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Description  |
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BACKGROUND OF THE INVENTION
The present invention relates to the preparation of liposomes adapted for
specific organ targeting and to the liposomes so prepared.
Liposomes are synthetic lipid vesicles whose lipid bilayers serve as a
model of biomembranes. Liposomes can be prepared by a various of
techniques to yield vesicles of varying size and lamellar structure. They
usually have a maximum diameter on the order of 100,000 .ANG. and most
often have a diameter between 110 to 10,000 .ANG., bounded by a wall
formed by at least one bimolecular layer (having a thickness on the order
100 .ANG.) of a compound of the general formula XY, where X is a
hydrophilic polar group and Y is a hydrophobic non-polar group, the
globules containing an aqueous liquid, for example and aqueous solution of
at least one biologically active substance, and existing generally in the
form of a colloidal dispersion in an aqueous medium such as an aqueous
saline solution, in particular a 0.9% by weight sodium chloride solution.
The preparation of liposomes provides a method of encapsulation which is
most practical and effective for aqueous materials as well as hydrophobic
and amphipathic material and which is particularly useful for
administration of biologically active substances, particularly
medicaments, into living organisms, while avoiding the destruction or
inactivation of the substance in the organism, for example by the action
of gastric or intestinal juices, before the substances reach the site
where they are required to act.
Central to this interest is an altered biodistribution of the agent to
various organs, tissues or inflammatory sites.
Targeting of encapsulated material in liposomes has the advantage of
increased specific activity of the agent to the specific target site,
lowered exposure of other areas to the agent thereby decreasing effective
toxicity of the agent and altered time course of agent delivery. Loaded
vesicles, therefore, hold promise of therapeutic and diagnostic use in
cancer patients. Multilamellar as well as unilamellar lipid vesicles
loaded with a radiopaque agent have been shown to enhance hepatic and
splenic imaging of the rat by X-ray computed tomography.
By selection of the compound of formula XY used to form the wall of the
liposomes, it is possible to produce liposomes having walls which resist
the degradation by various physiological processes.
Typical processes for the preparation of liposomes include placing a lipid
in contact with an aqueous liquid that is desired to be encapsulate and
then warming the heterogeneous mixture thus obtained at a temperature
slightly above ambient temperature and then submitting the mixture to
vigorous agitation following ultrasonic vibration.
Another process consists of dissolving a compound of formula XY (where X
and Y are defined above), for example a lipid, in a volatile solvent,
forming a film of the compound on the walls of a receptacle by evaporating
the solvent from the solution thus obtained, introducing in the same
receptacle the liquid which is desired to encapsulate in the liposomes,
and finally submitting the liquid in the receptacle to the action of
ultrasonic vibrations.
It would be highly desirable to provide a means for rendering liposomes
more selective for a particular organ in order to improve their
selectivity to deliver biologically active agents or contrast agents which
can be detected by conventional scanning apparatus.
SUMMARY OF THE INVENTION
The present invention is based upon the discovery that liposomes formed
with chemically modified cholesterol can be rendered selective for
targeting specific organs by adjusting the type of chemical modification
employed. The liposomes are formed by conventional means but with the
addition of the chemically modified cholesterol to the liposome-forming
composition. An aqueous composition containing the diagnostic or
therapeutic agent is admixed with a carrier liquid composition which is
insoluble or only slightly soluble in water. The resultant mixture may be
subjected to vigorous agitation such as ultrasonic agitation or prepared
by emulsifying aqueous droplets containing the desired material to be
encapsulated in organic solvent, forming a gel by evaporation of solvent
and addition of an aqueous phase to form the liposomes that can be
unilamellar or multilamellar, the interiors of which are filled with the
aqueous composition. Similarly natural membranes may be used as a target
vehicles containing various agents by introducing cholesterol derivatives
into their membranes. The cholesterol is chemically modified with a ligand
designed to be recognized by a particular organ or cell type such as a
long chain fatty acid, an amino acid, an oligosaccharide, a horomone, an
amino acid derivative, a protein, glycoprotein, or modified protein, or
the like. The resultant liposome is suitable for being targeted to a
specific organ or cell type.
DESCRIPTION OF SPECIFIC EMBODIMENTS
In accordance with this invention, liposomes are provided which contain a
tracer material, a cytotoxic agent or a therapeutic agent. The liposomes
of this invention are characterized by the inclusion in the monolayer or
bilayer a chemically modified cholesterol which is modified so that the
liposome is rendered more specific for rapid and preferential accumulation
in vivo to a specific desired organ. The liposomes can be unilamellar or
multilamellar and can be formed from any lipid material conventionally
utilized to form liposomes. Representative suitable lipid materials that
can be utilized to form liposomes include distearoyl phosphatidylcholine
and/or L-.alpha.-dipalmitoyl phosphatidylcholine or similar lipid
substances or naturally occurring cells such as red blood cells. The walls
of the liposomes can also be formed from soybean phospholipid, egg yolk
lecithin and L-60 -dimyristoyl phosphatidylcholine. The liposomes may be
prepared by simple sonication from liquid suspension, hydration of
crystallized lipids or any other conventional procedure well known in the
art. Generally, the liposomes have a size range of between about 0.001 and
about 10 microns.
* The modified cholesterol of the invention must be modified at the
hydroxyl group positioned at the 3 position on the cholesterol molecule
because this hydroxyl group is oriented on the outside surface of the
lipid bilayer.
For purposes of this invention, cholesterol is modified with a ligand such
as is set forth above. The following table lists the human organs, the
ligands are characterized by high uptake by the organ in accordance with
this invention and the cholesterol derivatives that would be utilized.
TABLE 1
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Organ Ligand Cholesterol Derivative
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Heart Fatty acids Chol-butyrate, chol-hexanate
Prostrate
Cadaverine N--cadaverinyl chol-carbamate
Liver Desialated glyco-
Carbamate or ester linkage
protein between chol and ligand
Pancreas
Phenylalanine
chol-phenylacetate
Pancreas
Procainamide N--procainamidyl-chol-carbamate
Adrenal
Cholesterol Cholesteric cholesterate
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The modified cholesterol is added to the lipids when forming the liposomes
and is generally added at a concentration of between about 0.1 to 5 mole
percent of total bilayer lipids preferably between about 1.0 and about 3.0
mole percent.
The aqueous portion of the liposome contains the tracer material, cytotoxic
agent or therapeutic agent which is to be delivered to the desired organ
by the liposome. Representative suitable agents which can be delivered by
the liposomes of this invention include radioactive tracers such as
iodine-131, iodine-123, iodine- 126, iodine-133, bromine-17, indium-111,
indium 113 m, gallium-67, gallium-68, ruthenium-95, ruthenium-97,
ruthenium-103, ruthenium-105, mercury-107, mercury-203, rhenium-99 m,
rhenium-105, rhenium-101, tellurium-121 m, technetium-99 m or the like
which are useful in radionuclide scintigraphy; fluorescent agents such as
fluoroscein, tetrachycline; radiographic contrast agents such as
diatrizoate, metrizamide, iothalamate or the like which are useful in
fluoroscopy, plain film X-ray, angiography, digital subtraction
angiography and computed tomography; diamagnetic and paramagnetic
substances such as perfluorohydrocarbons, nitroxide free radicals,
phosphates, magnesium, gadolinium or the like, which are useful in nuclear
magnetic resonance imaging or gaseous agents or gaseous percursors auch as
carbon dioxide, helium, argon, bicarbonates, aminomalonate carbonates,
xenon or the like which are useful in ultra-songraphic applications.
Representative suitable therapeutic or cytotoxic agents include
anti-cancer agents, anti-infection agents, anti-inflammatory agents,
enzymes or the like such as methotrexate, ricin-A chain, 5 FU, Adriamycin,
6 MP, Azaserine, asparaginase, dexamethasone, prostaglandins, ara-A- or
the like. Other agents which can be incorporated into the liposomes of
this invention include genetic material which is useful in treating inborn
errors of metabolism such as those used in glycogen storage diseases,
lipidoses or the like.
After formation of the liposomes, they can be utilized be being suspended
in a physiologically acceptable liquid such as saline and administered
parenterally, orally, intramuscularly, subcutaneously, intraperitoneally,
rectally, intralymphoatically and intrathecally.
The following example illustrates the present invention and are not
intended to limit the same.
EXAMPLE 1
A quanity of 126 mg of egg lecithin (egg phosphatidyl choline), 27 mg of
cholesterol and an amount of cholesterol phenylacetate (30 mg) are mixed
in 9 ml diethyl ether. 3 ml of diatrizoate sodium is added to the flask
and the mixtures sonicated until a homogeneous suspension is obtained.
Diethyl ether is removed by rotary evaporation under a water aspirator.
Saline is added to the residue to complete lipsome formation.
Unencapsulated diatrizoate is removed by centrifugation. Resuspension of
the pelletized liposomes in saline yields the final material; injection of
a portion of this material (20%) into a rat is followed by significant
contrast enhancement of the pancreas, liver and spleen on subsequent
computed tomographic scans of the rat.
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