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Description  |
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The use of liposomes as carriers for introducing drugs and macromolecules
into cells has been reported. Liposomes are vesicles comprising a
phospholipid bilayer enclosing an aqueous or partially aqueous volume
produced by hydration and mechanical dispersion of lipid material in an
aqueous medium. Drugs may be introduced into the aqueous volume by
suspension or dissolution in the aqueous medium. It is believed that
liposome-encapsulated drugs are transported essentially intact to targeted
tissues and organs in the body, where they are transferred into individual
cells and released. Advantages of liposome encapsulation lies in the
protection of the drug while it is delivered to the organ or tissue, the
liposome-mediated transport of the drug into the cell at an elevated
localized concentration, and protection of the untargeted cells from the
drug.
Scar tissue results from the formation of a hard layer of connective tissue
formed over a healing wound or cut. In many cases, scar tissue results in
tissue contraction which may result in disfiguration and, more seriously,
which may produce side effects which comprise tissue and organ function.
Scar tissue formation in the eye, referred to as proliferative
vitreoretinopathy, frequently results in retinal detachment. Current
methods for preventing such scar tissue formation in the eye, such as
corticosteroids, are not always effective. Other examples of scar tissue
formation which are detrimental include posterior tear capsule
opacification after cataract surgery, scar tissue over filtration sites
for glaucoma, scar tissue formation after skin grafting, and scar tissue
formation around breast implants. All of these might benefit from
fluoroorotate therapy.
It would be desirable to provide improved methods and compositions for
inhibiting the formation of scar tissue under a variety of circumstances
and particularly the formation of scar tissue in the eye. It is of
substantial importance to be able to inhibit scar tissue without adversely
affecting cells and cell processes.
2. Description of the Relevant Literature
5-Fluoroorotate is a derivative of 5-fluorouracil for which no cellular
transport system exists. Bosch et al. (1958) Cancer Res. 18:335-343. Both
5-fluorouracil and 5-fluoroorotate interfere with ribosomal maturation
(Wilkinson et al. (1971) J. Biol. Chem. 246:63-68 and J. Biol. Chem.
246:6418-6427) and may also be metabolized to fluorodeoxyuridine
monophosphate, an inhibitor of thymidylate synthetase (Hartman and
Heidelberger (1961) J. Biol. Chem. 236:3006-3013). Attempts to encapsulate
5-fluorouracil in liposomes have met with limited success. Gregoriadis et
al. (1974) Lancet 1:1313-1316 and Gregoriadis (1974) Biochem. Soc. Trans.
2:117. Heath et al., ARAVO Abstracts, p. 284, 8-10:15, May 10, 1985,
report advantages of encapsulating 5-fluoroorotate for treatment of
retinal detachment.
SUMMARY OF THE INVENTION
According the the present invention, physiologically active agents are
provided comprising negatively charged low molecular weight polar drugs in
the lumen of liposomes of predetermined composition and size range.
Paradigmatically, 5-fluoroorotate is encapsulated in relatively large
liposomes employing combinations of diacylphosphatidylglycerol and
cholesterol, particularly with acyl groups of from 16 to 18 carbon atoms.
The subject drug inhibits cell contraction and cell growth. Use of
specific compounds associated with the subject agents can be used for
site-directed treatment of diseased or other aberrant physiological state.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Novel compositions are provided for enchancing physiological activity of
low molecular weight drugs, particularly polar drugs having negative
charges and molecular weights of from about 150 to 350 Daltons. The drugs
are administered in the lumen of relatively large non-leaky vesicles
composed of high-phase-transition-temperature phospholipids and steroids,
particularly cholesterol. The vesicle reagents are prepared by
conventional techniques, preferably providing relatively large diameter
liposomes. Particular combinations of drugs and lipids provide for stable
vesicles, which may be employed in vitro and in vivo for enhanced drug
activity.
The drugs which find use are relatively low molecular weight polar drugs
having a negative charge, particularly carboxylates having a pKa in the
range of about 4-6. These drugs will generally be relatively small,
ranging from about 150 to 350 Daltons, having from about 25-65 weight
percent of heteroatoms, particularly polar atoms such as oxygen and
nitrogen. Other heteroatoms may include halogen, particularly fluoride.
The compounds may be cyclic or acyclic, usually cyclic, more usually
heterocyclic. Generally, the drugs will have from about 3 to 10, more
usually from about 3-8 heteroatoms. These compounds will provide for a
significant population, at least about 10%, preferably at least about 30%,
more preferably at least about 70%, which are charged at physiological pH,
namely a pH of about 7, while having a similar population which is neutral
at a pH of about 5. It is particularly preferred to have at least 99%
charged molecules at a pH of 7.4. Of particular interest are
pyrimidinylcarboxylates, more particularly halogenated
pyrimidinylcarboxylates, and 5-fluoroorotate is exemplary.
The liposome composition will be a combination of phospholipids,
particularly high-phase-transition-temperature phospholipids, in
combination with steriods, particularly cholesterol. Other phospholipids
or other lipids may also be employed. Individual or combinations of
phospholipids may be employed.
Illustrative lipids include phosphatidyl compounds, such as
phosphatidylglycerol, phosphatidylcholine, phosphatidylserine,
phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
Of particular interest are diacylphosphatidylglycerols, where the lipids
contain from 14-18 carbon atoms, particularly from 16-18 carbon atoms,
preferably being saturated. Exemplary phospholipids include egg
phosphatidylglycerol, dipalmitoylphosphatidylglycerol,
distearoylphosphatidylglycerol; particulary of interest is
dipalmitoylphosphatidylglycerol. Desirably, the phospholipids and steroids
will be in a ratio of about 55-75:45-25, usually 60-75:40-25, more
particularly 65-70:35-30 molar ratio. Other components of the vesicle
bilayer will usually be less than 10 mol %, more usually less than 5 mol
%, preferably less than 1 mol %.
In preparing the liposome-encapsulated drug agent, aqueous media will be
employed containing various buffers, e.g., phosphate, carbonate, acetate,
etc., to provide a pH of from about 6 to 9, more usually from about 6 to
8, preferably from about 6.5 to 7.5. The osmolarity of the medium will be
chosen for the ultimate use of the encapsulated drug, particularly being
isotonic with a physiological fluid, such as blood, lymph fluid, cerebral
spinal fluid, or the like. Generally, the osmolality will be in the range
of about 250 to 350, more usually about 275 to 300 mOsm/kg.
Ampholytic compounds employed for preparation of the vesicle of particular
interest include morpholinoalkylsulfonates, with alkyl from about 2-4
carbon atoms. Counter ions will be physiologically acceptable counter
ions, such as chloride, lithium and sodium.
The drug will have an aqueous solubility of at least 10 mM in the form in
which it is employed for encapsulation.
The vesicles are prepared by dispersing or dissolving the drug in an
aqueous medium having the appropriate ampholytes and lipids at the
appropriate molar ratio. The drug concentration will generally vary from
about 0.1 mM to 200 mM, more usually from about 10 mM to about 75 mM. The
total concentration of ampholytes will generally range from about 25 to
200 mM, more usually from about 50 to 150 mM. The phospholipid
concentration will generally be in the range of 1 to 200 mM, more usually
in the range of 5 to 50 mM, with the steroid concentration controlled by
the ratio of steroid to phospholipid. As already indicated, other lipids
may also be employed which will be substituted for the phospholipid or
steroid, according to their nature.
Various techniques may be employed for producing the liposome-encapsulated
drug. Of particular interest is a method described by Szoka and
Papahadjopoulos (1978) Proc. Nat'l. Acad. Sci. U.S.A., 75:4194-4198, which
describes the technique called reverse-phase evaporation. Optionally
extrusion is employed to control size (Szoka, et al., biochem, et Biophys.
Acta (1980) 601:559-571). Smaller liposomes may be made by sonication of
liquid suspensions. The liposomes will be mono- or polylamellar and will
be of a size in the range of 0.02 to 100 .mu.m diameter, usually 0.05 to 1
.mu.m diameter, preferably 0.1 to 0.7 .mu.m diameter. Unencapsulated drug
may be conveniently removed be gel filtration. The liposomes are then
ready to be used or may be modified prior to formulation.
The vesicle bilayers may be modified in a variety of ways. Non-lipid
material may be conjugated through a linking group to one or more
hydrophobic groups, e.g., alkyl chains from about 12-20 carbon atoms,
either prior or subsequent to vesicle formation. The lipid groups are
incorporated into the lipid bilayer, so as to maintain such compound in
stable association with the bilayer. Various linking groups can be used
for joining the lipid chains to the compound. Functionalities of
particular interest include thioethers, disulfides, carboxamides,
alkylamines, ethers, and the like, used individually or in combination.
The particular manner of linking the compound to a lipid group is not a
critical part of this invention, the literature providing a great variety
of methods. Alternatively, some compounds will have hydrophobic regions or
domains, which will allow for their incorporation into the bilayer,
without linking to one or more lipid groups.
The number of molecules (either ligand or receptor) bound to a liposome
will vary with the size of the liposome, as well as the size of the
molecule, the binding affinity of the molecule to the target cell receptor
or ligand, and the like. Usually, the bound molecules will be present on
the liposome in from about 0.05 to 2 mol %, more usually from about 0.1 to
1 mol %, based on the percent of bound molecules to the total number of
molecules in the outer membrane bilayer of the liposome.
For the most part, the compounds to be bound to the lipid bilayer will be
ligands and receptors. A ligand may be any compound of interest which will
specifically bind to another compound, referred to as a receptor, the
ligand and receptor forming an homologous pair. The compounds bound to the
bilayer may vary widely, from small haptens of from about 125 to 2,000
molecular weight or higher, to antigens which will generally be at least
about 6,000 molecular weight and generally less than about 1 million
molecular weight, more usually less than about 300,000 molecular weight.
Of particular interest are the proteinaceous ligands and receptors.
A wide variety of compounds which have specific receptors on cell surfaces
will be of interest. Illustrative compounds or fragments thereof may
include chorionic gonadotropin, enkephalin, .beta.-endorphin, luteinizing
hormone, epidermal growth factor, transforming growth factor, platelet
derived growth factor, interleukin-2, morphine, epinephrine, interferon,
ACTH, polyiodothyronines, etc.
For the most part, the surface membrane proteins which bind to specific
effector molecules are referred to as receptors. However, in the present
context, for the most part receptors will be antibodies or
immunoglobulins. The immunoglobulins may be monoclonal or polyclonal,
preferably monoclonal. Usually, the immunoglobulins will be IgG and IgM,
although the other immunoglobulins may also find use, such as IgA, IgD and
IgE. The intact immunoglobulins may be used or only fragments thereof,
such as Fab, F(ab').sub.2, F.sub.d, F.sub.v, the light chain and the heavy
chain.
For antibodies, antibodies of interest are those that bind to surface
membrane antigens such as those antigens comprising the major
histocompatibility complex, particularly HLA-A, -B, -C and -D. Other
surface antigens include thy-1, leu-5, Ia, etc.
The compositions of this invention provide enhanced drug activity. The
compositions may be used in vitro or in vivo. When 5-fluoroorotate is
employed, the subject compositions can be employed as cytotoxic agents,
inhibiting proliferation and providing for anticontractile activity. These
compositions may therefore be used to inhibit the proliferation of
particular cells in a mixture of cells, or in tissue, where there is
preferential binding and endocytosis of the liposome into the taget cell.
For in vivo application, the liposome-encapsulated drug may be administered
in a variety of ways to a host, particularly a mammalian host, such as
intravenously, intramuscularly, subcutaneously, intraperitoneally,
intravascularly, topically, or the like. Concentration of the drug will
vary upon the particular application, the nature of the disease, the
frequency of administration, or the like. When employing 5-fluoroorotate,
usually the amount of drug employed will be from about 0.05 to 2 mg per
administration, more usually from about 0.1 to 0.3 mg. The
liposome-encapsulated drug may be provided in a formulation comprising
other drugs as appropriate and an aqueous physiologically acceptable
medium, e.g., saline, phosphate buffered saline, or the like.
An exemplary of the subject invention is the use of the
liposome-encapsulated 5-fluoroorotate for the prevention of scar tissue by
inhibiting tissue contraction. Scar tissue formation may result in
impaired tissue and organ function, as well as disfiguration.
In particular, the treatment compositions of the present invention are
useful for inhibiting scar tissue formation in the eye, referred to as
proliferative vitreoretinopathy (PVR), which often results in retinal
detachment. Such PVR can result from traumatic injury of the eye, as well
as from surgery, such as glaucoma surgery, strabismus surgery, retinal
detachment surgery, and the like. Treatment is effected by topical
application of the treatment composition to the eye or by intraocular
injection. Treatment will usually be repeated daily or more frequently. It
has been found that the compositions of the invention including
5-fluoroorotate are highly effective and non-toxic even at elevated
concentrations.
The following experiments are offered by way of illustration, not by way of
limitation.
EXPERIMENTAL
Materials and Methods
Sodium 5-fluoroorotate (Pharmacia, Piscataway, N.J.) displays a maximum
aqueous solubility of 15 mM, while the lithium salt is soluble to at least
50 mM. For encapsulation, a 50 mM lithium 5-fluoroorotate, pH 7.4, 290
mOsm/kg solution was prepared, containing 50 mM morpholinoethanesulfonate
and 50 mM morpholinopropanesulfonate, with chloride and lithium
counterions. For gel chromatography and subsequent dilution of the
liposomes, an equivalent buffer lacking drug was prepared. All solutions
were sterilized by filtration prior to use.
All phospholipids (Avanti, Birmingham, Ala.) were used without further
purification. Cholesterol (Sigma, St. Louis, Mo.) was recrystallized four
times with methanol. All lipids were stored under argon in chloroform
solution in sealed ampoules until use. Liposomes were prepared by
reverse-phase evaporation (Szoka and Papahadjopoulos (1978) Proc. Natl.
Acad. Sci. U.S.A. 75:4194-4198), and extrusion (Szoka et al. (1980)
Biochem. et Biophys. Acta. 601:559-571). Small liposomes were made by
extensive sonication of lipid suspensions. The unencapsulated drug was
removed by gel filtration with Sephadex.RTM. G75 (Pharmacia). Lipid
concentration was measured by phosporous analysis as described by Bartlett
(1959) J. Biol. chem. 234:466-468. The encapsulated drug was measured
using a molar extinction coefficient of 7100 in 0.1N HCl. A liposome
sample was extracted (Bligh et al. (1959) Can. J. Biochem. Physiol.
37:9911-917), the upper phase was acidified with HCl, and its absorbance
was meausred. L929 murine fibroblasts and CV1-P cells were obtained and
grown as previously described by Heath et al. (1983) Proc. Natl. Acad.
Sci. U.S.A. 80:1377-1381 and Fraley et al. (1980) J. Biol. Chem.
255:10431-10435, respectively. The IC.sub.50 of the liposome preparations
was measured by growth inhibition as previously described by Heath et al.
(1983) supra. The cells were incubated for 48 hours (L929) or 72 hours
(CV1-P) before counting.
Results
The captured aqueous volumes (Szoka et al. (1980) supra.) are within the
expected range for the liposome preparations. This suggests that the drug
is encapsulated within the aqueous phase and does not leak rapidly from
the liposomes. The liposomes were stored for several weeks at 4.degree. C.
without any change in their potency, which further confirms the stability
of the preparations.
Unencapsulated 5-fluoroorotate has an IC.sub.50 of 7 micromolar for CV1-P
cells and 1 micromolar for L929 cells (Table 1). When encapsulated in egg
phosphatidylglycerol:cholesterol (67:33) liposomes, the potency of
5-fluoroorotate was increased by 2-3 fold. Drug potency was increased
14-35 fold by encapsulation in dipalmitoylphosphatidylglycerol:cholesterol
(67:33) liposomes. Sonicated liposomes of this composition were 10 fold
less effective than larger liposomes for drug delivery. Drug in
distearoylphosphatidylglycerol:cholesterol (67:33) unextruded liposomes
was 2-14 times more potent growth inhibitor than free drug. Sonicated
liposomes of this composition are less effective than unextruded large
liposomes, but the difference is not as great as is seen between sonicated
liposomes and unextruded liposomes that contain
dipalmitoylphosphatidylglycerol.
TABLE 1
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Growth Inhibition by 5-fluoroorotate.
Liposome Properties
Molar Size.sup.c
Captured.sup.d
IC.sub.50.sup.a (micromolar)
Lipid.sup.b
Ratio (.mu.m)
(mol.sup.-1)
CVl-P L929
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Free Drug.sup.e
-- -- -- 7 .+-. 2
1.1 .+-. 0.3
PG:Chol 67:33 U 9.0 2.0 0.6
0.1 2.8 2.2 0.6
DSPG:Chol
67:33 U 2.8 0.5 0.72
SUV 0.8 0.7 0.96
DPPG:Chol
67:33 U 5.2 0.2 0.08
0.1 3.2 0.2 0.13
SUV 1.2 1.7 0.8
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.sup.a The IC.sub.50 is the concentration of the drug that inhibits cell
growth by 50%.
.sup.b The lipids used were Chol: cholesterol, PG: egg
phosphatidylglycerol, DPPG: dipalmitoylphosphatidylglycerol, DSPG:
distearoylphosphatidylglycerol.
.sup.c The liposomes were prepared by reverse phase evaporation and were
either unextruded (U) (0.1 to 1 mM, mean = 0.5 mM) or extruded to 0.1
micrometer (0.1). Liposomes were also prepared by extensive sonication
(SUV) (0.03 to 0.07 mM).
.sup.d The theoretical aqueous capture is the drug:lipid ratio (mol/mol)
.times. the inverse of the orginal drug concentration (0.05 M).
.sup.e The IC.sub.50 of the free drug is the mean of 3 determinations for
CVlP cells and of 4 determinations for L929 cells. All other values are
derived from individual growth inhibition curves.
As evidenced from the above results, drugs which may otherwise be
relatively ineffective because of inability to penetrate the plasma
membrane can be encapsulated in liposomes having predetermined size and
composition and applied, directly or indirectly, to target cells. The drug
is thus found to have greatly enhanced activity as compared to the
unencapsulated drug. Furthermore, encapsulation which provides for
preferential binding of the liposome to target tissue diminishes adverse
effects to normal tissue, while concentrating the drug at the desired
site. The subject compositions are easily prepared, are stable for long
periods of time, and serve to reduce the drug load to the host by
concentrating the drug at a desired site.
Although the foregoing invention has been described in some detail by way
of illustration and example for purposes of clarity of understanding, it
will be obvious that certain changes and modifications may be practiced
within the scope of the appended claims.
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Description |
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