techniques or
the transmembrane potential loading technique described above), dehydrated
for purposes of storage, shipping, and the like, and then rehydrated at
the time of use; or (2) pre-formed liposomes can be dehydrated for
storage, etc., and then at or near the time of use, they can be rehydrated
and loaded with an ionizable antineoplastic agent using the transmembrane
potential loading technique described above.
When the dehydrated liposomes are to be used, rehydration is accomplished
by simply adding an aqueous solution, e.g., distilled water or an
appropriate buffer, to the liposomes and allowing them to rehydrate. The
liposomes can be resuspended into the aqueous solution by gentle swirling
of the solution. The rehydration can be performed at room temperature or
at other temperatures appropriate to the composition of the liposomes and
their internal contents.
If the antineoplastic agent which is to be administered was incorporated
into the liposomes prior to dehydration, and no further composition
changes are desired, the rehydrated liposomes can be used directly in the
cancer therapy following known procedures for administering liposome
encapsulated drugs.
Alternatively, using the transmembrane potential procedures described
above, ionizable antineoplastic agents can be incorporated into the
rehydrated liposomes just prior to administration. In connection with this
approach, the concentration gradient used to generate the transmembrane
potential can be created either before dehydration or after rehydration
using the external medium exchange techniques described above.
For example,, liposomes having the same internal and external media, i.e.,
no transmembrane potentials, can be prepared, dehydrated, stored,
rehydrated, and then the external medium can be replaced with a new medium
having a composition which will generate transmembrane potentials, and the
transmembrane potentials used to load ionizable antineoplastic agents into
the liposomes. Alternatively, liposomes having internal and external media
which will produce transmembrane potentials can be prepared, dehydrated,
stored, rehydrated, and then loaded using the transmembrane potentials.
Liposomes of the present invention may be administered to a subject such as
a mammal including humans. For administration to humans in the treatment
of afflictions, the prescribing physician will ultimately determine the
appropriate dose for a given human subject, and this can be expected to
vary according to the age, weight, and response of the individual as well
as the nature and severity of the patient's symptoms.
The mode of administration may determine the sites and cells in the
organism to which the compound will be delivered. For instance, delivery
to a specific site of infection may be most easily accomplished by topical
application (if the infection is external e.g., on areas such as eyes,
skin, in ears, or on afflictions such as wounds or burns) or by absorption
through epithelial or mucocutaneous linings (e.g., nasal, oral, vaginal,
rectal, gastrointestinal, mucosa, etc.). Such topical application may be
in the form of creams or ointments. The liposome-entrapped materials can
be administered alone but will generally be administered in admixture with
a pharmaceutical carrier selected with regard to the intended route of
administration and standard pharmaceutical practice. They may be injected
parenterally, for example, intravenously, intramuscularly, or
subcutaneously. For parenteral administration, they are best used in the
form of a sterile aqueous solution which may contain other solutes, for
example, enough salts or glucose to make the solution isotonic.
For the oral mode of administration, liposome composition of this invention
can be used in the form of tablets, capsules, lozenges, troches, powders,
syrups, elixirs, aqueous solutions and suspensions, and the like. In the
case of tablets, carriers which can be used include lactose, sodium
citrate, and salts of phosphoric acid. Various disintegrants such as
starch, and lubricating agents such as magnesium stearate, sodium lauryl
sulfate and talc, are commonly used in tablets. For oral administration in
capsule form, useful diluents are lactose and high molecular weight
polyethylene glycols. When aqueous suspensions are required for oral use,
certain sweetening and/or flavoring agents can be added.
The liposomes of this invention may also be used in diagnostic assays; in
this case the amount of the composition used will depend on the
sensitivity of the liposome-coupled antibody to the target components in
the sample.
MATERIALS AND METHODS
Egg phospatidylcholine (EPC) was isolated from hen egg yolk employing
established procedures. Egg phosphatidylethanolamine (EPE) was obtained
from EPC utilizing the headgroup exchange capacity of phospholipase D
(Kates et al., Methods in Enzymology, 14, Lavenstein, J. ed., 1969,
Academic Press, Inc., p. 197-211). Dioleoylphosphatidylcholine (DOPC) and
dipalmitoylphosphatidylcholine (DPPC) were obtained from Avanti Polar
Lipids. Strepavidin, cholesterol, trehalose, dithiothreitol (DTT), and
fluorescein isothiocyanate Celite (Celite FITC) were purchased from Sigma.
N-Succinimidyl 3-(2-pyridyldithio) propionate (SPDP), N-succinimidyl
4-(p-Maleimidophenyl)butyrate (SMPB), biotinyl-N-hydroxysuccinimide (BHS)
biotin-phosphatidylethanolasine, and
N-biotinoyldipalmitoylphosphatidylcholine were obtained from Molecular
Probes. Rabbit anti-human red blood cell IgG and Rabbit anti-rat red blood
cell IgG were supplied by Cooper Biomedical. Sephadex G-50 fine, Sepharose
4B-CL, and Sepharose C14B were purchased from Pharmacia. [.sup.3 H]-DPPC
and [.sup.3 H]-BHS were obtained from NEN, carrier free Na .sup.125 I (100
mCi/ml) was supplied by Amersham and iodogen was obtained from Pierce.
Adriamycin was obtained from B.C. Cancer Control Agency. Biotinylated
anti-rat erythrocyte IgG was obtained from Cappel. All other chemicals
were of analytical grade.
Lipid was estimated by the standard lipid phosphate assay or by
incorporation of trace quantities of [.sup.3 H-DPPC] introduced in the
original lipid film, and later monitored using a Packard Tri-Carb 4000
series scintillation counter. FITC was assayed by monitoring the
fluorescence at 520 nm using a SLM-Aminco SPF-500C spectrofluotometer with
an excitation wavelength of 495 nm. .sup.125 I was measured using a
Packard Auto-Gamma 5650 gamma counter. Vesicle size distributions were
determined by quasi-elastic light scattering (QELS) analysis utilizing a
Nicomp Model 270 submicron particle sizer operating at 632.8 nm and 5 mW.
EXAMPLE 1
Covalent Coupling
I. Synthesis of N-[4-(p-Maleimidophenyl)butyryl] phosphatidylethanolamine
(MPB-PE)
MPB-PE was prepared according to the procedure of Martin et al., J. Biol.
Chem., 257, 286-288, (1982). EPE (200 mg) was dissolved in 5 ml of freshly
distilled anhydrous methanol containing 200 umol of freshly distilled
triethylamine and 100 mg SMBP. The reaction was carried out at room
temperature under nitrogen and its progress followed using thin layer
chromatography (TLC, running solvent:chloroform/methanol/water, 65:25:4).
Following an 18 hour incubation, 95% of the EPE was converted to MPB-PE.
Methanol was removed under reduced pressure and redissolved in chloroform
and this mixture washed extensively with 1% NaCl to remove unreacted SMPB
and residual triethylamine. The product of this reaction was characterized
by two dimensional TLC and proton NMR. This analysis indicated the
presence of two components, one being MPB-PE and comprising approximately
60% of the product. The product of the reaction mixture described above
was incorporated into vesicles without additional Purification. The
product was stored at -20.degree. C. and was shown to be stable for at
least 6 months.
II. Synthesis of N-[3(2-Pyridyldithio)Proprionyl] phosphatidylethanolamine
(PDP-PE)
PDP-PE was prepared according to the procedure of Leserman et al., Liposome
Technology, III. Gregoriadis, ed., 1984, CRC Press, Inc., CA., p. 29-40.
Briefly, 50 umol EPE was dissolved in 3.5 ml chloroform/methanol (9:1) and
added to 1.5 ml methanol containing 60 umol SPDP and 100 umol
triethylamine. After a four hour incubation at room temperature, TLC
(solvent: chloroform/methanol/water, 65:25:4) analysis indicated 99%
conversion of EPE to a faster running product. This reaction mixture was
washed with 10 ml of phosphate buffered saline (0.1M NaCl, 0.1M potassium
phosphate, pH 7.4). This was repeated three additional times prior to
removal of the organic phase under reduced pressure. Further analysis by
two dimensional TLC and proton NMR indicated a single product which
appeared greater than 98% pure. PDP-PE was stored under N.sub.2 in
chloroform at -20.degree. C. and was stable for at least six months.
III. Vesicle Preparation
Large unilamellar vesicles (LUVs) were prepared employing the LUVET (LUV's
by extrusion techniques) procedure described by Hope et al., Biochim,
Biophys. Acta, 812, p. 55-65 (1985). Appropriate mixtures of lipid were
deposited as a dry lipid film by evaporation from chloroform under a
stream of nitrogen gas, placed under vacuum for at least two hours and
subsequently hydrated with appropriate buffer by vortex mixing to produce
multilamellar vesicles (MLVs). Frozen and thawed MLV (FATMLV) systems as
described in Mayer et.al, Biochim. et Biophys. Acta, 817, 193-196 (1985),
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