Liposomes containing modified cholesterol for organ targeting

Phospholipid liposomes are provided having an outer layer including a cholesterol derivative such as a cholesterol ester and an aqueous medium confined by the layer which includes a tracer agent, a cytoxic agent or a therapeutic agent. The liposomes are adapted for specific organ targeting.

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. 
FNT * 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 
______________________________________ 
Organ Ligand Cholesterol Derivative 
______________________________________ 
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 
______________________________________ 
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.