U.S. patent number 4,544,545 [Application Number 06/505,696] was granted by the patent office on 1985-10-01 for liposomes containing modified cholesterol for organ targeting.
This patent grant is currently assigned to Trustees University of Massachusetts. Invention is credited to Michael A. Davis, Donald L. Melchior, Patrick J. Ryan.
United States Patent |
4,544,545 |
Ryan , et al. |
October 1, 1985 |
Liposomes containing modified cholesterol for organ targeting
Abstract
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.
Inventors: |
Ryan; Patrick J. (Worcester,
MA), Davis; Michael A. (Westwood, MA), Melchior; Donald
L. (Framingham, MA) |
Assignee: |
Trustees University of
Massachusetts (Amherst, MA)
|
Family
ID: |
24011438 |
Appl.
No.: |
06/505,696 |
Filed: |
June 20, 1983 |
Current U.S.
Class: |
424/1.21;
424/178.1; 424/193.1; 424/450; 424/812; 424/9.3; 424/9.321;
424/9.4; 424/9.5; 424/94.3; 516/31 |
Current CPC
Class: |
A61K
9/1271 (20130101); Y10S 424/812 (20130101) |
Current International
Class: |
A61K
9/127 (20060101); A61K 049/00 (); A61K 043/00 ();
A61K 039/00 () |
Field of
Search: |
;424/1.1,9,16,7.1,85,88,94 ;252/315.3 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Ryan et al, Biochim. Biophys. Acta, 756, (1983), 106-110. .
Stoffel et al, Hoppe-Seyler's Z. Physiol. Chem. 357, (1976),
21-33..
|
Primary Examiner: Nucker; Christine M.
Attorney, Agent or Firm: Cook; Paul J.
Claims
We claim:
1. In a liposome formed by the incapsulation of an active agent by
at least one lipid containing bilayer, the improvement comprising a
ligand modified cholesterol compound in the lipid of said bilayer,
said ligand being positioned at the 3 carbon position of said
cholesterol, said ligand modified cholesterol compound rendering
the liposome more specific for accumulation at a pre-selected organ
in vivo.
2. The liposomes of claim 1 wherein said modified cholesterol is
cholesterol butyrate.
3. The liposomes of claim 1 wherein said modified cholesterol is
cholesterol phenylacetate.
4. The liposomes of any one of claims 1, 2 or 3 wherein said lipid
bilayer includes a radioactive cation.
5. The liposomes of any one of claims 1, 2 or 3 wherein said lipid
bilayer includes a fluorescent dye.
6. The liposomes of any one of claims 1, 2 or 3 wherein said lipid
bilayer includes gaseous material.
7. The liposomes of any one of claims 1, 2 or 3 wherein said lipid
bilayer includes a drug.
8. The liposomes of any one of claims 1, 2 or 3 wherein said lipid
bilayer includes an enzyme.
9. The liposomes of any one of claims 1, 2 or 3 wherein said lipid
bilayer includes metallic ions.
10. The liposomes of any one of claims 1, 2 or 3 wherein said lipid
bilayer includes a magnetic compound.
11. The liposomes of any one of claims 1, 2 or 3 wherein said lipid
bilayer includes genetic material.
12. The liposomes of any one of claims 1, 2 or 3 wherein said lipid
bilayer includes an antigen or antibody.
Description
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.
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.
* * * * *