U.S. patent application number 11/040615 was filed with the patent office on 2005-08-25 for novel cochleate formulations.
This patent application is currently assigned to University of Medicine and Dentistry of New Jersey, University of Medicine and Dentistry of New Jersey. Invention is credited to Jin, Tuo, Mannino, Raphael J., Segarra, Ignacio, Zarif, Leila.
Application Number | 20050186265 11/040615 |
Document ID | / |
Family ID | 22885334 |
Filed Date | 2005-08-25 |
United States Patent
Application |
20050186265 |
Kind Code |
A1 |
Zarif, Leila ; et
al. |
August 25, 2005 |
Novel cochleate formulations
Abstract
A process for producing a small-sized, lipid-based cochleates.
Cochleates are derived from liposomes which are suspended in an
aqueous two-phase polymer solution, enabling the differential
partitioning of polar molecule based-structures by phase
separation. The liposome-containing two-phase polymer solution,
treated with positively charged molecules such as Ca.sup.2+ or
Zn.sup.2+, forms a cochleate precipitate of a particle size less
than one micron. The process may be used to produce cochleates
containing biologically relevant molecules.
Inventors: |
Zarif, Leila; (Cannes,
FR) ; Jin, Tuo; (Highland Park, NJ) ; Segarra,
Ignacio; (South Orange, NJ) ; Mannino, Raphael
J.; (Annandale, NJ) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP.
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
University of Medicine and
Dentistry of New Jersey
Newark
NJ
|
Family ID: |
22885334 |
Appl. No.: |
11/040615 |
Filed: |
January 18, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11040615 |
Jan 18, 2005 |
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10421358 |
Apr 23, 2003 |
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10421358 |
Apr 23, 2003 |
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09613840 |
Jul 11, 2000 |
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6592894 |
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09613840 |
Jul 11, 2000 |
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09235400 |
Jan 22, 1999 |
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6153217 |
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Current U.S.
Class: |
424/450 ;
435/458 |
Current CPC
Class: |
A61K 9/1274 20130101;
A61K 31/20 20130101; A61K 36/54 20130101; A61K 31/496 20130101;
A61K 31/713 20130101; A61P 31/10 20180101; A61P 31/04 20180101;
A61P 9/10 20180101; A61K 31/11 20130101; Y10T 428/2984 20150115;
Y10S 514/966 20130101; A61K 31/7048 20130101; A61K 31/472 20130101;
A61P 37/06 20180101; A61K 31/07 20130101; A61P 25/20 20180101; Y10S
514/967 20130101; Y10S 436/829 20130101; A61P 31/12 20180101; A61P
35/00 20180101; A61P 29/00 20180101; A61P 23/00 20180101; A61K
31/704 20130101; A61K 38/13 20130101 |
Class at
Publication: |
424/450 ;
435/458 |
International
Class: |
A61K 009/127; C12N
015/88 |
Claims
1. A cochleate composition comprising a population of cochleates,
wherein the cochleates comprise: a negatively charged first lipid a
divalent cation or higher valency cation and an additional
biologically relevant molecule, wherein the mean particle size of
the cochleates is less than one micron.
2. The cochleate composition according to claim 1, wherein the
biologically relevant molecule bears a charge.
3. The cochleate composition according to claim 2, wherein the
biologically relevant molecule is positively charged.
4. The cochleate composition according to claim 2, wherein the
biologically relevant molecule is negatively charged.
5. The cochleate composition according to claim 1, wherein the
biologically relevant molecule is amphiphilic.
6. The cochleate composition according to claim 1, wherein the
biologically relevant molecule is hydrophobic.
7. The cochleate composition according to claim 1, wherein the
biologically relevant molecule is at least one member selected from
the group consisting of a drug, a polynucleotide, a polypeptide, an
antigen, a nutrient and a flavor substance.
8-22. (canceled)
23. The cochleate composition according to claim 1, wherein the
negatively charged lipid is comprised of phosphatidylserine.
24. The cochleate composition according to claim 1, wherein the
cochleates further comprise a minor amount of a second lipid.
25. The cochleate composition according to claim 24, wherein the
other lipid is a member selected from the group consisting of a
zwitterionic lipid, a PEGylated lipid, a cationic lipid, or a
polycationic lipid.
26. The cochleate composition according to claim 24, wherein the
second lipid comprises lipid capable of forming hydrogen bonds to
the biologically relevant molecule.
27. The cochleate composition according to claim 1, wherein the di-
or higher-valent ions are metal ions.
28. The cochleate composition according to claim 27, wherein the
di- or higher-valent metal ions are selected from the group
consisting of calcium, zinc, barium, and magnesium.
29. A cochleate composition according to claim 1 wherein the di-or
higher-valent cation is a di or higher-valent cationic lipid.
30-63. (canceled)
64. A pharmaceutical composition comprising an effective amount of
the cochleate composition of claim 1 and a pharmaceutically
acceptable carrier.
65. (canceled)
66. A method of treatment comprising administering to a human or
animal host a pharmaceutically effective amount of the
pharmaceutical composition according to claim 64.
67. (canceled)
68. The method of treatment according to claim 66, wherein the
administration is by a mucosal or a systemic route.
69. (canceled)
70. The method of treatment according to claim 66, wherein the
administration is a mucosal route selected from the group
consisting of oral, intranasal, intraocular, intraanal,
intravaginal, and intrapulmonary.
71. (canceled)
72. The method of treatment according to claim 66, wherein the
administration is by a systemic route selected from the group
consisting of intravenous, intramuscular, subcutaneous, transdermal
and intradermal.
73. The cochleate composition of claim 7, wherein the nutrient is a
vitamin.
74. The cochleate composition of claim 73, wherein the vitamin is
at least one member selected from the group consisting of: vitamin
D, vitamin E, and vitamin K.
75. The cochleate composition of claim 73, wherein the vitamin is
vitamin A.
76. The cochleate of claim 7, wherein the nutrient is a second
cation.
77. The cochleate composition of claim 76, wherein the second
cation is at least one member selected from the group consisting
of: calcium, magnesium, barium, iron and zinc.
78. The cochleate composition of claim 7, wherein the cochleate
comprises at least one vitamin and at least one mineral.
79. The cochleate composition according to claim 7, wherein the
nutrient is at least one member selected from the group consisting
of fatty acids, amino acids, and saccharides.
80. The cochleate composition according to claim 7, wherein the
nutrient comprises a fatty acid selected from the group consisting
of polyunsaturated fatty acids and saturated fatty acids.
81. The cochleate composition according to claim 7, wherein the
nutrient comrpises a saccharide selected from the group consisting
of glucose and sucrose.
82. The cochleate composition of claim 7, wherein the drug is an
antifungal agent.
83. The cochleate composition of claim 7, wherein the drug is a
non-steriodal anti-inflammatory agent.
84. The cochleate composition of claim 7, wherein the drug is an
anticancer agent.
85. The cochleate composition of claim 7, wherein the drug is
selected from the group consisting of an antiviral, an anesthetic,
or an anti-infectious agent, an immunosuppressant, a steroidal
anti-inflammatory, a tranquilizer, and a vasodilatory agent.
86. The cochleate composition of claim 7, wherein the drug is
Amphotericin B.
87. The cochleate composition of claim 7, wherein the drug is
naproxen.
88. The cochleate composition of claim 7, wherein the drug is
Vitamin A acid.
89. The cochleate composition of claim 7, wherein the drug is at
least one member selected from the group consisting of acyclovir,
adriamycin, carbamazepine, melphalan, nifedipine, indomethacin,
naproxen, phenytoin, ergotamines, rapamycin, propanidid, propofol,
alphadione, echinomycine, miconazole nitrate, teniposide, taxol,
taxotere, nystatin, and rifampin.
90. The cochleate composition of claim 7, wherein the drug is at
least one member selected from the group consisting of estrogens,
testosterones, steroids, cannabinoids, and vitamin A acid.
91. The cochleate composition of claim 7, wherein the
polynucleotide is a deoxyribonucletic acid (DNA) molecule
92. The cochleate composition according to claim 91, wherein the
DNA is transcribed to yield a ribonucleic acid.
93. The cochleate composition of claim 7, wherein the
polynucleotide is a ribonucleic acid (RNA) molecule.
94. The cochleate composition according to claim 93, wherein the
ribonucleic acid is translated to yield a biologically active
polypeptide.
95. The cochleate composition of claim 7, wherein the
polynucleotide is a plasmid or a ribozyme.
96. The cochleate composition of claim 7, wherein the
polynucleotide is an antisense molecule.
97. The cochleate composition of claim 7, wherein the polypeptide
is at least one member selected from the group consisting of
cyclosporin, angiotensin I, II, or III, enkephalins and their
analogs, ACTH, anti-inflammatory peptides I, II, or III,
bradykinin, calcitonin, beta-endorphin, dinorphin, leucokinin,
leutinizing hormone releasing hormone (LHRH), insulin, neurokinins,
somatostatin, substance P, thyroid releasing hormone (TRH), and
vasopressin.
98. The cochleate composition of claim 7, wherein the antigen is at
least one member selected from the group consisting of
carbohydrates, envelope glycoproteins from viruses,
polynucleotides, DNA, RNA, plasmid DNA, immunogens, ribozymes, and
antisense molecules.
99. The cochleate composition of claim 7, wherein the antigen is at
least one member selected from the group consisting of animal cell
membrane proteins, plant cell membrane proteins, bacterial membrane
proteins, envelope glycoproteins from viruses, and parasitic
membrane proteins.
100. The cochleate composition of claim 7, wherein the flavor agent
is a botanical essential oil or an extract.
101. The cochleate composition of claim 7, wherein the flavor agent
is at least one flavor agent selected from the group consisting of:
a cinnamon oil, a vanillin, an herb, a citrus, a spice and a
seed.
102. A cochleate composition comprising a population of cochleates,
wherein the cochleates comprise: a negatively charged lipid; a
divalent cation or higher valency cation; and, optionally, an
additional biologically relevant molecule, wherein the mean
particle size of the cochleates is less than one micron.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This is a Continuation-in-Part of U.S. application Ser. No.
09/235,400, filed Jan. 22, 1999 (now allowed).
FIELD OF THE INVENTION
[0002] The present invention relates to a novel method for
preparing a novel lipid-based cochleate delivery system, the
preparations derived from the lipid-based cochleate delivery
system, such as drugs, carbohydrates, vitamins, minerals,
polynucleotides, polypeptides, lipids and the like, and the use of
these preparations.
BACKGROUND OF THE INVENTION
[0003] The ability of biologically relevant molecules to be
administered via the oral route depends on several factors. The
biologically relevant molecule must be soluble in the
gastrointestinal fluids in order for the biologically relevant
molecule to be transported across biological membranes for an
active transport mechanism, or have suitable small particle size
that can be absorbed through the Peyer's Patches in the small
intestine and through the lymphatic system. Particle size is an
important parameter when oral delivery is to be achieved (see
Couvreur et al., Adv. Drug Delivery Rev., 10:141-162 (1993)).
[0004] The primary issue in the ability to deliver drugs orally is
the protection of the drug from proteolytic enzymes. An ideal
approach is to incorporate the drug in a hydrophobic material so
that the aqueous fluids cannot penetrate the system. Lipid-based
cochleates are an ideal system that can achieve this purpose.
[0005] The advantages of cochleates are numerous. The cochleates
have a nonaqueous structure and therefore they:
[0006] a) are more stable because of less oxidation of lipids;
[0007] b) can be stored lyophilized, which provides the potential
to be stored for long periods of time at room temperatures, making
them advantageous for worldwide shipping and storage prior to
administration;
[0008] c) maintain their structure even after lyophilization,
whereas liposome structures are destroyed by lyophilization;
[0009] d) exhibit efficient incorporation of biologically relevant
molecules into the lipid bilayer of the cochleate structure;
[0010] e) have the potential for slow release of a biologically
relevant molecule in vivo as cochleates dissociate;
[0011] f) have a lipid bilayer which serves as a carrier and is
composed of simple lipids which are found in animal and plant cell
membranes, so that the lipids are non-toxic;
[0012] g) are produced easily and safely;
[0013] h) can be produced as defined formulations composed of
predetermined amounts and ratios of drugs or antigens.
[0014] Cochleate structures have been prepared first by D.
Papahadjopoulos as an intermediate in the preparation of large
unilamellar vesicles (see U.S. Pat. No. 4,078,052). The use of
cochleates to deliver protein or peptide molecules for vaccines has
been disclosed in U.S. Pat. Nos. 5,840,707 and 5,643,574. The use
of cochleates to orally deliver drugs, nutrients, and flavors have
been described in U.S. Pat. No. 5,994,318.
[0015] However, the advantages of using small-sized cochleates have
only recently been explored. The effective oral delivery of drugs
that are mediated by hydrogel-isolated cochleates has been
described in U.S. application Ser. No. 09/235,400. However, the
effective delivery of hydrogel-isolated cochleates have not been
described for other biologically relevant molecules such as drugs,
polypeptides, polynucleotides, antigens, vitamins, minerals, amino
acids, saccharides, flavor oils, and the like.
SUMMARY OF THE INVENTION
[0016] Accordingly, it is an object of this invention to provide a
method for obtaining a hydrogel-isolated cochleate of a particle
size of less than one micron. The method further comprises the
steps required to encochleate at least one biologically relevant
molecule in the hydrogel-isolated cochleates in an effective
amount.
[0017] A "biologically relevant molecule" is one that has a role in
the life processes of a living organism. The molecule may be
organic or inorganic, a monomer or a polymer, endogenous to a host
organism or not, naturally occurring or synthesized in vitro, and
the like. Thus, examples include vitamins, minerals, flavors, amino
acids, toxins, microbicides, microbistats, co-factors, enzymes,
polypeptides, polypeptide aggregates, polynucleotides, lipids,
carbohydrates, nucleotides, starches, pigments, fatty acids,
hormones, cytokines, viruses, organelles, steroids and other
multi-ring structures, saccharides, metals, metabolic poisons,
drugs, and the like.
[0018] These and other objects have been obtained by providing an
encochleated biologically relevant molecule, wherein the
biologically relevant molecule-cochleate comprises the following
components:
[0019] a) a biologically relevant molecule,
[0020] b) a negatively charged lipid, and
[0021] c) a cation component,
[0022] wherein the particle size of the cochleate is less than one
micron.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIG. 1 is a schematic of the process by which the
hydrogel-isolated cochleates of the present invention, with or
without a biologically relevant molecule, are obtained.
[0024] FIGS. 2A and 2B illustrate a particle size distribution
(weight analysis) of hydrogel-isolated cochleates either loaded
with amphotericin B (AmB) (FIG. 2A) or empty (FIG. 2B) as measured
by laser light scattering.
[0025] FIGS. 3A and 3B illustrate microscopic images of a mixture
of liposomes in dextran dispersed into PEG gel solution. The small
black dots are dextran particles formed by dispersing the dextran
phase in the PEG phase. The large open circles are formed by the
fusion of small dextran particles. Partition of liposomes favors
the dextran phase as indicated by a yellow color of AmB. FIG. 3B:
Microscopic images of the sample shown in FIG. 3A after treatment
with CaCl.sub.2 solution. The black objects in circles, are
cochleates formed by the addition of Ca.sup.2+ ions.
[0026] FIGS. 4A-4F illustrate microscopic images of the sample
shown in FIGS. 3A and 3B after washing with a buffer containing 1
mM CaCl.sub.2 and 100 mM NaCl. Aggregates are formed by the
cochleate particles (FIG. 4B). A suspension shown in FIG. 4A
following the addition of EDTA. Cochleate particles opened to
liposomes with a diameter of 1-2 microns, indicating the intrinsic
size of the cochleate particles is in the sub-micron range (FIG.
4C). AmB hydrogel-isolated cochleates precipitated with zinc
according to the procedure described in Example 14 (FIG. 4D).
Cochleates displayed in FIG. 4C after treatment with EDTA (FIG.
4E). Empty hydrogel-isolated cochleates precipitated with zinc
according to the procedure described in Example 13 (FIG. 4F).
Cochleates displayed in FIG. 4F are after treatment with EDTA.
[0027] FIG. 5 illustrates micrographs of hydrogel-isolated
cochleates after freeze fracture.
[0028] FIG. 6 illustrates growth inhibition of Candida albicans by
hydrogel-isolated cochleates loaded with AmB at 0.625 .mu.g AmB/ml.
Comparison is made to AmB in DMSO and AmBisome.RTM..
[0029] FIG. 7 illustrates the effect of hydrogel-isolated
cochleates on the viability of Candida albicans after 30 hours.
[0030] FIGS. 8A and 8B illustrate the efficacy of Amphotericin
B-cochleates on macrophage cultures.
[0031] FIG. 9 illustrates Amphotericin B tissue levels after
administration of Amphotericin B-cochleates.
[0032] FIG. 10 illustrates the time profile tissue concentration of
AmB after a single dose administration of hydrogel-isolated
cochleates loaded with AmB.
[0033] FIG. 11 illustrates AmB tissue level 24 hrs after single
dose and 24 hrs after a multiple dose regime.
[0034] FIG. 12 illustrates correlation between Amphotericin B
tissue level and the level of Candida albicans after administration
of Amphotericin B cochleates.
DETAILED DESCRIPTION OF THE INVENTION
[0035] The present invention provides a solution to achieve
effective oral delivery of drugs and other biologically relevant
molecules by producing small-sized cochleates of less than one
micron using new methods. The new approach is based on the
incompatibility between two polymer solutions, both of which are
aqueous. Aqueous two-phase systems of polymers are well used for
protein purification due to a number of advantages such as freedom
from the need for organic solvents, mild surface tension and the
biocompatibility of aqueous polymers (see P. A. Albertsson,
"Partition of cell particles and macromolecules", 3.sup.rd edition,
Wiley N.Y. (1986); and "Separation using aqueous Phase System" D.
Fisher Eds, Plenum N.Y. (1989)). It is known, for example, that
large polar molecules such as proteins partition to a much higher
concentration in a polymer phase with the physical characteristics
similar to those of dextran than in a polymer phase with the
physical characteristics similar to those of PEG (see Forciniti et
al, Biotechnol. Bioeng., 38:986 (1991)).
[0036] According to the present invention there are provided
methods for preparing small-sized, lipid-based cochleate particles
and preparations derived therefrom, comprising a biologically
relevant molecule incorporated into the particles. The cochleate
particles are formed of an alternating sequence of lipid
bilayers/cation. The biologically relevant molecule is incorporated
either in the lipid bilayers or in the interspace between the lipid
bilayers. One of the methods for preparing the small-sized
cochleates comprises: 1) preparing a suspension of small
unilamellar liposomes or biologically relevant molecule-loaded
liposomes, 2) mixing the liposome suspension with polymer A, 3)
adding, preferably by injection, the liposome/Polymer A suspension
into another polymer B in which polymer A is nonmiscible, leading
to an aqueous two-phase system of polymers, 4) adding a solution of
cation salt to the two-phase system of step 3, such that the cation
diffuses into polymer B and then into the particles comprised of
liposome/polymer A allowing the formation of small-sized
cochleates, 5) washing the polymers out and resuspending the empty,
drug or other biologically relevant molecule-loaded cochleates into
a physiological buffer or any appropriate pharmaceutical
vehicle.
[0037] A second method for preparing the small-sized cochleates
comprises detergent and a biologically relevant molecule and
cation. The detergent is added to disrupt the liposomes. The method
comprises the following steps:
[0038] 1) providing an aqueous suspension containing a
detergent-lipid mixture;
[0039] 2) mixing the detergent-lipid suspension with polymer A;
[0040] 3) adding the detergent-lipid/polymer A suspension into a
solution comprising polymer B, wherein polymer A and polymer B are
immiscible, thereby creating a two-phase polymer system;
[0041] 4) adding a solution of a cationic moiety to the two-phase
polymer system; and
[0042] 5) washing the two-phase polymer system to remove the
polymer.
[0043] A lyophilization procedure can be applied and the
lyophilized biologically relevant molecule-cochleate complex can be
filled into soft or hard gelatin capsules, tablets or other dosage
form, for systemic, dermal or mucosal delivery.
[0044] Both methods described above lead to a small-sized particle
with a narrow size range that allows efficient oral delivery of
biologically relevant molecules. The biologically relevant molecule
partitions into either or both lipid bilayers and interspace, and
the biologically relevant molecule is released from the cochleate
particles by dissociation of the particles in vivo. Alternative
routes of administration may be systemic, such as intramuscular,
subcutaneous or intravenous, or mucosal such as intranasal,
intraocular, intravaginal, intraanal, or intrapulmonary.
Appropriate dosages are determinable by, for example, dose-response
experiments in laboratory animals or in clinical trials and taking
into account body weight of the patient, absorption rate,
half-life, disease severity and the like. The number of doses,
daily dosage and course of treatment may vary from individual to
individual. Other delivery routes can be dermal, transdermal or
intradermal.
[0045] The first step of either method of the present invention,
which is the preparation of small liposomes, can be achieved by
standard methods such as sonication or microfluidization or other
related methods (see, for example, Liposome Technology, Liposome
Preparation and Related Techniques, Edited by Gregory Gregoriadis,
Vol I, 2.sup.nd Edition, CRC Press (1993)).
[0046] The second step of either method comprises the addition,
preferably by injection, of polymer A/liposome suspension into
polymer B can be achieved mechanically by using a syringe pump at
an appropriate controlled rate, for example a rate of 0.1 ml/min to
50 ml/min, and preferably at a rate of 1 to 10 ml/min.
[0047] The formation of hydrogel-isolated cochleates (with or
without a biologically relevant molecule) is achieved in the third
step by adding a positively charged molecule to the aqueous
two-phase polymer solution containing liposomes. The positively
charged molecule can be a polyvalent cation and more specifically,
any divalent cation that can induce the formation of a cochleate.
In a preferred embodiment, the divalent cations include Ca.sup.++,
Zn.sup.++, Ba.sup.++ and Mg.sup.++ or other elements capable of
forming divalent ions or other structures having multiple positive
charges capable of chelating and bridging negatively charged
lipids, such as polycationic lipids. Addition of positively charged
molecules to liposome-containing solutions is also used to
precipitate cochleates from the aqueous solution.
[0048] To isolate the cochleate structures and to remove the
polymer solution, cochleate precipitates are repeatedly washed in a
fourth step with a buffer containing a positively charged molecule,
and more preferably, a divalent cation. Addition of a positively
charged molecule to the wash buffer ensures that the cochleate
structures are maintained throughout the wash step, and that they
remain as precipitates.
[0049] Finally, the medium in which the cochleates are suspended
can contain salt such as calcium chloride, zinc chloride, cobalt
chloride, sodium chloride, sodium sulfate, potassium sulfate,
ammonium sulfate, magnesium sulfate and sodium carbonate. The
medium can contain polymers, such as pluronics, and polyethylene
glycols. The biologically relevant molecule-cochleate is made by
diluting into an appropriate biologically acceptable carrier (e.g.,
a divalent cation-containing buffer).
[0050] The lipids of the present invention are non-toxic lipids and
include, but are not limited to simple lipids which are found in
animal and plant cell membranes. Preferably the lipid is a
negatively charged lipid, more preferably a negatively charged
phospholipid, and even more preferably a lipid from the group of
phosphatidylserine, phosphatidylinositol, phosphatidic acid, and
phosphatidyl glycerol. The lipids may also include minor amounts of
zwitterionic lipids, cationic lipids, polycationic lipids or
neutral lipids capable of forming hydrogen bonds to a biologically
relevant molecule such as PEGylated lipid.
[0051] The polymers A and B of the present invention can be of any
biocompatible polymer classes that can produce an aqueous two-phase
system. For example, polymer A can be, but is not limited to,
dextran 200,000-500,000, Polyethylene glycol (PEG) 3,400-8,000;
polymer B can be, but is not limited to, polyvinylpyrrolidone
(PVP), polyvinylalcohol (PVA), Ficoll 30,000-50,000, polyvinyl
methyl ether (PVMB) 60,000-160,000, PEG 3,400-8,000. The
concentration of polymer A can range from between 2-20% w/w as the
final concentration depending on the nature of the polymer. The
same concentration range can be applied for polymer B. Examples of
suitable two-phase systems are Dextran/PEG, 5-20% w/w Dextran
200,000-500,000 in 4-10% w/w PEG 3,400-8,000; Dextran/PVP 10-20%
w/w Dextran 200,000-500,000 in 10-20% w/w PVP 10,000-20,000;
Dextran/PVA 3-15% w/w Dextran 200,000-500,000 in 3-15% w/w PVA
10,000-60,000; Dextran/Ficoll 10-20% w/w Dextran 200,000-500,000 in
10-20% w/w Ficoll 30,000-50,000; PEG/PVME 2-10% w/w PEG
3,500-35,000 in 6-15% w/w PVME 60,000-160,000.
[0052] The biologically relevant molecule is a molecule that has a
role in the life processes of a living organism. The molecule may
be organic or inorganic, a monomer or a polymer, charged, either
positively or negatively, hydrophilic, amphiphilic or hydrophobic
in aqueous media, endogenous to a host organism or not, naturally
occurring or synthesized in vitro and the like. The biologically
relevant molecule may be a drug, and the drug may be an antiviral,
an anesthetic, an anti-infectious, an antifungal, an anticancer, an
immunosuppressant, a steroidal anti-inflammatory, a non-steroidal
anti-inflammatory, a tranquilizer or a vasodilatory agent. Examples
include Amphotericin B, acyclovir, adriamycin, carbamazepine,
melphalan, nifedipine, indomethacin, naproxen, estrogens,
testosterones, steroids, phenytoin, ergotamines, cannabinoids,
rapamycin, propanidid, propofol, alphadione, echinomycine,
miconazole nitrate, teniposide, taxol, taxotere, nystatin,
rifampin, and vitamin A acid.
[0053] The biologically relevant molecule may be a polypeptide such
as cyclosporin, angiotensin I, II and III, enkephalins and their
analogs, ACTH, anti-inflammatory peptides I, II, III, bradykinin,
calcitonin, beta-endorphin, dinorphin, leucokinin, leutinizing
hormone releasing hormone (LHRH), insulin, neurokinins,
somatostatin, substance P, thyroid releasing hormone (TRH) and
vasopressin.
[0054] The biologically relevant molecule may be an antigen, but
the antigen is not limited to a protein antigen. The antigen can
also be a carbohydrate or a polynucleotide. Examples of antigenic
proteins include envelope glycoproteins from viruses, animal cell
membrane proteins, plant cell membrane proteins, bacterial membrane
proteins and parasitic membrane proteins. Examples of a
polynucleotide include a DNA or an RNA molecule. The polynucleotide
can also be in the form of a plasmid DNA. The polynucleotide can be
one that expresses a biologically active polypeptide, for example,
an enzyme or a structural or housekeeping protein. Further, the
polynucleotide need not be expressed, but may be an immunogen, a
ribozyme or an antisense molecule.
[0055] The biologically relevant molecule may also be a nutrient
such as vitamins, minerals, fatty acids, amino acids, and
saccharides. Specific examples include vitamins A, D, E, or K;
minerals such as calcium, magnesium, barium, iron or zinc;
polyunsaturated fatty acids or essential oils; amino acids; and
saccharides such as glucose and sucrose.
[0056] The biologically relevant molecule may also be a flavor
substance. Examples include flavor substances generally associated
with essential oils, such as cinnamon oil, and extracts obtained
from botanical sources such as herbs, citrus, spices and seeds.
Oils/extracts are sensitive to degradation by oxidation, and
because the processing of the natural oils and extracts often
involves multi-step operations, costs are generally considered to
be higher. The advantage of an oil/extract-cochleate would be in
the stabilization of these otherwise volatile and expensive flavor
substances. Flavor-cochleates can also be incorporated into
consumable food preparations as flavor enhancers.
[0057] The biologically relevant molecule is extracted from the
source particle, cell, tissue, or organism by known methods.
Biological activity of biologically relevant molecules need not be
maintained. However, in some instances (e.g., where a protein has
membrane fusion or ligand binding activity or a complex
conformation which is recognized by the immune system), it is
desirable to maintain the biological activity. In these instances,
an extraction buffer containing a detergent which does not destroy
the biological activity of the membrane protein is used. Suitable
detergents include ionic detergents such as cholate salts,
deoxycholate salts and the like or heterogeneous polyoxyethylene
detergents, such as Tween, BRIG or Triton.
[0058] Utilization of this method allows reconstitution of
antigens, more specifically proteins, into the liposomes with
retention of biological activities, and eventually efficient
association with the cochleates. This avoids organic solvents,
sonication, or extreme pH, temperature, or pressure all of which
may have an adverse effect upon efficient reconstitution of the
antigen in a biologically active form.
[0059] Hydrogel-isolated cochleates may contain a combination of
various biologically relevant molecules as appropriate.
[0060] The cochleate particles can be enteric. The cochleate
particles can be placed within gelatin capsules and the capsule can
be enteric coated.
[0061] In the preparations of the present invention certain
hydrophobic materials can be added to provide enhanced absorption
properties for oral delivery of biologically relevant molecules.
These materials are preferably selected from the group consisting
of long chain carboxylic acids, long chain carboxylic acid esters,
long chain carboxylic acid alcohols and mixtures thereof. The
hydrophobic materials can be added either initially to the lipid
prior to the formation of liposomes or in a later step in the form
of a fat vehicle such as an emulsion.
[0062] The skilled artisan can determine the most efficacious and
therapeutic means for effecting treatment practicing the instant
invention. Reference can also be made to any of numerous
authorities and references including, for example, "Goodman &
Gillman's, The Pharmaceutical Basis for Therapeutics", (6.sup.th
Ed., Goodman et al, eds., MacMillan Publ. Co., New York
(1980)).
[0063] The invention will now be described by examples which are
not to be considered as limiting the invention. In the examples,
unless otherwise indicated, all ratios, percents and amounts are by
weight.
EXAMPLES
Example 1
Preparation of Empty Hydrogel-Isolated Cochleates from
Dioleoylphosphatidylserine Precipitated with Calcium
[0064] Step 1: Preparation of Small Unilamellar Vesicles from
Dioleoylphosphatidylserine
[0065] A solution of dioleoyl phosphatidylserine (DOPS, Avanti
Polar Lipids, Alabaster, Ala., USA) in chloroform (10 mg/ml) was
placed in a round-bottom flask and dried to a film using a Buchi
rotavapor at 35.degree. C. The rotavapor was sterilized by flashing
nitrogen gas through a 0.2 .mu.m filter. The following steps were
carried out in a sterile hood. The dried lipid film was hydrated
with de-ionized water at the concentration of 10 mg lipid/ml. The
hydrated suspension was purged and sealed with nitrogen, then
sonicated in a cooled bath sonicator (Laboratory Supplies Corn.,
Inc.). Sonication was continued (for several seconds to several
minutes depending on lipid quantity and nature) until the
suspension became clear (suspension A) and there were no liposomes
apparently visible under a phase contrast microscope with a
1000.times. magnification. Laser light scattering (weight analysis,
Coulter N4 Plus) indicated that the mean diameter was 35.7.+-.49.7
nm.
[0066] Step 2: Preparation of Hydrogel-Isolated Cochleates
[0067] The liposome suspension obtained in step 1 was mixed with
40% w/w dextran-500,000 (Sigma) in a suspension of 2/1 v/v
Dextran/liposome. This mixture was injected with a syringe into 15%
w/w PEG-8,000 (Sigma) (PEG 8000/(suspension A)) under magnetic
stirring to result in suspension B. The rate of the stirring was
800-1,000 rpm. A CaCl.sub.2 solution (100 mM) was added to the
suspension to reach the final concentration of 1 mM.
[0068] Stirring was continued for one hour, and then a washing
buffer containing 1 mM CaCl.sub.2 and 150 mM NaCl was added to
suspension B at the volumetric ratio of 1:1. The suspension was
vortexed and centrifuged at 3000 rpm, 2-4.degree. C., for 30 min.
After the supernatant was removed, additional washing buffer was
added at the volumetric ratio of 0.5:1, followed by centrifugation
under the same conditions. A schematic of this new method of
obtaining cochleates is detailed in FIG. 1. The resultant pellet
was reconstituted with the same buffer to the desired
concentration. Laser light scattering (weight analysis, Coulter N4
Plus) indicates that the mean diameter for the cochleate is
407.2.+-.85 nm (FIG. 2B).
Example 2
Preparation of Empty Hydrogel-Isolated Cochleates from a Mixture of
Dioleoylphosphatidylserine and
1,2-Distearoyl-sn-glyceroI-3-phosphoethano- lamine-n-(poly(ethylene
glycol)-5000, DSPE-PEG) Precipitated with Calcium
[0069] Step 1: Preparation of Small Unilamellar Vesicles
[0070] A solution of dioleoylphosphatidylserine (DOPS) and
1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-n-(poly(ethylene
glycol)-5000), (DSPE-PEG, Avanti Polar Lipids, Alabaster, Ala.,
USA) in chloroform (ratio of DOPS:DSPS-PEG=100:1, w:w) was placed
in a round-bottom flask and dried to a film using a Buchi rotavapor
at 35.degree. C. The rotavapor was sterilized by flashing nitrogen
gas through a 0.2 .mu.m filter. The following steps were carried
out in a sterile hood. The dried lipid film was hydrated with
de-ionized water to a concentration of 10 mg lipid/ml. The hydrated
suspension was purged and sealed with nitrogen, then sonicated in a
cooled bath sonicator (Laboratory Supplies Corn., Inc.). Sonication
was continued (for several seconds to several minutes depending on
lipid quantity and nature) until the suspension became clear
(suspension A) and there were no liposomes apparently visible under
a phase contrast optical microscope with a 1000.times.
magnification.
[0071] Step 2: Preparation of Hydrogel-Isolated Cochleates
[0072] The liposome suspension obtained in Step 1 was mixed with
40% w/w dextran-500,000 in a suspension of 2/1 v/v
Dextran/liposome. This mixture was injected via a syringe into 15%
w/w PEG-8,000 (PEG 8000/(suspension A)) under magnetic stirring to
result in suspension B. The rate of the stirring was 800-1,000 rpm.
A CaCl.sub.2 solution (100 mM) was added to the suspension to reach
the final concentration of 1 mM.
[0073] Stirring was continued for one hour, and then a washing
buffer containing 1 mM CaCl.sub.2 and 150 mM NaCl was added to
suspension B at the volumetric ratio of 1:1. The suspension was
vortexed and centrifuged at 3000 rpm, 2-4.degree. C., for 30 min.
After the supernatant was removed, additional washing buffer was
added at the volumetric ratio of 0.5:1, followed by centrifugation
under the same conditions. (see FIG. 1). The resulting pellet was
reconstituted with the same buffer to the desired concentration.
Phase contrast optical microscopy indicates the formation of
uniform, very small, needle-like cochleates.
Example 3
Preparation of Empty Hydrogel-Isolated Cochleates from a Mixture of
Dioleoylphosphatidylserine and n-octyl-beta-D-gluco-pyranoside
Precipitated with Calcium
[0074] Step 1: Preparation of Small Unilamellar Vesicles
[0075] A solution of dioleoylphosphatidylserine (DOPS) in
chloroform was placed in a round-bottom flask and dried to a film
using a Buchi rotavapor at 35.degree. C. The rotavapor was
sterilized by flashing nitrogen gas through a 0.2 jim filter. The
following steps were carried out in a sterile hood. The dried lipid
film was hydrated with a solution of
n-octyl-beta-D-gluco-pyranoside (OCG) at 1 mg/ml at a ratio of
DOPSrOCG of 10:1 w:w. The hydrated suspension was purged and sealed
with nitrogen, then sonicated briefly in a cooled bath
sonicator.
[0076] Step 2: Preparation of Hydrogel-Isolated Cochleates
[0077] The suspension obtained in Step 1 was mixed with 40% w/w
dextran-500,000 in a suspension of 2/1 v/v Dextran/liposome. This
mixture was injected via a syringe into 15% w/w PEG-8,000 (PEG
8000/(suspension A)) under magnetic stirring to result in
suspension B. The rate of the stirring was 800-1,000 rpm. A
CaCl.sub.2 solution (100 mM) was added to the suspension to reach
the final concentration of 1 mM.
[0078] Stirring was continued for one hour, and then a washing
buffer containing 1 mM CaCl.sub.2 and 150 mM NaCl was added to
suspension B at the volumetric ratio of 1:1. The suspension was
vortexed and centrifuged at 3000 rpm, 2-4.degree. C., for 30 min.
After the supernatant was removed, additional washing buffer was
added at the volumetric ratio of 0.5:1, followed by centrifugation
under the same conditions (see FIG. 1). The resulting pellet was
reconstituted with the same buffer to the desired concentration.
Phase contrast optical microscopy indicates the formation of
uniform, very small, needle-like cochleates.
Example 4
Preparation of Amphotericin B-loaded Hydrogel-Isolated Cochleates
Precipitated with Calcium
[0079] Step 1: Preparation of Small Unilamellar AmB-Loaded,
Vesicles from Dioleoylphosphatidylserine
[0080] A mixture of dioleoyl phosphatidylserine (DOPS) in
chloroform (10 mg/ml) and AmB in methanol (0.5 mg/ml) at a molar
ratio of 10:1 was placed in a round-bottom flask and dried to a
film using a Buchi rotavapor at 40.degree. C. The rotavapor was
sterilized by flashing nitrogen gas through a 0.2 .mu.m filter. The
following steps were carried out in a sterile hood. The dried lipid
film was hydrated with de-ionized water at the concentration of 10
mg lipid/ml. The hydrated suspension was purged and sealed with
nitrogen, then sonicated in a cooled bath sonicator. Sonication was
continued (for several seconds to several minutes depending on
lipid quantity and nature) until the suspension became clear yellow
(suspension A) and there were no liposomes apparently visible under
a phase contrast microscope with a 1000.times. magnification.
[0081] Step 2: Preparation of AmB-loaded. Hydrogel-Isolated
Cochleates.
[0082] The liposome suspension obtained in Step 1 was then mixed
with 40% w/w dextran-500,000 in a suspension of 2/1 v/v
Dextran/liposome. This mixture was then injected via a syringe into
15% w/w PEG-8,000 (PEG 8000/(suspension A)) under magnetic stirring
to result in suspension B. The rate of the stirring was 800-1,000
rpm. A CaCl.sub.2 solution (100 mM) was added to the suspension to
reach the final concentration of 1 mM.
[0083] Stirring was continued for one hour, and then a washing
buffer containing 1 mM CaCl.sub.2 and 150 mM NaCl was added to
suspension B at the volumetric ratio of 1:1. The suspension was
vortexed and centrifuged at 3000 rpm, 2-4.degree. C., for 30 min.
After the supernatant was removed, additional washing buffer was
added at the volumetric ratio of 0.5:1, followed by centrifugation
under the same conditions (see FIG. 1). The resulting pellet was
reconstituted with the same buffer to the desired concentration.
Laser light scattering (weight analysis, Coulter N4 Plus) indicated
that the AmB-cochleates mean diameter was 407.3.+-.233.8 nm (FIG.
2A).
Example 5
Preparation of Doxorubicin (DXR)-Loaded Hydrogel-Isolated
Cochleates Precipitated with Calcium
[0084] Step 1: Preparation of Small Unilamellar DXR-Loaded Vesicles
from Dioleoylphosphatidylserine
[0085] A mixture of dioleoylphosphatidylserine (DOPS) in chloroform
(10 mg/ml) and (DXR) in methanol (0.5 mg/ml) at a molar ratio of
10:1 was placed in a round-bottom flask and dried to a film using a
Buchi rotavapor at room temperature. The rotavapor was sterilized
by flashing nitrogen gas through a 0.2 .mu.m filter. The following
steps were carried out in a sterile hood. The dried lipid film was
hydrated with de-ionized water at the concentration of 25 mg
lipid/ml. The hydrated suspension was purged and sealed with
nitrogen, then sonicated in a cooled bath sonicator. Sonication was
continued (for several seconds to several minutes depending on
lipid quantity and nature) until the suspension became clear pink
(suspension A) and there were no liposomes apparently visible under
phase contrast microscope with a 1000.times. magnification.
[0086] Step 2: Preparation of DXR-Loaded. Hydrogel-Isolated
Cochleates
[0087] Five milliliters of the liposome suspension obtained in step
1 was mixed with 40% w/w dextran-500,000 (Sigma) in a suspension of
2/1 v/v Dextran/liposome. This mixture was injected via a syringe
into 15% w/w PEG-8,000 (PEG 8000/(suspension A)) under magnetic
stirring to result in suspension B. The rate of the stirring was
800-1,000 rpm. A CaCl.sub.2 solution (100 mM) was added to the
suspension to reach the final concentration of 1 mM.
[0088] Stirring was continued for one hour, and then a washing
buffer containing 1 mM CaCl.sub.2 and 150 mM NaCl was added to
suspension B at the volumetric ratio of 1:1. The suspension was
vortexed and centrifuged at 6400 rpm, 2-4.degree. C., for 30 min
(see FIG. 1). The resulting pellet was reconstituted with the same
buffer to the desired concentration. Laser light scattering (weight
analysis, Coulter N4 Plus) confirmed the formation of small
DXR-cochleates.
Example 6
Preparation of Cyclosporin A (CSPA)-Loaded Hydrogel-Isolated
Cochleates Precipitated with Calcium
[0089] Step 1: Preparation of Small Unilamellar CSPA-Loaded
Vesicles from Dioleoylphosphatidylserine
[0090] A mixture of dioleoylphosphatidylserine (DOPS) in chloroform
(10 mg/ml) and CSPA in methanol (0.5 mg/ml) at a molar ratio of
10:1 was placed in a round-bottom flask and dried to a film using a
Buchi rotavapor at room temperature. The rotavapor was sterilized
by flashing nitrogen gas through a 0.2 .mu.m filter. The following
steps were carried out in a sterile hood. The dried lipid film was
hydrated with de-ionized water at the concentration of 10 mg
lipid/ml. The hydrated suspension was purged and sealed with
nitrogen, then sonicated in a cooled bath sonicator. Sonication was
continued (for several seconds to several minutes depending on
lipid quantity and nature) until the suspension became clear
(suspension A) and there were no liposomes apparently visible under
a phase contrast microscope with a 1000.times. magnification.
[0091] Step 2: Preparation of CSPA-Loaded Hydrogel-Isolated
Cochleates
[0092] The liposome suspension obtained in Step 1 was mixed with
40% w/w dextran-500,000 in a suspension of 2/1 v/v
Dextran/liposome. This mixture was injected via a syringe into 15%
w/w PEG-8,000 (Sigma) (PEG 8000/(suspension A)) under magnetic
stirring to result in suspension B. The rate of the stirring was
800-1,000 rpm. A CaCl.sub.2 solution (100 mM) was added to the
suspension to reach the final concentration of 1 mM.
[0093] Stirring was continued for one hour, and then a washing
buffer containing 1 mM CaCl.sub.2 and 150 mM NaCl was added to
suspension B at the volumetric ratio of 1:1. The suspension was
vortexed and centrifuged at 3000 rpm, 2-4.degree. C., for 30 min.
After the supernatant was removed, additional washing buffer was
added at the volumetric ratio of 0.5:1, followed by centrifugation
under the same conditions (see FIG. 1). The resulting pellet was
reconstituted with the same buffer to the desired concentration.
Laser light scattering (weight analysis, Coulter N4 Plus) confirmed
the formation of small CSPA-cochleates.
Example 7
Preparation of Nelfinavir (NVIR)-Loaded Hydrogel-Isolated
Cochleates Precipitated with Calcium
[0094] Step 1: Preparation of Small Unilamellar NVIR-Loaded
Vesicles from Dioleoylphosphatidylserine
[0095] A mixture of dioleoylphosphatidylserine (DOPS) in chloroform
(10 mg/ml) and NVIR in methanol (0. 5 mg/ml) at a molar ratio of
10:1 was placed in a round-bottom flask and dried to a film using a
Buchi rotavapor at RT. The rotavapor was sterilized by flashing
nitrogen gas through a 0.2 .mu.m filter. The following steps were
carried out in a sterile hood. The dried lipid film was hydrated
with de-ionized water at the concentration of 10 mg lipid/ml. The
hydrated suspension was purged and sealed with nitrogen, then
sonicated in a cooled bath sonicator. Sonication was continued (for
several seconds to several minutes depending on lipid quantity and
nature) until the suspension became clear (suspension A) and there
were no liposomes apparently visible under a phase contrast
microscope with a 1000.times. magnification.
[0096] Step 2: Preparation of NVIR-Loaded, Hydrogel-Isolated
Cochleates
[0097] The liposome suspension obtained in Step 1 was mixed with
40% w/w dextran-500,000 in a suspension of 2/1 v/v
Dextran/liposome. This mixture was injected via a syringe into 15%
w/w PEG-8,000 (PEG 8000/(suspension A)) under magnetic stirring to
result in suspension B. The rate of the stirring was 800-1,000 rpm.
A CaCl.sub.2 solution (100 mM) was added to the suspension to reach
the final concentration of 1 mM.
[0098] Stirring was continued for one hour, and then a washing
buffer containing 1 mM CaCl.sub.2 and 150 mM NaCl was added to
suspension B at the volumetric ratio of 1:1. The suspension was
vortexed and centrifuged at 3000 rpm, 2-4.degree. C., for 30 min.
After the supernatant was removed, additional washing buffer was
added at the volumetric ratio of 0.5:1, followed by centrifugation
under the same conditions (see FIG. 1). The resulting pellet was
reconstituted with the same buffer to the desired concentration.
Laser light scattering (weight analysis, Coulter N4 Plus) confirmed
the formation of small NVIR-cochleates.
Example 8
Preparation of Rifampin (RIF)-Loaded Hydrogel Isolated Cochleates
Precipitated with Calcium
[0099] Step 1: Preparation of Small Unilamellar RIF-Loaded Vesicles
from Dioleoylphosphatidylserine
[0100] A mixture of dioleoylphosphatidylserine (DOPS) in chloroform
(10 mg/ml) and RIF in methanol (0.5 mg/ml) at a molar ratio of 10:1
was placed in a round-bottom flask and dried to a film using a
Buchi rotavapor at RT. The rotavapor was sterilized by flashing
nitrogen gas through a 0.2 .mu.m filter. The following steps were
carried out in a sterile hood. The dried lipid film was hydrated
with de-ionized water at the concentration of 10 mg lipid/ml. The
hydrated suspension was purged and sealed with nitrogen, then
sonicated in a cooled bath sonicator. Sonication was continued (for
several seconds to several minutes depending on lipid quantity and
nature) until the suspension became clear (suspension A) and there
were no liposomes apparently visible under a phase contrast
microscope with a 1000.times. magnification.
[0101] Step 2: Preparation of RIF-Loaded. Hydrogel-Isolated
Cochleates
[0102] The liposome suspension obtained in step 1 was mixed with
40% w/w dextran-500,000 (Sigma) in a suspension of 2/1 v/v
Dextran/liposome. This mixture was injected via a syringe into 15%
w/w PEG-8,000 (Sigma) (PEG 8000/(suspension A)) under magnetic
stirring to result in suspension B. The rate of the stirring was
800-1,000 rpm. A CaCl.sub.2 solution (100 mM) was added to the
suspension to reach the final concentration of 1 mM.
[0103] Stirring was continued for one hour, and then a washing
buffer containing 1 mM CaCl.sub.2 and 150 mM NaCl was added to
suspension B at the volumetric ratio of 1:1. The suspension was
vortexed and centrifuged at 3000 rpm, 2-4.degree. C., for 30 min.
After the supernatant was removed, additional washing buffer was
added at the volumetric ratio of 0.5:1, followed by centrifugation
under the same conditions (see FIG. 1). The resulting pellet was
reconstituted with the same buffer to the desired concentration.
Laser light scattering (weight analysis, Coulter N4 Plus) confirmed
the formation of small RIF-cochleates.
Example 9
Preparation of Vitamin A Acid-Loaded Hydrogel Isolated Cochleates
Precipitated with Calcium
[0104] Step 1: Preparation of Small Unilamellar Vitamin A-Loaded
Vesicles from Dioleoylphosphatidylserine
[0105] Vitamin A acid (retinoic acid) is sensitive to air oxidation
and is inactivated by UV light. Vitamin A is protected when
embedded into lipid bilayers. The incorporation is achieved as
follows:
[0106] A mixture of dioleoylphosphatidylserine (DOPS) in chloroform
(10 mg/ml) and Vitamin A in methanol (0.5 mg/ml) at a molar ratio
of lipid/vitamin A of 10:1 was placed in a round-bottom flask and
dried to a film using a Buchi rotavapor at RT. The rotavapor was
sterilized by flashing nitrogen gas through a 0.2 .mu.m filter. The
following steps were carried out in a sterile hood. The dried lipid
film was hydrated with de-ionized water at the concentration of 10
mg lipid/ml. The hydrated suspension was purged and sealed with
nitrogen, then sonicated in a cooled bath sonicator. Sonication was
continued (for several seconds to several minutes depending on
lipid quantity and nature) until the suspension became clear
(suspension A) and there were no liposomes apparently visible under
a phase contrast microscope with a 1000.times. magnification.
[0107] Step 2: Preparation of Vitamin A-Loaded. Hydrogel-Isolated
Cochleates
[0108] The liposome suspension obtained in Step 1 was mixed with
40% w/w dextran-500,000 in a suspension of 2/1 v/v
Dextran/liposome. This mixture was injected via a syringe into 15%
w/w PEG-8,000 in a ratio of suspension A/PEG of 1/2 v/v (PEG
8000/(suspension A)) under magnetic stirring to result in
suspension B. The rate of the stirring was 800-1,000 rpm. A
solution (100 mM) was added to the suspension to reach the final
concentration of 1 mM.
[0109] Stirring was continued for one hour, and then a washing
buffer containing 1 mM CaCl.sub.2 and 150 mM NaCl was added to
suspension B at the volumetric ratio of 1:1. The suspension was
vortexed and centrifuged at 3000 rpm, 2-4.degree. C., for 30 min.
After the supernatant was removed, additional washing buffer was
added at the volumetric ratio of 0.5:1, followed by centrifugation
under the same conditions (see FIG. 1). The resulting pellet was
reconstituted with the same buffer to the desired concentration.
The amount of vitamin A encapsulated in the cochleates was
determined by UV absorption at 346 nm and it was found that more
than 90% of the initial vitamin A was associated with the
cochleates.
Example 10
Preparation of Polyunsaturated Fatty Acid (PFA)-Loaded
Hydrogel-Isolated Cochleates Precipitated with Calcium
[0110] PFA's are biologically relevant molecules involved in the
control of the level of cholesterol in blood and are the precursors
of prostaglandins. PFA's are sensitive to oxidation which limits
their incorporation into food. PFA's undergo, in the presence of
oxygen, a series of reactions called autoxidation, leading to
aldehydes and then ketones which have a fishy unpleasant odor and
flavor. Embedding PFA in rigid, rolled-up, lipid bilayers helps
prevent the autoxidation cascade. A general method of preparing
PFA-cochleates is as follows:
[0111] Step 1: Preparation of Small Unilamellar PFA-Loaded Vesicles
from Dioleoylphosphatidylserine
[0112] A mixture of dioleoylphosphatidylserine in chloroform (10
mg/ml) and PFA in methanol (0.5 mg/ml) at a molar ratio of 10:1 was
placed in a round-bottom flask and dried to a film using a rotary
evaporator at RT. The rotary evaporator was sterilized by flashing
nitrogen gas through a 0.2 .mu.m filter. The following steps were
carried out in a sterile hood. The dried lipid film was hydrated
with de-ionized water at the concentration of 10 mg lipid/ml. The
hydrated suspension was purged and sealed with nitrogen, then
sonicated in a cooled bath sonicator. Sonication was continued (for
several seconds to several minutes depending on lipid quantity and
nature) until the suspension became clear (suspension A) and there
were no liposomes apparently visible under a phase contrast
microscope with a 1000.times. magnification.
[0113] Step 2: Preparation of PFA-Loaded, Hydrogel-Isolated
Cochleates
[0114] The liposome suspension obtained in Step 1 was mixed with
40% w/w dextran-500,000 in a suspension of 2/1 v/v
Dextran/liposome. This mixture was injected via a syringe into 15%
w/w PEG-8,000 (PEG 8000/(suspension A)) under magnetic stirring to
result in suspension B. The rate of the stirring was 800-1,000 rpm.
A CaCl.sub.2 solution (100 mM) was added to the suspension to reach
the final concentration of 1 mM.
[0115] Stirring was continued for one hour, and then a washing
buffer containing 1 mM CaCl.sub.2 and 150 mM NaCl was added to
suspension B at the volumetric ratio of 1:1. The suspension was
vortexed and centrifuged at 3000 rpm, 2-4.degree. C., for 30 min.
After the supernatant was removed, additional washing buffer was
added at the volumetric ratio of 0.5:1, followed by centrifugation
under the same conditions (see FIG. 1). The resulting pellet was
reconstituted with the same buffer to the desired
concentration.
Example 11
Preparation of Vanillin-Loaded Hydrogel-Isolated Cochleates
Precipitated with Calcium
[0116] Step 1: Preparation of Small Unilamellar Vitamin A-Loaded
Vesicles from Dioleoylphosphatidylserine
[0117] A mixture of dioleoylphosphatidylserine (DOPS) in chloroform
(10 mg/ml) and Vanillin in methanol (0.5 mg/ml) at a molar ratio of
lipid/vanillin of 10:1 was placed in a round-bottom flask and dried
to a film using a Buchi rotavapor at RT. The rotavapor was
sterilized by flashing nitrogen gas through a 0.2 jam filter. The
following steps were carried out in a sterile hood. The dried lipid
film was hydrated with de-ionized water at the concentration of 10
mg lipid/ml. The hydrated suspension was purged and sealed with
nitrogen, then sonicated in a cooled bath sonicator. Sonication was
continued (for several seconds to several minutes depending on
lipid quantity and nature) until the suspension became clear
(suspension A) and there were no liposomes apparently visible under
a phase contrast microscope with a 1000.times. magnification.
[0118] Step 2: Preparation of Vanillin-Loaded. Hydrogel-Isolated
Cochleates
[0119] The liposome suspension obtained in Step 1 was mixed with
40% w/w dextran-500,000 in a suspension of 2/1 v/v
Dextran/liposome. This mixture was injected via a syringe into 15%
w/w PEG-8,000 in a ratio of suspension A/PEG of 54 v/v (PEG
8000/(suspension A)) under magnetic stirring to result in
suspension B. The rate of the stirring was 800-1,000 rpm. A
CaCl.sub.2 solution (100 mM) was added to the suspension to reach
the final concentration of 1 mM.
[0120] Stirring was continued for one hour, and then a washing
buffer containing 1 mM CaCl.sub.2 and 150 mM NaCl was added to
suspension B at the volumetric ratio of 1:1. The suspension was
vortexed and centrifuged at 3000 rpm, 2-4.degree. C., for 30 min.
After the supernatant was removed, additional washing buffer was
added at the volumetric ratio of 0.5:1, followed by centrifugation
under the same conditions (see FIG. 1). The resulting pellet was
reconstituted with the same buffer to the desired concentration.
The amount of vanillin encapsulated in the cochleates was
determined by UV absorption at 239 nm.
Example 12
Preparation of Cinnamon Oil (CinO)-Loaded Hydrogel-Isolated
Cochleates Precipitated with Calcium
[0121] Step 1: Preparation of Small Unilamellar CinO-Loaded
Vesicles from Dioleoylphosphatidylserine.
[0122] A mixture of dioleoylphosphatidyl serine (DOPS) in
chloroform (10 mg/ml) and CinO in methanol (0.5 mg/ml) at a molar
ratio of 10:1 was placed in a round-bottom flask and dried to a
film using a Buchi rotavapor at 40.degree. C. The rotavapor was
sterilized by flashing nitrogen gas through a 0.2 .mu.m filter. The
following steps were carried out in a sterile hood. The dried lipid
film was hydrated with de-ionized water at the concentration of 10
mg lipid/ml. The hydrated suspension was purged and sealed with
nitrogen, then sonicated in a cooled bath sonicator. Sonication was
continued (for several seconds to several minutes depending on
lipid quantity and nature) until the suspension became clear
(suspension A) and there were no liposomes apparently visible under
a phase contrast microscope with a 1000.times. magnification.
[0123] Step 2: Preparation of CinO-Loaded. Hydrogel-Isolated
Cochleates
[0124] The liposome suspension obtained in Step 1 was mixed with
40% w/w dextran-500,000 in a suspension of 2/1 v/v
Dextran/liposome. This mixture was injected via a syringe into 15%
w/w PEG-8,000 (PEG 8000/(suspension A)) under magnetic stirring to
result in suspension B. The rate of the stirring was 800-1,000 rpm.
A CaCl.sub.2 solution (100 mM) was added to the suspension to reach
the final concentration of 1 mM.
[0125] Stirring was continued for one hour, and then a washing
buffer containing 1 mM CaCl.sub.2 and 150 mM NaCl was added to
suspension B at the volumetric ratio of 1:1. The suspension was
vortexed and centrifuged at 3000 rpm, 2-4.degree. C., for 30 min.
After the supernatant was removed, additional washing buffer was
added at the volumetric ratio of 0.5:1, followed by centrifugation
under the same conditions (see FIG. 1). The resulting pellet was
reconstituted with the same buffer to the desired
concentration.
Example 13
Preparation of DNA-Loaded Hydrogel-Isolated Cochleates Precipitated
with Calcium
[0126] Step 1: Preparation of Small Unilamellar DNA-Loaded Vesicles
from Dioleoylphosphatidylserine
[0127] A solution of dioleoylphosphatidylserine in chloroform (10
mg/ml) was placed in a round-bottom flask and dried to a film using
a Buchi rotavapor at RT. The rotavapor was sterilized by flashing
nitrogen gas through a 0.2 filter. The following steps were carried
out in a sterile hood. The dried lipid film was hydrated with a
solution of pCMV-beta-gal-DNA in TE buffer (at 1 mg/ml) to reach a
concentration of DOPS:DNA of 10:1 and a concentration of 10 mg
lipid/ml. The hydrated suspension was purged and sealed with
nitrogen, then vortexed for several minutes.
[0128] Step 2: Preparation of DNA-Loaded. Hydrogel-Isolated
Cochleates
[0129] The DNA/liposome mixture was mixed with 40% w/w
dextran-500,000 in a suspension of 2/1 v/v Dextran/liposome. This
mixture was injected via a syringe into 15% w/w PEG-8,000 (PEG
8000/(suspension A)) under magnetic stirring to result in
suspension B. The rate of the stirring was 800-1,000 rpm. A
CaCl.sub.2 solution (100 mM) was added to the suspension to reach
the final concentration of 1 mM.
[0130] Stirring was continued for one hour, and then a washing
buffer containing 1 mM CaCl.sub.2 and 150 mM NaCl was added to
suspension B at the volumetric ratio of 10:1. The suspension was
vortexed and centrifuged at 3000 rpm, 2-4 C, for 30 min. After the
supernatant was removed, additional washing buffer was added at the
volumetric ratio of 5:1, followed by centrifugation under the same
conditions (see FIG. 1). The resulting pellet was reconstituted
with the same buffer to the desired concentration.
Example 14
Preparation of Empty Hydrogel-Isolated Cochleates Precipitated with
Zinc
[0131] Step 1: Preparation of Small Unilamellar Vesicles from
Dioleoylphosphatidylserine
[0132] A solution of dioleoylphosphatidylserine (DOPS) in
chloroform (10 mg/ml) was placed in a round-bottom flask and dried
to a film using a Buchi rotavapor at 35.degree. C. The rotavapor
was sterilized by flashing nitrogen gas through a 0.2 .mu.m filter.
The following steps were carried out in a sterile hood. The dried
lipid film was hydrated with de-ionized water at the concentration
of 10 mg lipid/ml. The hydrated suspension was purged and sealed
with nitrogen, then sonicated in a cooled bath sonicator.
Sonication was continued (for several seconds to several minutes
depending on lipid quantity and nature) until the suspension became
clear (suspension A) and there were no liposomes apparently visible
under a phase contrast microscope with a 1000.times.
magnification.
[0133] Step 2: Preparation of Hydrogel-Isolated Cochleates
[0134] The liposome suspension obtained in step 1 was mixed with
40% w/w dextran-500,000 in a suspension of 2/1 v/v
Dextran/liposome. This mixture was injected via a syringe into 15%
w/w PEG-8,000 (PEG 8000/(suspension A)) under magnetic stirring to
result in suspension B. The rate of the stirring was 800-1,000 rpm.
A ZnCl.sub.2 solution (100 mM) was added to the suspension to reach
the final concentration of 1 mM.
[0135] Stirring was continued for one hour, and then a washing
buffer containing 1 mM ZnCl2 and 150 mM NaCl was added to
suspension B at the volumetric ratio of 1:1. The suspension was
vortexed and centrifuged at 3000 rpm, 2-4.degree. C., for 30 min.
After the supernatant was removed, additional washing buffer was
added at the volumetric ratio of 0.5:1, followed by centrifugation
under the same conditions (see FIG. 1). The resulting pellet was
reconstituted with the same buffer to the desired concentration.
Laser light scattering (weight analysis, Coulter N4 Plus) confirmed
the formation of small cochleates.
Example 15
Preparation of Amphotericin B-Loaded Hydrogel-Isolated Cochleates
Precipitated with Zinc
[0136] Step 1: Preparation of Small Unilamellar AmB-Loaded Vesicles
from Dioleoylphosphatidylserine
[0137] A mixture of dioleoyl phosphatidylserine (DOPS) in
chloroform (10 mg/ml) and AmB in methanol (0.5 mg/ml) at a molar
ratio of 10:1 was placed in a round-bottom flask and dried to a
film using a Buchi rotavapor at 40.degree. C. The rotavapor was
sterilized by flashing nitrogen gas through a 0.2 .mu.m filter. The
following steps were carried out in a sterile hood. The dried lipid
film was hydrated with de-ionized water at the concentration of 10
mg lipid/ml. The hydrated suspension was purged and sealed with
nitrogen, then sonicated in a cooled bath sonicator. Sonication was
continued (for several seconds to several minutes depending on
lipid quantity and nature) until the suspension became clear yellow
(suspension A) and there were no liposomes apparently visible under
a phase contrast microscope with a 1000.times. magnification.
[0138] Step 2: Preparation of AmB-Loaded, Hydrogel-Isolated
Cochleates
[0139] The liposome suspension obtained in Step 1 was mixed with
40% w/w dextran-500,000 in a suspension of 2/1 v/v
Dextran/liposome. This mixture was injected via a syringe into 15%
w/w PEG-8,000 (PEG 8000/(suspension A)) under magnetic stirring to
result in suspension B. The rate of the stirring was 800-1,000 rpm.
A ZnCl.sub.2 solution (100 mM) was added to the suspension to reach
the final concentration of 1 mM.
[0140] Stirring was continued for one hour, and then a washing
buffer containing 1 mM ZnCl.sub.2 and 150 mM NaCl was added to the
suspension B at the volumetric ratio of 1:1. The suspension was
vortexed and centrifuged at 3000 rpm, 2-4.degree. C., for 30 min.
After the supernatant was removed, additional washing buffer was
added at the volumetric ratio of 0.5:1, followed by centrifugation
under the same conditions (see FIG. 1). The resultant pellet was
reconstituted with the same buffer to the desired concentration.
Laser light scattering (weight analysis, Coulter N4 Plus) confirmed
the formation of small AmB-Zn-cochleates.
Example 16
Microscopic Observation of Hydrogel-Isolated Cochleates
[0141] Optical microscopic study was performed stepwise alone with
the preparation procedure in order to gain some mechanistic details
of the formation of the hydrogel-isolated cochleates.
[0142] The microscopic images seen in FIGS. 3A, 3B and 4A-4F show
the morphological changes at each preparation step of AmB loaded
hydrogel-isolated cochleates precipitated with Ca2+ ions. When the
AmB/liposome-dextran mixture was dispersed into PEG solution, phase
separation resulted as shown by FIG. 3A. Partition of the liposomes
favored the dispersed dextran phase as indicated by a yellow color
of AmB. This partitioning ensures that liposomes are isolated in
each dextran particle. Addition of Calcium ions into the continued
phase (PEG) resulted in formation of precipitates in the dispersed
phase. As the final product, small needle-shape cochleates were
formed and observed under the microscope, these cochleates opened
into unilamellar vesicles upon addition of EDTA and chelation of
the calcium (FIGS. 4A and 4B). The needle-shaped morphology was
confirmed by scanning electron microscopy after freeze-fracture
(FIG. 5). Similar microscopic images were obtained for empty and
AmB-Zn-precipitated hydrogel-isolated cochleates (FIGS. 4C and 4D)
and empty Zn-precipitated hydrogel-isolated cochleate (FIGS. 4E and
4F).
Example 17
Antifungal Activity of Hydrogel-Isolated Cochleates Loaded with
Amphotericin B, In Vitro
[0143] Growth Inhibition of Candida albicans
[0144] An in vitro yeast susceptibility assay was performed
comparing the inhibitory and lethal effects of AmB-cochleates,
AmBisomes (liposomal formulation of AmB) and AmB/DMSO. Five
colonies of freshly growing Candida albicans were selected from a
YPD agar plate (from a 48 hour culture) and added to 2 ml of
2.times. YPD broth, pH 5.7. The OD590 of this stock culture was
measured and the yeast density was adjusted to OD590=0.1 and 0.1 ml
of this suspension added to each well of a 96 well plate.
[0145] AmB/cochleates, AmB/DMSO and AmBisomes were added to 96 well
plates to a final concentration of 0.078, 0.156, 0.3125, 0.625,
1.25 and 2.5 .mu.g/ml of AmB. The 96 well plates were incubated at
37.degree. C. with gentle shaking and cell density was measured on
a 96 well plate reader (Molecular Devices Spectramax 340) at 0, 2,
4, 6, 24 and 30 hours. FIG. 6 shows that AmB-cochleates have a
greater growth inhibitory effect than AmBisomes (liposomal
formulation of AmB).
[0146] Fungicidal Effect of Hydrogel-Isolated Cochleates Loaded
with Amphotericin B
[0147] Aliquots of yeast cells (50 .mu.l) were removed from the 96
well plates and serially diluted (up to 1:10000 for plating onto
agar plates) and counted using a hemocytometer. Fifty .mu.l of the
diluted yeast cells were plated onto YPD agar plates and incubated
for 24 hours at 37.degree. C. Yeast colonies were counted using a
BioRad Fluor-S Multi-Imager equipped with Quanitity One.TM.
software.
[0148] Yeast cells treated with AmBisome, AmB/DMSO and
AmB/cochleates (0.625 .mu.g AmB/ml) were examined for the ratio of
colony forming units to total cell number after 30 hours of
incubation. The results show that the AmB/cochleates had the
greatest lethal effect on the yeast cells compared to the other
antifungal agents tested. There was nearly 0% yeast viability after
treatment with the AmB-cochleates and 12% yeast viability after
treatment with AmB/DMSO. The AmBisome was not as effective,
resulting in 52% yeast viability (FIG. 7).
[0149] Macrophage Protection with AmB Cochleates
[0150] Particle scavenging cells, such as macrophage, are the first
line of defense against many microbial infections. However, many
microbes, which induce severe human clinical infections, have been
shown to infect macrophage and avoid destruction.
[0151] It is possible that in vivo, macrophage play an important
role in the uptake of cochleates, via an endocytotic mechanism.
Since macrophage also play an important role in the host defense
and clearance of fungi and parasites, it is important to study the
interaction between macrophage and cochleates.
[0152] The following examples indicate that the cochleates are
taken up by macrophage. Large doses of AmB delivered to the
macrophage were found to be non-toxic and remained within the
macrophage in a biologically active form. AmB cochleates provided
protection for the macrophage against infection by Candida albicans
when administered prior to or after fungal infection.
[0153] Prophylactic Dose Regime:
[0154] J774A. 1 macrophage (M) were subcultured into a 96-well
plate at a concentration of 1.times.10.sup.5 cells/ml in DMEM+10%
FBS. One-hundred .mu.l AmB cochleates (AmBc 0.2, 0.6, 1.25, and 2.5
.mu.g AmB/ml), Fungizone, or empty cochleates (EC at 2, 6, 12.5,
and 25 .mu.g lipid/ml) were added at the specified concentration.
Plates were incubated overnight at 37.degree. C. and 5% CO.sub.2.
24 hours later, the medium was replaced. This step was performed
twice. Candida albicans (CA) was added to the plate at a
concentration of 2.5.times.10.sup.3 cells/ml, a ratio of 1:200 with
respect to the macrophage. Plates were incubated overnight under
the conditions stated above.
[0155] Following the 24 hr incubation, the plates were removed and
observed. Medium was pipetted vigorously to remove and disrupt the
cells, 25 .mu.l of this suspension was placed onto Sabouraud
Dextrose Agar plates, and then placed in a dry incubator overnight
at 37.degree. C. Candida albicans CFU's were counted the following
day. The data in FIG. 8A suggest that AmB cochleate loaded
macrophage are very effective at killing the fungal cells.
[0156] Post-Infection Dose Regime:
[0157] J774A.1 macrophage (M) were subcultured into a 96-well plate
and then incubated overnight. Following incubation, the macrophage
were infected with CA at a ratio of 200:1, then subsequently AmBc,
Fungizone or EC was added at the specified concentrations.
Twenty-four hours later, the cell cultures were observed and CFU's
determined as described above.
[0158] When M were challenged with CA and subsequently dosed with
AmB cochleates, the CFU count was again nearly zero. These results
indicate that macrophage engulf and concentrate AmB cochleate, as
macrophage were protected against Candida albicans challenge after
AmB cochleate had been washed off (FIG. 8B).
[0159] In contrast, Fungizone, (AmB in deoxycholate), the most
popular clinical form of AmB was extremely toxic and lethal to the
macrophage in vitro. Within 5 hours of administration, there was a
large amount of cellular debris found in the petri dish, with no
signs of viable macrophage.
[0160] Microscopic observation reveals the AmB cochleates are not
toxic to the macrophage even at the highest doses studied. The AmB
cochleates are accumulated at high levels resulting in large
distended vacuoles. After washing of the macrophage and incubating
again for 24 hours, most of the vacuoles had returned to the normal
shape and size, yet a few were noticeably enlarged. A few
macrophage were even noticed to be "moving" with the enlarged
vacuoles. AmB cochleates are concentrated within the vacuoles and
it is probable that AmB is released gradually over time.
Example 18
Evaluation of Tissue Penetration of AmB after IV Administration of
Amphotericin B Hydrogel-Isolated Cochleates
[0161] Tissue penetration of amphotericin B has been evaluated
after IV administration. Groups (n=5) of C57BL/6 mice (20-23 g)
were given IV (0.625 mg/kg) AmB cochleates (0.05 ml/20 g) with a
1/2 cc U 100 insulin syringe with a 18 g 1/2 needle size. At
predetermined sacrifice times (2, 5, 10, 20 and 40 min, 1, 2, 3, 4,
6, 8, 12, 24, 36 and 48 hrs), animals were given anesthesia, their
blood was collected via cardiac puncture, and then, the animals
were euthanized and dissected. Tissues of interest were removed
(brain, lung, liver, spleen, kidneys, heart, fat, stomach, stomach
contents, intestine and intestinal contents) and weighed. For
analysis of AmB, samples were mixed with extraction solvent (10%
methanol, 35% water, 55% ethanol), homogenized, sonicated and
centrifuged. A 90 .mu.l aliquot of supernatant was transferred into
a micro vial, injected into the HPLC system in a Nova-Pak C-18
column (3.9.times.150 mm, 4 .mu.m particle size), and kept at
40.degree. C.
[0162] Amphotericin B was eluted at a flow rate of 0.5 ml/min with
29% methanol, 30% acetonitrile and 41% 2.5 mM EDTA and then
detected at 408 nm. The concentration of AmB was calculated with
the help of an external standard curve.
[0163] In FIG. 9 the tissue exposure after a single IV dose of AmB
cochleates is shown. Large penetration of key tissues like liver,
spleen and kidney can be observed.
Example 19
Oral Delivery of AmB Mediated by Hydrogel-Isolated Cochleates
Loaded with AmB
[0164] Single Dose Regime
[0165] Oral availability of the hydrogel-isolated cochleates loaded
with AmB has been examined by intragastric administration of the
formulation of Example 4 to overnight fasting, C57BL16 mice (20-23
g). {fraction (1/10)} ml of the formulation at the dose of 10 mg/kg
was administrated to 9 mice. Three mice from each group were
sacrificed at 1, 6 and 24 hrs post administration followed by
analysis of AmB level in organs and tissues.
[0166] Tissue and blood samples were processed as follows: tissues
were diluted {fraction (1/20)} or {fraction (1/10)} by addition of
extraction solvent (H.sub.2O 35%, methanol 10%, ethanol 55% w/w/w
nv/v/v) and homogenized with an Ultra-Turrex.RTM. device. A 0.5 ml
aliquot was taken, sonicated for 1 min and centrifuged at 7260 rpm
for 12 min at 4.degree. C. Supernatant was transferred to an HPLC
micro-vial and 30 .mu.l was injected on a C-18, 3.9.times.150 mm, 4
.mu.m particle sized analytical column with a flow rate of 0.5 ml,
at 40.degree. C. Concentration of AmB detected at 408 nm was
calculated with the help of an external calibration curve.
[0167] FIG. 10 shows the time profile of AmB in the tissues over a
period of time of 24 hrs. Although only three time points are
plotted, accumulation in key tissues (liver, lungs, spleen and
kidneys) can be seen.
[0168] Multiple Dose Regime
[0169] Two other groups of mice received a 10 mg/kg/day oral
multiple dose regime for ten days and one group was sacrificed 24
hrs after the last dose and the other group 20 days after the last
dose received. At the predetermined time points mice were
anesthetized, sacrificed and dissected for tissue collection.
Tissues were processed as in the single dose regime and the AmB
level was determined by HPLC. Results from 24 hr after the
10.sup.th dose are depicted in FIG. 11 and show that
hydrogel-isolated cochleates allow the delivery of AmB from the
gastrointestinal tract at therapeutic levels.
Example 20
Correlation Between Biodistribution in Healthy and Infected Mice
and the Level of Candida albicans in Tissue after Oral
Administration
[0170] FIG. 12 shows the relationship between tissue levels of
Amphotericin B (.mu.g/g tissue on left scale) and efficacy as
decrease of Candida albicans infection (CFU/g on the right scale)
after oral administration of AmB-cochleates.
[0171] After oral administration of 10 mg/kg/day for 10 consecutive
days to healthy mice, AmB presented high levels in kidneys followed
by lungs, spleen, liver and brain, which shows much lower levels
than the other tissues. It has been shown that disease state
affects pharmacokinetics of drugs at different levels. This
phenomenon can be seen clearly in the graph: AmB in tissue reaches
lower levels in Candida albicans infected mice after oral
administration of 10 mg/kg/day (same dose) for 15 days, 5 more
doses than the healthy group. It also shows a change in the
distribution pattern where the lungs are the target tissue with
lowest levels.
[0172] Oral administration of an AmB cochleate formulation at 10
mg/kg/day for 15 days provided high efficacy. The decrease in CFU/g
in kidney tissue is about 3.5 logs for the cochleate formulation.
In lungs, AmB cochleate formulations completely eradicate Candida
albicans and clear the lungs of fungal infection. It is clear that
the cochleate delivery system provides a high level of AmB in
infected animals, this correlates with the higher efficiency seen
in the cochleate formulation, indicating that AmB-cochleates are a
suitable vehicle for oral treatment of systemic Candidiasis.
[0173] In addition, orally administered AmB-cochleates were
non-toxic even at the highest dose of 50 mg/kg (no lesions were
found in kidneys, GI tract and other organs of mice given 10, 20
and 50 mg/kg of AmB-cochleates). This high dose (50 mg/kg) is
equivalent to 100 times the lowest dose (0.5 mg/kg) that showed
100% of survival in the Candida infected mouse model.
* * * * *