 |
Claims  |
|
|
What is claimed is:
1. A method for preparing lipid vesicles comprising:
(a) forming a solution of an amphipathic lipid in at least one organic
solvent plus a first aqueous component in amounts sufficient to form a
monophase;
(b) evaporating the organic solvent or solvents of the monophase at a
temperature and pressure which maintains the monophase and facilitates
evaporation until a film forms; and
(c) adding a second aqueous component to the film and agitating the second
aqueous component with the film in order to resuspend the film and to form
lipid vesicles.
2. The method according to claim 1 in which the monophase is sonicated
during evaporation.
3. The method according to claim 1 in which the monophase is vortexed
during evaporation.
4. The method according to claim 1 in which the monophase is shaken by hand
during evaporation.
5. The method according to claim 1 in which the monopase is rotoevaporated.
6. The method according to claim 1 in which at least one organic solvent
comprises an alcohol.
7. The method according to claim 6 in which the alcohol comprises ethanol.
8. The method according to claim 6 in which the alcohol comprises
2-propanol.
9. The method according to claim 6 in which the alcohol comprises methanol.
10. The method according to claim 1 in which at least one organic solvent
comprises acetone.
11. The method according to claim 1 in which at least one organic solvent
comprises tetrahydrofuran.
12. The method according to claim 1 in which at least one organic solvent
comprises glyme.
13. The method according to claim 1 in which at least one organic solvent
comprises dioxane.
14. The method according to claim 1, in which at least one organic solvent
comprises pyridine.
15. The method according to claim 1, in which at least one organic solvent
comprises diglyme.
16. The method according to claim 1, in which at least one organic solvent
comprises 1-methyl-2-pyrrolidone.
17. The method according to claim 1, in which at least one organic solvent
comprises butanol-2.
18. The method according to claim 1, in which at least one organic solvent
comprises butanol-1.
19. The method according to claim 1, in which at least one organic solvent
comprises isoamyl alcohol.
20. The method according to claim 1, in which at least one organic solvent
comprises isopropanol.
21. The method according to claim 1, in which at least one organic solvent
comprises 2-methoxyethanol.
22. The method according to claim 1, in which at least one organic solvent
comprises chloroform/methanol in a 1:1 ratio.
23. The method according to claim 7, in which the volume of organic solvent
or solvents and the volume of the first aqueous component are in a ratio
of about 25:1 to about 1:1.
24. The method according to claim 7, in which the amphipathic lipid
comprises phosphatidylcholine.
25. The method according to claim 7, wherein the temperature at which the
evaporation is performed is 54.degree. C.
26. The method according to claim 1 in which the organic solvent or
solvents contains an anti-oxidant.
27. The method according to claim 26, in which the anti-oxidant comprises
butylated hydroxytoluene.
28. The method according to claim 27, in which the anti-oxidant comprises
alpha-tocopherol.
29. The method according to claim 1, in which an agent to be entrapped in
the lipid vesicles is added to the monophase before evaporation.
30. The method according to claim 1, in which an agent to be entrapped in
the lipid vesicles is added to film with the second aqueous component.
31. The method according to claim 29 or 30, in which the agent to be
entrapped comprises a biologically active agent.
32. The method according to claim 31, in which the biologically active
agent comprises an antibacterial compound, an antifungal compound, an
antiparasitic compound, or an antiviral compound.
33. The method according to claim 31, in which the biologically active
agent comprises a tumoricidal compound, a toxin, a cell receptor binding
molecule, or an globulin.
34. The method according to claim 31, in which the biologically active
agent comprises an anti-inflammatory compound, an anti-glaucoma compound,
a mydriatic compound, or a local anesthetic compound.
35. The method according to claim 31, in which the biologically active
agent comprises an enzyme, a hormone, a neurotransmitter, an
immunomodulator, a nucleotide or a cyclic adenosine monophosphate.
36. The method according to claim 31, in which the biologically active
agent comprises a dye, a fluorescent compound, a radioactive compound, or
a radio-opaque compound.
37. The method according to claim 31, in which the biologically active
agent comprises an antibiotic.
38. The method according to claim 37, in which the antibiotic comprises an
aminoglycoside antibiotic.
39. The method according to claim 38, in which the aminoglycoside
antibiotic comprises gentamicin.,
40. The method according to claim 37, in which the antibiotic comprises a
penicillin.
41. The method according to claim 40, in which the penicillin comprises
nafcillin.
42. The method according to claim 32, in which the antibiotic comprises a
tetracycline.
43. The method according to claim 42, in which the tetracycline comprises
doxycycline.
44. The method according to claim 37, in which the antibiotic comprises
chloramphenicol.
45. Lipid vesicles produced by a method comprising:
(a) forming a solution of an amphipathic lipid in at one organic solvent
plus a first aqueous component in amounts sufficient to form a monophase;
(b) evaporating the organic solvent or solvents of the monophase at a
temperature and pressure which maintains the monophase and facilitates
evaporation until a film forms;
(c) adding a second aqueous component to the film and agitating the second
aqueous component with the film in order to resuspend the film and to form
lipid vesicles.
46. Lipid vesicles according to claim 45, in which the major lipid
component of the vesicles comprises phosphatidycholine.
47. Lipid vesicles according to claim 45, in which the major lipid
component of the vesicles comprises egg phosphatidylcholine.
48. Lipid vesicles according to claim 45, in which an anti-oxidant
comprises a component of the vesicle.
49. Lipid vesicles according to claim 48, in which the anti-oxidant
comprises butylated hydroxytoluene.
50. Lipid vesicles according to claim 48, in which the anti-oxidant
comprises alpha-tocopherol.
51. Lipid vesicles prepared according to the method of claim 45, in which a
biologically active agent is entrapped within the vesicles.
52. Lipid vesicles according to claim 51, in which the biologically active
agent was added to the monophase.
53. Lipid vesicles according to claim 51, in which the biologically active
agent was added to the film with the second aqueous component.
54. Lipid vesicles according to claim 51, in which the bioogically avtice
agent entrapped within the vesicles comprises an antibacterial compound,
an antifungal compound, an antiparasitic compound, or an antiviral
compound.
55. Lipid vesicles according to claim 51, in which the biologically active
agent entrapped within the vesicles comprises a tumoricidal compound, a
toxin, a cell receptor binding compound or an immunoglobulin.
56. Lipid vesicles according to claim 51, in which the biologically active
agent entrapped within the vesicles comprises an anti-inflammatory
compound, an anti-glaucoma compound, a mydriatic compound or a local
anesthetic.
57. Lipid vesicles according to claim 51, in which the biologically active
agent entrapped within the vesicles comprises an enzyme, a hormone, a
neurotransmitter, an immunomodulator, a nucleotide or a cyclic adenosine
monophosphate.
58. Lipid vesicles according to claim 51, in which the biologically active
agent entrapped within the vesicles comprises a dye, a fluorescent
compound, a radioactive compound, or a radio-opaque compound.
59. Lipid vesicles according to claim 51, in which the biologically active
agent comprises an antibiotic.
60. Lipid vesicles according to claim 59, in which the antibiotic comprises
an aminoglycoside antibiotic.
61. Lipid vesicles according to claim 60, in which the aminoglycoside
antibiotic comprises gentamicin.
62. Lipid vesicles according to claim 59, in which the antibiotic comprises
a penicillin.
63. Lipid vesicles according to claim 62, in which the penicillin comprises
nafcillin.
64. Lipid vesicles according to claim 59, in which the antibiotic comprises
a tetracycline.
65. Lipid vesicles according to claim 64, in which the tetracycline
comprises doxycycline.
66. Lipid vesicles according to claim 59, in which the antibiotic comprises
chloramphenicol.
67. A method for delivery of a biologically active agent in vivo
comprising: administering to an organism the lipid vesicles of claim 51.
68. The method according to claim 67, in which the lipid vesicles are
administered topically, intraperitoneally, intravenously, intramuscularly,
intraarticularly, subcutaneously, intraauricularly or orally.
69. A method for treatment of infections in animals or plants, comprising:
administering lipid vesicles of claim 45 containing a compound effect for
treating the infection.
70. The method according to claim 69, in which the infection is
intracellular.
71. the method according to claim 69, in which the infection is
extracellular.
72. The method according to claim 69, in which the infection is caused by a
parasite.
73. The method according to claim 69, in which the infection is caused by a
bacteria.
74. The method according to claim 73, in which the bacteria comprises
Brucella spp.
75. The method according to claim 74, in which the administration is
intraperitoneal.
76. The method according to claim 73, in which the bacteria comprises
Salmonella spp.
77. The method according to claim 76, in which the administration is
intraperitoneal.
78. The method according to claim 69, in which the infection comprises an
ocular infection.
79. The method according to claim 78, in which the administration is
topical.
80. The method for the treatment of afflictions in animals or plants
comprising: administering the lipid vesicles of claim 45 containing a
compound effective for treating the affliction.
81. The method according to claim 80, in which the affliction comprises an
ocular affliction.
82. The method according to claim 81, in which the ocular affliction
comprises glaucoma.
83. The method according to claim 82, in which the administration is
topical. |
|
 |
|
 |
Claims |
|
|
|
Description |
|
|
FIELD OF THE INVENTION
This invention relates to liposomes and their uses as carriers in delivery
systems. More specifically, it discloses a new process for making a new
type of lipid vesicle having unique properties which confer special
advantages such as increased stability, high percentage of drug entrapment
and ability to combine incompatible drugs in the lipid vesicle.
The practice of the present invention is demonstrated herein by way of
example for the treatment of Brucella canis infection and the treatment of
Salmonella typhirium infection.
BACKGROUND OF THE INVENTION
Liposomes
Liposomes are completely closed bilayer membranes containing an entrapped
aqueous phase. Liposomes may be any variety of unilamellar vesicles
(possessing a single membrane bilayer) or multilamellar vesicles
(onion-like structures characterized by concentric membrane bilayers, each
separated from the next by an aqueous layer).
The original liposome preparation of Bangham et al. (1965, J. Mol. Biol.
13:238-252) involves suspending phospholipids in an organic solvent which
is then evaporated to dryness leaving a phospholipid film on the reaction
vessel. Then an appropriate amount of aqueous phase is added, the mixture
is allowed to "swell", and the resulting liposomes which consist of
multilamellar vesicles (hereinafter referred to as MLVs) are dispersed by
mechanical means. The structure of the resulting membrane bilayer is such
that the hydrophobic (non-polar) "tails" of the lipid orient toward the
center of the bilayer while the hydrophilic (polar) "heads" orient towards
the aqueous phase. This technique provides the basis for the development
of the small sonicated unilamellar vesicles (hereinafter referred to as
SUVs) described by Papahadjapoulos and Miller (1967, Biochim. Biophys.
Acta. 135:624-638) and large unilamellar vesicles (hereinafter referred to
as LUVs). These "classical liposomes" (MLVs, SUVs and LUVs), however, have
a number of drawbacks not the least of which is a low volume of entrapped
aqueous space per mole of lipid and a restricted ability to encapsulate
large macromolecules.
Efforts to increase the entrapped volume involved first forming inverse
micelles or liposome precursors, i.e., vesicles containing an aqueous
phase surrounded by a monolayer of lipid molecules oriented so that the
polar head groups are directed towards the aqueous phase. Liposome
precursors are formed by adding the aqueous solution to be entrapped to a
solution of polar lipid in an organic solvent and sonicating. The organic
solvent is then evaporated in the presence of excess lipid. The resultant
liposomes, consisting of an aqueous phase entrapped by a lipid bilayer are
dispersed in an aqueous phase (see U.S. Pat. No. 4,224,179 issued Sept.
23, 1980 to Schneider).
In another attempt to maximize the efficiency of entrapment,
Papahaduopoulos (U.S. Pat. No. 4,235,871 issued Nov. 25, 1980) describes a
"reverse-phase evaporation process" for making oligolamellar lipid
vesicles also known as reverse-phase evaporation vesicles (hereinafter
referred to as REVs). According to this procedure, the aqueous material to
be entrapped is added to a mixture of polar lipid in an organic solvent.
Then a homogeneous water-in-oil type of emulsion is formed and the organic
solvent is evaporated until a gel is formed. The gel is then converted to
a suspension by dispersing the gel-like mixture in an aqueous media. The
REVs produced consist mostly of unilamellar vesicles and some
oligolamellar vesicles which are characterized by only a few concentric
bilayers with a large internal aqueous space. Certain permeability
properties of REVs were reported to be similar to those of MLVs and SUVs
(see Szoka and Papahadjopoulos, 1978, Proc. Natl. Acad. Sci. U.S.A.
75:4194-4198).
Batzri and Korn (1973, Biochim.Biophys. Acta. 298:1015-1019) describe a
process for the preparation of liposomes by an ethanol-infusion method.
This method yields SUVs which have to be separated from a carrier liquid
and then resuspended in an aqueous phase. All procedures used to effect
this have been uneconomical. Furthermore, the SUVs produced are unstable.
Additional disadvantages of this method are that it produces liposomes
with a low entrapment efficiency and it is limited to using lipids which
are soluble in ethanol.
Liposomes which entrap a variety of compounds can be prepared; however,
stability of the liposomes during storage is invariably limited. This loss
in stability results in leakage of the entrapped compound from the
liposomes into the surrounding media, and can also result in contamination
of the liposome contents by permeation of materials from the surrounding
media into the liposome itself. As a result the storage life of classical
liposomes is very limited. Attempts to improve stability involved
incorporating into the liposome membrane certain substances (hereinafter
called stabilizers) which affect the physical properties of the lipid
bilayers (e.g., steroid groups). However, many of these substances are
relatively expensive and the production of such liposomes is not
cost-effective.
In addition to the storage problems of classical liposomes a number of
compounds cannot be incorporated into these vesicles. For example, MLVs
can only be prepared under conditions above the phase-transition
temperature of the lipid membrane. This precludes the incorporation of
heat labile molecules within liposomes that are composed of phospholipids
which exhibit desirable properties but possess long and highly saturated
side chains.
Uses of Liposomes
Much has been written regarding the possibilities of using liposomes for
drug delivery systems. In a liposome drug delivery system the medicament
is entrapped during liposome formation and then administered to the
patient to be treated. Typical of such disclosures are U.S. Pat. No.
3,993,754 issued on Nov. 23, 1976, to Rahman and Cerny, and U.S. Pat. No.
4,145,410 issued on Mar. 20, 1979, to Sears, U.S. Pat. No. 4,235,871
issued Nov. 25, 1980, to Papahadjopoulos and Szoka and U.S. Pat. No.
4,224,179, , issued Sept. 23, 1980 to Schneider.
Desirable features of drug delivery systems depend upon the condition being
treated. For example, when treating conditions which require maintenance
doses of medication, resistance to rapid clearance of the drug accompanied
by a sustained release of the drug which will prolong the drug's action
increases the effectiveness of the drug and allows the use of fewer
administrations. However, if one is treating an intracellular infection,
the maintenance of stability in biological fluids, until the point that
the liposome is internalized by the infected cell, is critical as is
release of the liposome entrapped drug in its bio-active form. Some of the
problems encountered in using liposome preparations in vivo include the
following:
(1) Liposome-entrapped materials leak when the liposomes are in contact
with body fluids. This has been attributed to the removal of the liposomal
phospholipids by plasma high density lipoproteins (HDLs), or to the
degradation of the liposome membrane by phospholipases, among other
reasons. A result of the degradation of the liposomes in vivo is that
almost all the liposomal contents are released in a short period of time,
therefore, sustained release and resistance of the drug to clearance are
not achieved.
(2) On the other hand, if a very stable liposome is used in vivo (i.e.,
liposomes which do not leak when in contact with body fluids in vivo or in
vitro), then the liposomal contents will not be released as needed. As a
result, these stable liposomes are ineffective as carriers of therapeutic
substances in vivo because the sustained release or the ability to release
the liposomal contents when necessary is not accomplished.
(3) Liposomes are internalized by the phagocytic cells of the
reticuloendothelial system (RES), and, therefore, are cleared from the
system rapidly, rendering the entrapped drug largely ineffective against
diseases involving cells other than the RES. On the other hand, because
cells of the RES phagocytose liposomes, liposome entrapped drugs may be
very useful in treating intracellular infections of the RES. However,
after phagocytosis, the liposomal contents are packaged within lysosomes
of the phagocytic cell and very often the degradative enzymes contained
within the lysosome will degrade the entrapped compound or render the
compound inactive by altering its structure or modifying the compound at
its active site.
(4) The liposome carriers normally used in delivery systems are expensive
and production is not cost-effective. For example, an improved method for
the chemotherapy of leishmanial infections using liposome encapsulated
anti-leishmanial drugs has been reported by Steck and Alving in U.S. Pat.
No. 4,186,183 issued on Jan. 29, 1980. The liposomes used in the
chemotherapy contained a number of stabilizers which increased the
stability of the liposomes in vivo. However, as previously mentioned,
these stabilizers are expensive and the production of liposomes containing
these stabilizers is not cost-effective.
(5) Ultimately, the problem encountered in the use of liposomes as carriers
in drug delivery systems is the inability to effect a cure of the disease
being treated. In addition to rapid clearance and degradation of the
entrapped compound, a number of other explanations for the inability to
cure diseases are possible. For instance, the liposomes may not deliver a
dose which is effective due to the low percentage of entrapment of active
compound into the vesicles when prepared.
Liposomes have been used by researchers as model membrane systems and have
been employed as the "target cell" in complement-mediated immunoassays.
However, when used in such assays, it is important that the liposome
membrane does not leak when incubated in sera because these assays measure
the release of the liposome contents as a function of serum complement
activation by immune complex formation involving certain immunoglobulin
classes (e.g., IgM and certain IgG molecules).
SUMMARY OF THE INVENTION
This invention presents a new and improved method of preparation of a new
type of lipid vesicle prepared in a monophasic solvent system, which
hereinafter will be referred to as monophasic vesicles (MPVs). These
vesicles are different from other lipid vesicles in that MPVs possess
unique properties when compared to multilamellar vesicles (MLVs),
sonicated unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs)
and reverse phase evaporation vesicles (REVs). As a result of these
differences, MPVs overcome many of the problems presented by classical
liposomes heretofore available.
Advantages of the present process include: the use of less toxic solvents
for the preparation of the liposome; the ability to incorporate
incompatible drugs in the lipid vesicles; the appropriateness for
injection; and the reduced possibility of a health hazard to workers.
The properties of MPVs include: (1) the ability to cure certain diseases
which other methodologies cannot cure; (2) greatly increased stability of
the MPVs over classical liposomes during storage in buffer; (3) the
increased ability of MPVs to withstand physiologic environments; (4) the
entrapment of materials at high efficiency both in the drying and the
rehydration step; and (5) the release of compounds in their bioactive
form.
Methods for preparing MPVs, and for the use of MPVs for the delivery of
bioactive compounds in vivo and in the treatment of pathologies, such as
infections, are described.
DETAILED DESCRIPTION OF THE INVENTION
Preparation of MPV
MPVs are lipid vesicles possessing a plurality of bilayers. The membrane
bilayer is composed of a bimolecular layer of an amphipathic lipid in
which the non-polar hydrophobic hydrocarbon "tails" point inward towards
the center of the bilayer and the polar, hydrophilic "heads" point towards
the aqueous phase. Occluded by the bilayers is an aqueous compartment,
part of which makes up the lumen of the vesicle, and part of which lies
between adjacent layers. Complexed with the lipid bilayers can be a
variety of proteins, glycoproteins, glycolipids, polysaccharides, and any
other hydrophobic and/or amphipathic substance.
MPVs are prepared by a unique process as follows: a lipid or a mixture of
lipids and an aqueous component are added to an organic solvent or a
combination of organic solvents in amounts sufficient to form a monophase.
The solvent or solvents are evaporated until a film forms. Then an
appropriate amount of aqueous component is added, and the film is
resuspended and agitated in order to form the MPVs.
The organic solvent or combination of solvents used in the process must be
miscible with water and once mixed with water should solubilize the lipids
used to make the MPVs.
For example, an organic solvent or mixture of solvents which satifies the
following criteria may be used in the process: (1) 5 ml of the organic
solvent forms a monophase with 0.2 ml of aqueous component and (2) the
lipid or mixture of lipids is soluble in the monophase.
Solvents which may be used in the process of the present invention include
but are not limited to ethanol, acetone, 2-propanol, methanol,
tetrahydrofuran, glyme, dioxane, pyridine, diglyme,
1-methyl-2-pyrrolidone, butanol-2, butanol-1, isoamyl alcohol,
isopropanol, 2-methoxyethanol, or a combination of chlorform:methanol
(e.g., in a 1:1 ratio).
According to the present invention the evaporation should be accomplished
at suitable temperatures and pressures which maintain the monophase and
facilitate the evaporation of the solvents. In fact, the temperatures and
pressures chosen are not dependent upon the phase-transition temperature
of the lipid used to form the MPVs. The advantage of this latter point is
that heat labile products which have desirable properties can be
incorporated in MPVs prepared from phospholipids such as
distearoylphosphatidylcholine, which can be formed into conventional
liposomes only at temperatures above the phase-transition temperature of
the phospholipids. The process usually allows more than 30-40% of the
available water-soluble material to be entrapped during evaporation and
2-15% of the available water-soluble material to be entrapped during the
resuspension; and up to 70-80% of the available lipid-soluble material can
be if the lipid:drug ratio is increased significantly. With MLVs the
entrapment of aqueous phase, which only occurs during the rehydration step
since no aqueous phase is present during the drying step, usually does not
exceed 10%.
Most amphipathic lipids may be constituents of MPVs. Suitable hydrophilic
groups include but are not limited to: phosphato, carboxylic, sulphato and
amino groups. Suitable hydrophobic groups include but are not limited to:
saturated and unsaturated aliphatic hydrocarbon groups and aliphatic
hydrocarbon groups substituted by at least one aromatic and/or
cycloaliphatic group. The preferred amphipathic compounds are
phospholipids and closely related chemical structures.
Specific examples of suitable lipids useful in the production of MPVs are
phospholipids which include but are not limited to the natural lecithins
or phosphatidylcholines (e.g., egg lecithin or soybean lecithin) and
synthetic lecithins, such as saturated synthetic lecithins (e.g.,
dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine or
distearoylphosphatidylcholine) and unsaturated synthetic lecithins (e.g.,
dioleoylphosphatidylcholine or dilinoleoylphosphatidylcholine). Other
phospholipids include but are not limited to phosphatidylethonolamine,
lysolecithin, lysophosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, sphingomyelin, cardiolipin, phosphatidic acid,
ceramides and the cerebrosides. The MPV bilayers can contain a steroid
component such as cholesterol, coprostanol, cholestanol, cholestane and
the like. When using compounds with acidic hydrophilic groups (phosphato,
sulfato, etc.) the obtained MPVs will be anionic; with basic groups such
as amino, cationic liposomes will be obtained.
MPVs may advantageously be used in delivery systems wherein a bioactive
agent is entrapped within the MPV ("entrapped" is defined as entrapment
within the aqueous compartment or within the membrane bilayer). In order
to entrap one or more agents in MPVs, the agent or agents may be added to
the monophase prior to evaporation and formation of the film.
Alternatively, the agent or agents may be added with the aqueous component
used to resuspend the film and form the MPVs. In fact, to obtain a high
entrapment efficiency, the agent or agents may be added to both the
monophase and to the aqueous component used to resuspend the film. Two or
more agents can also be entrapped in one MPV preparation by adding one
agent to the monophase and the other to the aqueous component used to
resuspend the film.
Virtually any bioactive compound can be entrapped within an MPV. Such
compounds include but are not limited to: nucleic acids, polynucleotides,
antibacterial compounds, antiviral compounds, antifungal compounds,
anti-parasitic compounds, tumoricidal compounds, proteins, toxins,
enzymes, hormones, neurotransmitters, glycoproteins, immunoglobulins,
immunomodulators, dyes, radiolabels, radio-opaque compounds, fluorescent
compounds, polysaccharides, cell receptor binding molecules,
anti-inflammatories, antiglaucomic agents, mydriatic compounds,
anesthetics, etc.
Also suitable for entrapment are combinations of incompatible drugs.
Concurrent therapy with certain antimicrobial agents can be complicated
because some agents which are particularly effective when used together in
vitro cannot be formulated in a single mixture at therapeutic
concentration for use in vivo due to a number of constraints. For example,
mixtures of gentamicin and nafcillin at therapeutic concentrations result
in the formation of complexes that precipitate out of solution and,
therefore, are not administered in vivo simultaneously. In fact, certain
drug combinations are not recommended for use in vivo due to drug
incompatibility (i.e., either inactivation of the drug or formation of a
precipitate). For example, it has been recommended that the following
antibiotics not be mixed with any other drug: gentamicin, kanamycin,
lincomycin, cephalothin, and ampicillin (Davis and Abbitt, 1977, JAVMA
170(2): 204-207). Moreover, certain agents cannot be solubilized in the
same medium due to chemical restraints (e.g., a lipid soluble compound and
a water soluble compound). These limitations reduce the possible
combinations of agents that may be used to obtain enhancement of
biological activity in combined therapy. For a review of the topic see
Goodman and Gilman, 1980, The Pharmacological Basis of Therapeutics Sixth
Edition, pp. 1080-1106 and Davis et al., 1980, Microbiology, pp. 574-583.
However, as seen from Examples, infra, incompatible drugs (i.e., nafcillin
and gentamicin) can be combined in MPVs to yield concurrent therapeutic
results.
The following is an illustrative example of the proportions that may be
used in MPV synthesis: MPVs may be formed by adding 127 micromoles of
phospholipid to 5 ml of ethanol and then adding 0.2 ml of aqueous
component containing the active substance to be encapsulated. The
resultant solution which comprises the material to be entrapped and the
entrapping lipid is sonicated (sonication is an optional step) while
streaming an inert gas over the mixture, thus removing most of the solvent
and forming a film. To the resulting film is added 5-10 ml of aqueous
component. The resuspended film is agitated in order to produce stable
MPVs.
Characterization of MPVs
MPVs are clearly distinct in their properties from liposomes with a single
or several lamellae (e.g., SUVs, MLVs and REVs). They have some physical
properties in common with lipid vesicles referred to as stable
plurilamellar vesicles (SPLVs). SPLVs are described in U.S. patent
application Ser. No. 476,496, filed Mar. 24, 1983, which is herein
incorporated by reference. SPLVs are prepared as follows: an amphipathic
lipid or mixture of lipids is dissolved in an organic solvent to which are
added an aqueous phase and the active ingredient to be entrapped. The
aqueous material is emulsified into the solvent while the solvent is being
evaporated. The resulting lipid vesicles exhibit increased stability in
storage and greater medicament entrapment capacity than classical
liposomes.
MPVs exhibit greater stability in urea than do SPLVs. The following
detailed comparison is focused on distinguishing MPVs from SPLVs and MLVs.
Stability of MPVs in storage
Stability of a lipid vesicle refers to the ability of the vesicle to
sequester its occluded space from the external environment over a long
period of time. For a lipid vesicle to be useful it is paramount that it
be stable in storage and handling. For some applications, however, it is
desirable that the vesicle leak its contents slowly when applied. For
other applications it is desirable that the vesicle remain intact after
administration until it reaches its desired site of action. It will be
seen that MPVs demonstrate many of these desirable characteristics.
There are two factors that cause vesicles to leak during storage. One is
auto-oxidation of the lipids whereby the hydrocarbon chains form peroxides
which destabilize the bilayers. Vesicles can also leak because agents in
the exterior environment disrupt the bilayer organization of the lipids
such that the lipids remain intact, but the membrane develops a pore.
In the following experiments vesicles were prepared which contained
radioactive tracer molecules within the occluded aqueous compartments.
When placed in a buffer containing isotonic saline at neutral pH, MPVs
containing antibiotic exhibit prolonged stability in storage. The vesicles
were prepared, each containing one of the following radio-labeled drugs:
.sup.125 I-p-hydroxypropionic acid-derived gentamicin sulfate, .sup.14
C-indomethacin, and .sup.3 H-inulin. After storage at various temperatures
for 14 days the vesicles were separated from the medium by centrifugation,
and the relative amount of radioactivity that escaped from the vesicles
into the medium was determined. The results demonstrated that both MPVs
and SPLVs were more stable during storage than were MLVs.
The shelf-life of an MPV preparation can be considerably lengthened by
storing the dried film containing the lipids and agent to be entrapped.
When fully formed MPVs are desired, the dried film can be resuspended by
adding an appropriate amount of aqueous component (e.g., buffer) and
agitating the resuspension.
Stability of MPVs in other environments
Placing lipid vesicles in a medium which contains membrane perturbing
agents is a way to probe different molecular organizations. Depending on
how the membrane is organized, different vesicles will respond differently
to such agents.
In the following experiments vesicles were prepared which contained a
radioactive tracer molecule (.sup.3 H-inulin) within the occluded aqueous
compartment. Inulin, a polysaccharide, partitions into the aqueous phase,
and thus when radiolabeled may be used to trace the aqueous contents of
lipid vesicles. After an appropriate interval of exposure to a given
agent, the vesicles were separated from the medium by centrifugation, and
the relative amount of radioactivity that escaped from the vesicles into
the medium was determined. These results are reported in Table I.
MPVs respond differently than SPLVs when exposed to urea. Urea is a
molecule with both a chaotropic effect (disrupts the structure of water)
and a strong dipole moment. It is observed that SPLVs are far more
susceptible to urea than MPVs. (See Table I).
TABLE I
______________________________________
STABILITY OF LIPID VESICLES IN 1 MOLAR UREA
% Leakage.sup.a
1 hour 3 hours 5 hours
______________________________________
MPVs 8.3 4.9 10.8
SPLVs 4.4 29.7 49.7
______________________________________
.sup.a Values are expressed as percent leaked, meaning the proportion of
radioactive material in the surrounding medium (cpm) relative to the
starting amount encapsulated in the vesicles (cpm).
Entrapment of active material by MPVs
MPVs were prepared to which the radioactive tracer molecules were added
prior to the drying step. The MPV entrapment efficiency of the
biologically active compounds was compared to that of SPLVs prepared with
the same components. The vesicles were separated from the suspending
preparation medium by centrifugation, and the relative amount of
radioactivity retained by the vesicles was determined. These results are
reported in Table II.
TABLE II
______________________________________
COMPARISON OF SPLVs AND MPVs
% Available Material
Entrapped In Aqueous Phase.sup.a
Encapsulation of:
SPLVs MPVs.sup.b
______________________________________
.sup.125 I--Gentamicin
32.8 38.3
.sup.3 H--Inulin
36.8 36.7
.sup.14 C--Indomethacin
21.9 15.3
______________________________________
.sup.a Values are expressed as percent entrapped meaning the proportion o
radioactive material in the liposome pellet (cpm) relative to the startin
amount (cpm) added to the preparation.
.sup.b Radiolabeled material to be entrapped was added to the monophase.
After evaporation to a film and resuspension with aqueous buffer to form
MPVs, the preparation was pelleted and the radioactivity of the
supernatant was determined.
MPVs have similar superiority over traditional MLVs as do SPLVs in the
percentage of entrapment of biologically active material. This affords the
benefit of conserving material.
Uses of MPVs
MPVs are particularly useful in systems where the following factors are
important: stability during storage and contact with body fluids; a
relatively high degree of encapsulation. Therefore, MPVs may be used to
enhance the therapeutic efficacy of medications; to cure infections; to
enhance topical drug delivery; for the production of vaccines; or as
diagnostic reagents for clinical tests following release of entrapped
"reporter" molecules. The MPVs can also be employed to encapsulate
cosmetic preparations, pesticides, compounds for sustained slow release to
effect the growth of plants and the like.
The methods which follow, while described in terms of the use of MPVs,
contemplate the use of MPVs or any other liposome or lipid vesicle having
functional characteristics similar to those of MPVs.
Delivery of bioactive compounds
Delivery of compounds to cells in vitro (e.g., animal cells, plant cells,
protists, etc.) generally requires the addition of the MPVs containing the
compound to the cells in culture. In one scheme MPVs and SPLVs containing
gentamicin were plated onto lawns of Staphylococcus aureus and Salmonella
typhimurium (See Table III). The results demonstrate that MPVs have drug
delivery properties similar to SPLVs.
TABLE III
______________________________________
ZONES OF INHIBITION.sup.a
Staphylococcus
Salmonella
aureus typhimurium
______________________________________
SPLV 0.29 cm 0.58 cm
MPV 0.36 cm 0.82 cm
______________________________________
.sup.a Liposomes were prepared as previously described and 10 microliter
aliquots were plated onto the lawns as indicated. Zones of inhibition wer
measured after 24 hours.
MPVs can also be used to deliver compounds in animals (including man),
plants and protists. Depending upon the purpose of delivery, the MPVs may
be administered by a number of routes: in man and animals this includes
but is not limited to injection (e.g., intravenous, intraperitoneal,
intramuscular, subcutaneous, intraarticular, intraauricular, intramammary,
intraurethrally, etc.), topical application (e.g., on afflicted areas),
and by absorption through epithelial or mucocutaneous linings (e.g.,
ocular epithelia, oral mucosa, rectal and vaginal epithelial linings,
respiratory tract linings, nasopharyngeal mucosa, intestinal mucosa,
etc.); in plants and protists this includes but is not limited to direct
application to organism, dispersion in the organism's habitat, addition to
the surrounding environment or surrounding water, etc.
The mode of application may also determine the sites and cells in the
organism to which the compound will be delivered. Delivery to the
circulatory system (and hence reticuloendothelial cells), may be most
easily accomplished by intravenous or intraperitoneal injections.
The sections which follow describe some overall schemes in which MPVs may
be used and demonstrate, but do not limit, the scope of the present
invention.
Treatment of pathologies
A number of pathological conditions which occur in man, animals and plants
may be treated effectively by encapsulating the appropriate compound or
compounds in MPVs. These pathologic conditions include but are not limited
to infections (intracellular and extracellular), cysts, tumors and tumor
cells, allergies, etc.
Many strategies are possible for using MPVs in the treatment of such
pathologies; in one scheme, MPVs are used to deliver therapeutic agents to
sites of intracellular infections. Certain diseases involve an infection
of cells of the reticuloendothelical system, e.g., brucellosis. These
intracellular infections are difficult to cure for a number of reasons:
(1) because the infectious organisms reside within the cells of the
reticuloendothelial system, they are sequestered from circulating
therapeutic agents which cannot cross the cell membrane in therapeutically
sufficient concentrations, and, therefore, are highly resistant to
treatment; (2) often the administration of toxic levels of therapeutic
agents are required in order to combat such infections; and (3) the
treatment has to be completely effective because any residual infection
after treatment can reinfect the host organism or can be transmitted to
other hosts.
According to one mode of the present invention, MPVs containing an
appropriate biologically active compound are administered (preferably
intraperitoneally or intravenously) to the host organism or potential host
organism (e.g., in animal herds, the uninfected animals as well as
infected animals may be treated). Since phagocytic cells internalize MPVs,
the administration of an MPV-encapsulated substance that is biologically
active against the infecting organism will result in directing the
bioactive substance to the site of infection. Thus, the method of the
present invention may be used to eliminate infection caused by a variety
of microorganisms, bacteria, parasites, fungi, mycoplasmas, and viruses,
including but not limited to: Brucella spp., Mycobacterium spp.,
Salmonella spp., Listeria spp., Francisella spp., Histoplasma spp.,
Corynebacterium spp., Coccidiodes spp. and lymphocytic choriomeningitis
virus.
The therapeutic agent selected will depend upon the organism causing the
infection. For instance, bacterial infections may be eliminated by
encapsulating an antibiotic or combination of antibiotics. The antibiotic
can be contained within the aqueous fluid of the MPV and/or inserted into
the vesicle bilayer. Suitable antibiotics include but are not limited to:
penicillin, ampicillin, hetacillin, carbencillin, tetracycline,
tetracycline hydrochloride, oxytetracycline hydrochloride,
chlortetracycline hydrochloride, 7-chloro-6-dimethyltetracycline,
doxycycline monohydrate, methacycline hydrochloride, minocycline
hydrochloride, rolitetracycline, dihydrostreptomycin, streptomycin,
gentamicin, kanamycin, neomycin, erythromycin, carbomycin, oleandomycin,
troleandomycin, Polymyxin B collistin, cephalothin sodium, cephaloridine,
cephaloglycin dihydrate, and cephalexin monohydrate.
We have demonstrated the effectiveness of such treatments in curing
brucellosis and salmonellosis (see Examples, infra). By the procedure of
this invention, the effectiveness and duration of action are prolonged.
This system is effective for treating infections which do not respond to
known treatments such as antibiotics entrapped in MLVs.
Of course, the invention is not limited to treatment of intracellular
infections. The MPVs can be directed to a variety of sites of infection
whether intracellular or extracellular.
MPVs are also useful in the treatment of any affliction requiring prolonged
contact with the active treating substance. For example, glaucoma is a
disorder characterized by a gradual rise in intraocular pressure causing
progressive loss of peripheral vision, and, when uncontrolled, loss of
central vision and ultimate blindness. Drugs used in the treatment of
glaucoma may be applied topically as eyedrops. However, in some cases
treatment requires administering drops every 15 minutes due to the rapid
clearing of the drug from the eye socket. If an affliction such as
glaucoma is to be treated by this invention therapeutic substances such as
pilocarpine, Floropryl, physostigmine, carcholin, acetazolamide,
ethozolamide, dichlorphenamide, carbachol, demecarium bromide,
diisopropylphosphofluoridate, ecothioplate iodide, physostigmine, or
neostigmine, etc. can be entrapped within MPVs which are then applied to
the affected eye.
Other agents which may be encapsulated in MPVs and applied topically
include but are not limited to: mydriatics (e.g., epinephrine,
phenylepinephrine, hydroxy amphetamine, ephedrine, atropine, homatropine,
scopolamine, cyclopentolate, tropicamide, encatropine, etc.); local
anesthetics; antiviral agents (e.g., idoxuridine, adenine arabinoside,
etc.); antimycotic agents (e.g., amphoteracin B, natamycin, pimaricin,
flucytosine, nystantin, thimerosal, sulfamerazine, thiobendazole,
tolnaftate, grisiofulvin, etc.); antiparasitic agents (e.g., sulfonamides,
pyrimethamine, clindamycin, etc.); and anti-inflammatory agents (e.g.,
corticosteriods such as ACTH, hydrocortisone, prednisone, medrysone, beta
methasone, dexamethasone, fluoromethalone, triamcinalone, etc.).
EXAMPLE
Preparation of MPVs
In the subsectio | | |
