U.S. patent application number 10/822230 was filed with the patent office on 2005-01-20 for novel encochleation methods, cochleates and methods of use.
Invention is credited to Delmarre, David, Gould-Fogerite, Susan, Krause-Elsmore, Sara L., Lu, Ruying, Mannino, Raphael J..
Application Number | 20050013854 10/822230 |
Document ID | / |
Family ID | 34084917 |
Filed Date | 2005-01-20 |
United States Patent
Application |
20050013854 |
Kind Code |
A1 |
Mannino, Raphael J. ; et
al. |
January 20, 2005 |
Novel encochleation methods, cochleates and methods of use
Abstract
Disclosed are novel methods for making cochleates and cochleate
compositions that include introducing a cargo moiety to a liposome
in the presence of a solvent. Also disclosed are cochleates and
cochleate compositions that include an aggregation inhibitor, and
optionally, a cargo moiety. Additionally, anhydrous cochleates that
include a protonized cargo moiety, a divalent metal cation and a
negatively charge lipid are disclosed. Methods of using the
cochleate compositions of the invention, including methods of
administration, are also disclosed.
Inventors: |
Mannino, Raphael J.;
(Annandale, NJ) ; Gould-Fogerite, Susan;
(Annandale, NJ) ; Krause-Elsmore, Sara L.;
(Kearny, NJ) ; Delmarre, David; (Jersey City,
NJ) ; Lu, Ruying; (New Providence, NJ) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP.
28 STATE STREET
BOSTON
MA
02109
US
|
Family ID: |
34084917 |
Appl. No.: |
10/822230 |
Filed: |
April 9, 2004 |
Related U.S. Patent Documents
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Application
Number |
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Patent Number |
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60461483 |
Apr 9, 2003 |
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60463076 |
Apr 15, 2003 |
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60502557 |
Sep 11, 2003 |
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60537252 |
Jan 15, 2004 |
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60499247 |
Aug 28, 2003 |
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60532755 |
Dec 24, 2003 |
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60556192 |
Mar 24, 2004 |
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Current U.S.
Class: |
424/450 ;
435/458 |
Current CPC
Class: |
A61K 31/395 20130101;
A61K 31/00 20130101; G01N 33/5091 20130101; A61K 31/7048 20130101;
A61K 31/167 20130101; A61K 31/135 20130101; A61K 31/7036 20130101;
A61K 31/60 20130101; A61K 38/12 20130101; A61K 9/1274 20130101;
A61K 38/14 20130101; A61K 9/1277 20130101 |
Class at
Publication: |
424/450 ;
435/458 |
International
Class: |
A61K 031/70; A61K
009/127; C12N 015/88 |
Goverment Interests
[0002] Portions of the subject matter disclosed herein were
supported by Federal Grant No. NIAID SBIR PI R43 AI46040-01,
awarded by the National Institutes of Health. The U.S. Government
may have certain rights in the invention.
Claims
1. A method for forming a cargo moiety-cochleate comprising:
introducing a cargo moiety to a liposome in the presence of a
solvent such that the cargo moiety associates with the liposome;
and precipitating the liposome to form a cargo
moiety-cochleate.
2. The method of claim 1, comprising the step of introducing a
solution of the solvent and the cargo moiety to an aqueous
liposomal suspension.
3. The method of claim 2, wherein the solution is added by dropwise
addition, continuous flow addition, or in a bolus.
4. The method of claim 1, comprising the step of introducing the
cargo moiety to a liposomal suspension comprising the solvent.
5. The method of claim 4, wherein the cargo moiety introduced in
the form of a powder or a liquid.
6. The method of claim 1, where an antioxidant is introduced to the
liposomal suspension.
7. The method of claim 1, wherein the liposomal suspension
comprises a plurality of unilamellar and multilamellar
liposomes.
8. The method of claim 7, comprising the step of filtering or
mechanically extruding through a small aperture the liposomal
suspension such that a majority of the liposomes are
unilamellar.
9. The method of claim 1, comprising precipitating the liposome
with a multivalent cation to form a cargo moiety-cochleate.
10. The method of claim 1, wherein the solvent is a water miscible
solvent.
11. The method of claim 1, wherein the solvent is at least one
solvent selected from the group consisting of dimethylsulfoxide
(DMSO), a methylpyrrolidone, N-methylpyrrolidone (NMP),
acetonitrile, alcohol, ethanol, dimethylformamide (DMF), ethanol
(EtOH), tetrahydrofuran (THF), and combinations thereof.
12. The method of claim 1, comprising the step of removing solvent
from the liposome by dialysis and/or removing solvent from the
cochleate by washing.
13. The method of claim 1, wherein the ratio of the lipid to the
cargo moiety is between about 0.5:1 and about 20:1.
14. The method of claim 1, wherein the ratio of the lipid to the
cargo moiety is between about 20:1 and about 20,000:1.
15. The method of claim 1, wherein the cargo moiety is hydrophobic
or hydrophilic or hydrosoluble.
16. The method of claim 1, wherein the cargo moiety is
amphipathic.
17. The method of claim 1, wherein the cargo moiety is an
antifungal agent.
18. The method of claim 1, wherein the cargo moiety is at least one
member selected from the group consisting of a vitamin, a mineral,
a nutrient, a micronutrient, an amino acid, a toxin, a microbicide,
a microbistat, a co-factor, an enzyme, a polypeptide, a polypeptide
aggregate, a polynucleotide, a lipid, a carbohydrate, a nucleotide,
a starch, a pigment, a fatty acid, a saturated fatty acid, a
monounsaturated fatty acid, a polyunsaturated fatty acid, a
flavoring, an essential oil or extract, a hormone, a cytokine, a
virus, an organelle, a steroid or other multi-ring structure, a
saccharide, a metal, a metabolic poison, an antigen, an imaging
agent, a porphyrin, a tetrapyrrolic pigment, and a drug.
19. The method of claim 18, wherein the drug is at least one member
selected from the group consisting of a protein, a small peptide, a
bioactive polynucleotide, an antibiotic, an antiviral, an
anesthetic, antipsychotic, an anti-infectious, an antifungal, an
anticancer, an immunosuppressant, an immunostimulant, a steroidal
anti-inflammatory, a non-steroidal anti-inflamamatory, an
antioxidant, an antidepressant which can be synthetically or
naturally derived, a substance which supports or enhances mental
function or inhibits mental deterioration, an anticonvulsant, an
HIV protease inhibitor, a non-nucleophilic reverse transcriptase
inhibitor, a cytokine, a tranquilizer, a mucolytic agent, a
dilator, a vasoconstrictor, a decongestant, a leukotriene
inhibitor, an anti-cholinergic, an anti-histamine, a cholesterol
lipid metablolism modulating agent and a vasodilatory agent.
20. The method of claim 18, wherein the drug is at least one member
selected from the group consisting of Amphotericin B, acyclovir,
adriamycin, carbamazepine, ivermectin, melphalen, nifedipine,
indomethacin, curcumin, aspirin, ibuprofen, naproxen,
acetaminophen, rofecoxib, diclofenac, ketoprofin, meloxicam,
nabumetone, estrogens, testosterones, steroids, phenytoin,
ergotamines, cannabinoids, rapamycin, propanadid, propofol,
alphadione, echinomycin, miconazole, miconazole nitrate,
ketoconazole, itraconazole, fluconazole, griseofulvin,
clotrimazole, econazole, terconazole, butoconazole, oxiconazole,
sulconazole, saperconazole, voriconazole, ciclopirox olamine,
haloprogin, tolnaftate, naftifine, terbinafine hydrochloride,
morpholine, flucytosine, natamycin, butenafine, undecylenic acid,
Whitefield's ointment, propionic acid, caprylic acid, clioquinol,
selenium sulfide, teniposide, hexamethylmelamine, taxol, taxotere,
18-hydroxydeoxycorticost- erone, prednisolone, dexamethazone,
cortisone, hydrocortisone, piroxicam, diazepam, verapamil,
vancomycin, tobramycin, teicoplanin, bleomycin, peptidolglycan,
ristocetin, sialoglycoproteins, orienticin, avaporcin,
helevecardin, galacardin, actinoidin, gentamycin, netilmicin,
amikacin, kanamycin A, kanamycin B, neomycin, paromomycin, neamine,
streptomycin, dihydrostreptomycin, apramycin, ribostamycin,
spectinomycin, caspofungin, echinocandin B, aculeacin A,
micafungin, anidulafungin, cilofungin, pneumocandin, geldanamycin,
nystatin, rifampin, tyrphostin, a glucan synthesis inhibitor,
vitamin A acid, mesalamine, risedronate, nitrofurantoin,
dantrolene, etidronate, nicotine, amitriptyline, clomipramine,
citalopram, dothepin, doxepin, fluoxetine, imipramine, lofepramine,
mirtazapine, nortriptyline, paroxetine, reboxitine, sertraline,
trazodone, venlafaxine, dopamine, St. John's wort,
phosphatidylserine, phosphatidic acid, amastatin, antipain,
bestatin, benzamidine, chymostatin, 3,4-dichloroisocoumarin,
elastatinal, leupeptin, pepstatin, 1,10-phenanthroline,
phosphoramidon, ethosuximide, ethotoin, felbamate, fosphenytoin,
lamotrigine, levitiracetam, mephenytoin, methsuximide,
oxcarbazepine, phenobarbital, phensuximide, primidone, topirimate,
trimethadione, zonisamide, saquinavir, ritonavir, indinavir,
nelfinavir, and amprenavir.
21. The method of claim 18, wherein the polynucleotide is at least
one member selected from the group consisting of a
deoxyribonucletic acid (DNA) molecule, a ribonucleic acid (RNA)
molecule, small interfering RNA (siRNA), a ribozyme, an antisense
molecule, a morpholino and a plasmid.
22. The method of claim 21, wherein the DNA is transcribed to yield
a ribonucleic acid.
23. The method of claim 22, wherein the ribonucleic acid is
translated to yield a biologically active polypeptide.
24. The method of claim 18, 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.
25. The method of claim 18, wherein the antigen is at least one
member selected from the group consisting of a membrane protein, a
carbohydrate, envelope glycoproteins from viruses, an animal cell
protein, a plant cell protein, a bacterial protein and a parasitic
protein.
26. The method of claim 18, wherein the nutrient is at least one
member selected from the group consisting of lycopene, vitamins,
minerals, fatty acids, amino acids, fish oils, fish oil extracts,
resveratrol, biotin, choline, inositol, ginko, saccharides, a
phytochemical or zoochemical, beta-carotene, lutein, zeaxanthine,
quercetin, silibinin, perillyl alcohol, genistein, sulfurophane,
eicosapentanoic acid, gamma-3, omega-3, gamma 6 and omega-6 fatty
acids.
27. The method of claim 18, wherein the vitamin is at least-one
member selected from the group consisting of vitamins A, B, B1, B2,
B3, B12, B6, B-complex, C, D, E, and K, vitamin precursors,
caroteniods, and beta-carotene.
28. The method of claim 18, wherein the mineral is at least one
member selected from the group consisting of boron, chromium,
colloidal minerals, colloidal silver, copper, manganese, potassium,
selenium, vanadium, vanadyl sulfate, calcium, magnesium, barium,
iron and zinc.
29. The method of claim 18, wherein the saccharide or sweetener is
at least one member selected from the group consisting of
saccharine, isomalt, maltodextrine, aspartame, glucose, maltose,
dextrose, fructose and sucrose.
30. The method of claim 18, wherein the flavor substance is an
essential oil or an extract.
31. The method of claim 30, wherein the flavor substance is
selected from the group consisting of oils and extracts of
cinnamon, vanilla, almond, peppermint, spearmint, chamomile,
geranium, ginger, grapefruit, hyssop, jasmine, lavender, lemon,
lemongrass, marjoram, lime, nutmeg, orange, rosemary, sage, rose,
thyme, anise, basil, black pepper and tea or tea extracts.
32. The method of claim 30, wherein the extract is from at least
one member selected from the group consisting of an herb, a citrus,
a spice and a seed.
33. The method of claim 1, further comprising introducing an
aggregation inhibitor to the liposomes.
34. The method of claim 33, wherein the aggregation inhibitor is at
least one aggregation inhibitor selected from the group of casein,
methylcellulose, albumin, serum albumin, bovine serum albumin and
rabbit serum albumin.
35. The method of claim 1, further comprising introducing an
aggregation inhibitor to the cochleates.
36. The method of claim 35, wherein the aggregation inhibitor is at
least one aggregation inhibitor selected from the group of casein,
methylcellulose, albumin, serum albumin, bovine serum albumin and
rabbit serum albumin.
37. A composition comprising one or more cochleates made by the
method of claim 1.
38. A method of treating a subject that can benefit from the
administration of a cargo moiety, comprising the step of:
administering the composition of claim 37, such that the cargo
moiety is administered to the subject such that the subject is
benifited.
39. The method of treatment according to claim 38, wherein the
administration is by a mucosal or a systemic route.
40. The method of treatment according to claim 39, wherein the
administration is at least one mucosal route selected from the
group consisting of oral, intranasal, intraocular, intrarectal,
intravaginal, topical, buccal, and intrapulmonary.
41. The method of treatment according to claim 39, wherein the
administration is by at least one systemic route selected from the
group consisting of intravenous, intramuscular, intrathecal,
subcutaneous, transdermal, and intradermal.
42. The method of claim 38, wherein the cargo moiety is
administered to treat at least one disease or disorder selected
from the group consisting of inflammation, pain, infection, fungal
infection, bacterial infection, viral infection, parasitic
disorders, an immune disorder, genetic disorders, degenerative
disorders, cancer, proliferative disorders, obesity, depression,
hair loss, impotence, hypertension, hypotension, dementia, senile
dementia, malnutrition, acute and chronic leukemia and lymphoma,
sarcoma, adenoma, carcinomas, epithelial cancers, small cell lung
cancer, non-small cell lung cancer, prostate cancer, breast cancer,
pancreatic cancer, hepatocellular carcinoma, renal cell carcinoma,
biliary cancer, colorectal cancer, ovarian cancer, uterine cancer,
melanoma, cervical cancer, testicular cancer, esophageal cancer,
gastric cancer, mesothelioma, glioma, glioblastoma, pituitary
adenomas, schizophrenia, obsessive compulsive disorder (OCD),
bipolar disorder, Alzheimer's disease, Parkinson's disease, cell
proliferative disorders, blood coagulation disorders,
Dysfibrinogenaemia and hemophilia (A and B), autoimmune disorders,
systemic lupus erythematosis, multiple sclerosis, myasthenia
gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenia,
Grave's disease, allogenic transplant rejection, ankylosing
spondylitis, psoriasis, scleroderma, uveitis, eczema,
dermatological disorders, hyperlipidemia, hyperglycemia,
hypercholesterolemia, cystic fibrosis, muscular dystrophy,
headache, arthritis, rheumatoid arthritis, osteoarthritis,
atherosclerosis, acute gout, acute or chronic soft tissue damage,
asthma, chronic rhinosinusitis, allergic fungal sinusitis, sinus
mycetoma, non-invasive fungus induced mucositis, non-invasive
fumgus induced intestinal mucositis, chronic otitis media, chronic
colitis, inflammatory bowel diseases, ulcerative colitis, and
Crohn's disease.
43. The method of claim 38, wherein the subject can benefit from
administration of a nutrient and the cargo moiety is a
nutrient.
44. An article of manufacture comprising packaging material and a
lipid contained within the packaging material, wherein the
packaging material comprises a label or package insert indicating
the use of the lipid for forming cochleates or cochleate
compositions of the invention.
45. The article of manufacture of claim 44, further comprising
instructions or guidelines for the formation of cochleates or
cochleate compositions of the invention.
46. The article of manufacture of claim 45, wherein one of the
instructions involves mixing a cargo moiety with a solvent and
dripping it into a solution of the lipids.
47. The article of manufacture of claim 44, further comprising a
solvent.
48. The article of manufacture of claim 44, further comprising a
cargo moiety.
49. The article of manufacture of claim 44, further comprising a
multivalent cation.
50. The article of manufacture of claim 44, further comprising a
control cargo moiety.
51. The article of manufacture of claim 44, further comprising a
chelating agent.
52. The article of manufacture of claim 44, further comprising an
aggregation inhibitor.
53. A composition comprising an anhydrous cochleate.
54. The composition of claim 53, wherein the cochleate comprises a
negatively charged lipid, a protonized cargo moiety, and a divalent
metal cation.
55. The composition of claim 54, wherein the protonized cargo
moiety is water soluble.
56. The composition of claim 54, wherein the protonized cargo
moiety is a protonized weakly basic cargo moiety.
57. The composition of claim 54, wherein the protonized cargo
moiety is a multivalent cation.
58. The composition of claim 54, wherein the protonized cargo
moiety is a protonized peptide.
59. The composition of claim 58, wherein the protonized cargo
moiety is a protonized protein.
60. The composition of claim 54, wherein the protonized cargo
moiety is a protonized nucleotide.
61. The composition of claim 60, wherein the protonized nucleotide
is at least one member selected from the group consisting of a
protonized DNA, a protonized RNA, a protonized morpholino, a
protonized siRNA molecule, a protonized ribozyme, a protonized
antisense molecule, and a protonized plasmid.
62. The composition of claim 54, wherein the protonized cargo
moiety is an aminoglycoconjugate.
63. The composition of claim 54, wherein the protonized cargo
moiety is a protonized aminoglycoside or a protonized
aminoglycopeptide.
64. The composition of claim 63, wherein the protonized cargo
moiety is at least one member selected from the group consisting of
protonized vancomycin, teicoplanin, bleomycin, peptidolglycan,
ristocetin, sialoglycoproteins, orienticin, avaporcin,
helevecardin, galacardin, actinoidin, gentamycin, netilmicin,
tobramycin, amikacin, kanamycin A, kanamycin B, neomycin,
paromomycin, neamine, streptomycin, dihydrostreptomycin, apramycin,
ribostamycin, spectinomycin, and combinations thereof.
65. The composition of claim 54, wherein the protonized cargo
moiety is a protonized echinocandin.
66. The composition of claim 65, wherein the protonized cargo
moiety is at least one member selected from the group consisting of
protonized caspofungin, echinocandin B, aculeacin A, micafungin,
anidulafungin, cilofungin, pneumocandin and combinations
thereof.
67. The composition of claim 54, wherein the ratio of protonized
cargo moiety to lipid is about 2:1 by weight.
68. The composition of claim 54, wherein the ratio of protonized
cargo moiety to lipid is between about 4:1 and about 10:1 by
weight.
69. The composition of claim 53, further comprising a second
protonized cargo moiety.
70. The composition of claim 53, further comprising a cargo
moiety.
71. The composition of claim 70, wherein the cargo moiety is a
nutrient.
72. The composition of claim 71, wherein the nutrient is Vitamin
E.
73. The composition of claim 54, wherein the divalent metal cation
is barium or calcium.
74. The composition of claim 53, further comprising an aggregation
inhibitor.
75. The composition of claim 74, wherein the aggregation inhibitor
comprises at least one aggregation inhibitor selected from the
group consisting of casein, methylcellulose, albumin, serum
albumin, bovine serum albumin and rabbit serum albumin.
76. The composition of claim 54, wherein the lipid comprises a
phospholipid.
77. The composition of claim 54, wherein the lipid comprises at
least one phospholipid selected from the group consisting of a
dioleoylphosphatidylserine (DOPS) and a phosphatidylserine
(PS).
78. A pharmaceutical composition comprising the composition of
claim 53 and a pharmaceutically acceptable carrier.
79. A method for forming the anhydrous cochleate composition of
claim 53 comprising the step of contacting a negatively charged
lipid, a protonized cargo moiety and a divalent metal cation, such
that a cochleate is formed.
80. The method of claim 79, comprising the step of acidifying a
cargo moiety to form a protonized cargo moiety.
81. The method of claim 79, comprising the step of adjusting the pH
of a solution of the cochleate to maintain a protonized cargo
moiety.
82-101. (canceled)
102. The method of claim 79, further comprising introducing an
aggregation inhibitor to the cochleate.
103. The method of claim 102, wherein the aggregation inhibitor is
introduced to the cochleate before and after the cochleate is
formed.
104. The method of claim 103, wherein the aggregation inhibitor
comprises casein and methylcellulose, and the casein is introduced
before the cochleate is formed and the methylcellulose is
introduced after the cochleate is formed.
105. The method of claim 79, wherein the lipid comprises a
phospholipid.
106. A method for treating a bacterial infection in a host
comprising the step of administering the composition of claim 53 to
a host such that the bacterial infection is treated.
107. The method of claim 106, wherein the host of the bacterial
infection is a cell, a tissue or an organ.
108. A method for treating a fungal infection in a host comprising
the step of administering the composition of claim 53 to a host
such that the fimgal infection is treated.
109. The method of claim 108, wherein the host of the fungal
infection is a cell, a tissue or an organ.
110. A method of treating a subject that can benefit from the
administration of a protonized cargo moiety, comprising the step
of: administering the composition of claim 53 comprising a
protonized cargo moiety, such that the protonized cargo moiety is
administered to the subject and such that the subject is
benefited.
111-114. (canceled)
115. A cochleate composition comprising: a plurality of cochleates;
and an aggregation inhibitor.
116. The composition of claim 115, further comprising a cargo
moiety.
117. The composition of claim 115, wherein the aggregation
inhibitor coats the cochleate.
118. The composition of claim 115, wherein the aggregation
inhibitor is at least one aggregation inhibitor selected from the
group consisting of a protein, a peptide, a polysaccharide, a milk
or milk product, a polymer, a gum, a wax and a resin.
119. The composition of claim 115, wherein the aggregation
inhibitor comprises at least one aggregation inhibitor selected
from the group consisting of: casein, .kappa.-casein, milk,
albumin, serum albumin, bovine serum albumin, rabbit serum albumin,
methylcellulose, ethylcellulose, propylcellulose, hydroxycellulose,
hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl
cellulose, hydroxypropylmethyl cellulose, polyvinyl pyrrolidone,
carboxymethyl cellulose, carboxyethyl cellulose, pullulan,
polyvinyl alcohol, sodium alginate, polyethylene glycol,
polyethylene oxide, xanthan gum, tragacanth gum, guar gum, acacia
gum, arabic gum, polyacrylic acid, methylmethacrylate copolymer,
carboxyvinyl polymer, amylose, high amylose starch,
hydroxypropylated high amylose starch, dextrin, pectin, chitin,
chitosan, levan, elsinan, collagen, gelatin, zein, gluten,
carrageenan, carnauba wax, shellac, latex polymers, milk protein
isolate, soy protein isolate, and whey protein isolate.
120. The composition of claim 115, wherein the aggregation
inhibitor comprises at least one aggregation inhibitor selected
from the group consisting of casein, methylcellulose, albumin,
serum albumin, bovine serum albumin and rabbit serum albumin.
121. The composition of claim 115, wherein the plurality of
cochleates has a mean diameter of less than about 600 nm.
122. The composition of any one of claim 115, wherein the plurality
of cochleates has a mean diameter of less than about 500 nm.
123. The composition of any one of claim 115, wherein the size
distribution of the plurality of cochleates is less than about 700
nm.
124. The composition of any one of claim 115, wherein the size
distribution of the plurality of cochleates is less than about 550
nm.
125-141. (canceled)
142. The composition of claim 116, wherein the cochleate further
comprises an antifungal drug.
143. The composition of claim 142, wherein the antifungal drug is
at least one member selected from the group consisting of
Amphotericin B, miconazole nitrate, ketoconazole, itraconazole,
fluconazole, griseofulvin, clotrimazole, econazole, terconazole,
butoconazole, oxiconazole, sulconazole, saperconazole,
voriconazole, ciclopirox olamine, haloprogin, tolnaftate,
naftifine, terbinafine hydrochloride, morpholines, flucytosine,
natamycin, butenafine, undecylenic acid, Whitefield's ointment,
propionic acid, caprylic acid, clioquinol, nystatin, selenium
sulfide, caspofungin, echinocandin B, aculeacin A, micafungin,
anidulafungin, cilofungin, and pneumocandin.
144. The composition of claim 142, wherein the aggregation
inhibitor comprises at least one aggregation inhibitor selected
from the group consisting of casein, methylcellulose, albumin,
serum albumin, bovine serum albumin and rabbit serum albumin.
145. The composition of claim 142, wherein the antifungal is
Amphotericin B and the aggregation inhibitor comprises
methylcellulose.
146. The composition of claim 142, wherein the composition is in
the form of a nasal spray.
147. A cochleate composition comprising a first plurality of
cochleates with a first mean particle size and a second plurality
of cochleates with a second mean particle size, wherein the second
mean particle size is different from the first mean particle
size.
148. The composition of claim 147, further comprising at least one
cargo moiety.
149. The composition of claim 147, wherein the first plurality of
cochleates and the second plurality of cochleates comprise the same
cargo moiety. claim 150. The composition of claim 147, wherein the
first plurality of cochleates contains a different cargo moiety
than the second plurality of cochleates.
151. The composition of claim 147, further comprising a third
plurality of cochleates with a third mean particle size, wherein
the third mean particle size is different from both the first and
the second mean particle sizes.
152. The composition of claim 151, further comprising a cargo
moiety.
153. (canceled)
154. A pharmaceutical composition comprising the cochleate or
cochleate composition of claim 115 and a pharmaceutically
acceptable carrier.
155. A method of treating a subject that can benefit from the
administration of a cargo moiety, comprising the step of:
administering the cochleate composition of claim 116, such that the
cargo moiety is administered to the subject such that the subject
is benefited.
156. The method of treatment according to claim 155, wherein the
aggregation inhibitor comprises at least one aggregation inhibitor
selected from the group consisting of casein, methylcellulose,
albumin, serum albumin, bovine serum albumin and rabbit serum
albumin.
157. The method of treatment according to claim 155, wherein the
administration is by a mucosal or a systemic route.
158. The method of treatment according to claim 155, wherein the
cochleate composition is delivered in a form selected from the
group consisting of a solid, a capsule, a cachet, a pill, a tablet,
a gelcap, a crystalline substance, a lozenge, a powder, a granule,
a dragee, an electuary, a pastille, a pessary, a tampon, a
suppository, a patch, a gel, a paste, an ointment, a salve, a
cream, a foam, a lotion, a partial liquid, an elixir, a mouth wash,
a syrup, a spray, a nebulae, a mist, an atomized vapor, an
irrigant, an aerosol, a tincture, a wash, an inhalant, a solution
or a suspension in an aqueous or non-aqueous liquid, and an
oil-in-water or water-in-oil liquid emulsion.
159. The method of treatment according to claim 155, wherein the
administration is a mucosal route selected from the group
consisting of oral, intranasal, intraocular, intrarectal,
intravaginal, topical, buccal and intrapulmonary.
160. The method of treatment according to claim 155, wherein the
administration is intranasal.
161. The method of treatment according to claim 160, wherein the
cochleate composition is delivered in a form selected from the
group consisting of a spray, a nebulae, a mist, an atomized vapor,
an irrigant, an aerosol, a wash, and an inhalant.
162-163. (canceled)
164. The method of treatment according to claim 155, wherein the
aggregation inhibitor comprises methylcellulose, the cargo moiety
is Amphotericin B, and the cochleate composition is delivered in
the form of a nasal spray.
165. The method of claim 164, wherein the cochleate composition is
used to treat rhinosinusitis
166. The method of treatment according to claim 155, wherein the
administration is by a systemic route selected from the group
consisting of intravenous, intramuscular, intrathecal,
subcutaneous, transdermal and intradermal.
167-168. (canceled)
169. A method of making the cochleate composition of claim 115
comprising the step of: introducing an aggregation inhibitor to a
cochleate composition.
170. The method of claim 169, comprising the step of introducing
the aggregation inhibitor to a composition of cochleates.
171. The method of claim 169, comprising the step of introducing
the aggregation inhibitor to a composition of aggregated
cochleates.
172. The method of claim 169, comprising the steps of: introducing
the aggregation inhibitor to a composition of liposomes; and
inducing formation of the cochleate composition.
173. The method of claim 169, comprising the steps of: introducing
the aggregation inhibitor to a solution of lipids; forming a
liposomes; and inducing formation of the cochleate composition.
174. The method of claim 169, wherein the aggregation inhibitor is
added in an aggregation inhibitor to lipid ratio of between about
4:1 and about 0.1:1 by weight.
175. The method of claim 169, wherein the aggregation inhibitor is
added in an aggregation inhibitor to lipid ratio of about 1:1 by
weight.
176. The method of claim 169, wherein the aggregation inhibitor is
added in an amount suitable for modulating the resulting cochleate
to the desired size range.
177-200. (canceled)
201. A kit for the manufacture of cochleates, comprising: an
aggregation inhibitor; and an instruction for formation of
cochleates with the aggregation inhibitor.
202. The kit of claim 201, comprising at least one component
selected from the group consisting of: a lipid, a phospholipid, a
cation, a cargo moiety, and a solvent.
203. A method for forming a cargo moiety-cochleate comprising:
introducing a cargo moiety to a lipid in the presence of a solvent;
adding an aqueous solution to form a liposome; and precipitating
the liposome to form a cargo moiety-cochleate.
204. The method of claim 203, wherein the lipid is in an aqueous
solution comprising a solvent.
205. The method of claim 203, wherein the cargo moiety introduced
in the form of a powder or a liquid.
206. The method of claim 203, wherein the lipid is in the form of a
powder.
207. The method of claim 203, wherein the cargo moiety is in a
solution comprising the solvent.
208. The method of claim 207, wherein the solution is added by
dropwise addition, continuous flow addition, or in a bolus.
209. The method of claim 203, comprising precipitating the liposome
with a multivalent cation to form a cargo moiety-cochleate.
210. The method of claim 203, wherein the ratio of the lipid to the
cargo moiety is between about 0.5:1 and about 20:1.
211. The method of claim 203, wherein the ratio of the lipid to the
cargo moiety is between about 20:1 and about 20,000:1.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S.
Provisional Application No. 60/461,483 filed Apr. 9, 2003; U.S.
Provisional Application Ser. No. 60/463,076, filed Apr. 15, 2003;
U.S. Provisional Application Ser. No. 60/502,557, filed Sep. 11,
2003; U.S. Provisional Application Ser. No. 60/537,252, filed Jan.
15, 2004; U.S. Provisional Application No. 60/499,247 filed Aug.
28, 2003; U.S. Provisional Application No. 60/532,755, filed Dec.
24, 2003; and U.S. Provisional Application No. 60/______, filed
Mar. 24, 2004. The entire contents of each of the aforementioned
applications are hereby expressly incorporated herein by reference
in their entireties.
TECHNICAL FIELD
[0003] The invention generally relates to cochleate delivery
vehicles. More specifically, the invention relates to novel methods
of making and using cochleates employing a solvent to encochleate a
cargo moiety, to cochleates including one or more aggregation
inhibitors, and to cochleates including a protonized cargo moiety,
divalent cation and negatively charged lipid.
BACKGROUND
[0004] The advantages of cochleates are numerous. For example,
cochleates are more stable than aqueous structures such as
liposomes, they can be stored lyophilized which provides the
potential to be stored for long periods of time at room
temperatures, they maintain their structure even after
lyophilization (whereas liposome structures are destroyed by
lyophilization), and they are non-toxic.
[0005] Cochleate structures have been prepared first by D.
Papahadjopoulos as an intermediate in the preparation of large
unilamellar vesicles. U.S. Pat. No. 4,078,052. Methods of making
and using cochleates to deliver a variety of molecules have been
disclosed, e.g., in U.S. Pat. Nos. 5,994,318 and 6,153,217.
[0006] In these methods, prior to precipitation of the cochleates,
the material to be encochleated is introduced into liposomes by
solubilization of the lipid and material in solvent, removal of the
solvent to form a dry lipid film, then by hydration of the lipid
and components to be encochleated. Alternatively, material and
lipid may be solublized in detergent which may be removed by
dialysis or other methods. These steps are time consuming,
represent added expense in manufacturing and product costs, and can
in some instances affect the activity and/or stability of the
encochleated material.
[0007] Additionally, cochleates are highly susceptible to
aggregation, and thus particle size and particle size distribution
can be highly variable and unstable after preparation and removal
from the two-phase polymer system. The ability of drugs to be
administered via the oral route depends on several factors. The
drug typically must be sufficiently soluble in the gastrointestinal
fluids in order for the drug to be transported across biological
membranes for an active transport mechanism, or have a sufficiently
small particle size, such that it 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 P. et al, Adv. Drug Delivery
Reviews 10:141-162, 1993). Thus, it would be advantageous to be
able to control and stabilize the particle size and particle size
distribution of encochleated materials.
[0008] There also exists a need for delivery vehicles that can
safely and effectively deliver cargo moieties that are poorly
absorbed by the body (e.g., weakly basic drugs). For example,
aminoglycopeptides (e.g., vancomycin), are poorly absorbed through
the GI tract and are difficult to deliver to cells harboring
bacteria. Accordingly, in order to administer an effective amount
of drug against a bacterial infection, large amounts of drug are
ingested to not only account for poor absorption through the GI
tract, but also poor delivery to the site of infection.
Consequently, a toxic level of drug can accumulate in the GI tract
(e.g., in the kidneys) or the blood stream and can lead to serious
illness, such as erythematous or urticarial reactions, flushing,
tachycardia, and hypotension. Aminoglycosides (e.g., streptomycin
and tobramycin) are similarly problematic because of the risk of
nephrotoxicity and ototoxicity due to poor absorption, which can
lead to acute, renal, vestibular and auditory toxicity. While these
drugs can be delivered intravenously to bypass the issue of poor GI
tract absorption, uptake by the cells is still problematic. That
is, even at high concentrations, aminoglycopeptides and
aminoglycosides can not penetrate the cell membrane in order to
contact the bacteria. Additionally, echinocandins (e.g.,
caspofungin), a new, less toxic class of antifungal drugs, still
have unwanted side effects and poor oral bioavailability. As such,
they are generally administered intravenously.
[0009] The present invention addresses each of these drawbacks.
SUMMARY OF THE INVENTION
[0010] The present invention provides novel methods of forming
cochleates, which methods can be efficiently and easily scaled up.
Additionally, the present invention provides an anhydrous cochleate
including a protonized cargo moiety, e.g., an aminoglycoside, and
methods for making and administering the same. The present
invention also provides a cochleate composition which includes an
aggregation inhibitor, and methods for making an administering the
same.
[0011] In one aspect, the present invention provides a method for
forming a cargo moiety-cochleate, which includes introducing a
cargo moiety to a liposome in the presence of a solvent such that
the cargo moiety associates with the liposome and precipitating the
liposome to form a cargo moiety-cochleate. The cargo moiety can be
any cargo moiety described herein, including protonized cargo
moieties. In certain embodiments, the cargo moiety is hydrophobic,
hydrophilic, hydrosoluble or amphipathic. In other embodiments, the
cargo moiety is an antifungal agent.
[0012] In preferred embodiments, the solvent is a water miscible
solvent, more preferably the solvent is dimethylsulfoxide (DMSO), a
methylpyrrolidone, N-methylpyrrolidone (NMP), acetonitrile,
alcohol, ethanol, dimethylformamide (DMF), ethanol (EtOH),
tetrahydrofuran (THF), or combinations thereof. In certain
embodiments, the method can further involve introducing a solution
of the solvent and the cargo moiety to an aqueous liposomal
suspension. In preferred embodiments, the solution is introduced
dropwise, by continuous flow addition, or in a bolus. Additionally
or alternatively, the method can further involve introducing the
cargo moiety to a liposomal suspension comprising the solvent. In
preferred embodiments, the cargo moiety is introduced as a powder
or a liquid. In other embodiments, an antioxidant can be introduced
to the liposomal suspension.
[0013] In yet other embodiments, the liposomal suspension comprises
a plurality of unilamellar and multilamellar liposomes. In
preferred embodiments, the method additionally includes the step of
filtering or mechanically extruding through a small aperture the
liposomal suspension such that a majority of the liposomes are
unilamellar.
[0014] In still other embodiments, the method further involves
precipitating the liposome with a multivalent cation to form a
cargo moiety-cochleate. In yet other embodiments, the solvent can
be removed from the liposome by dialysis and/or washing.
[0015] In some embodiments, the ratio of the lipid to the cargo
moiety is between about 0.5:1 and about 20:1. In other embodiments,
the ratio of the lipid to the cargo moiety is between about 20:1
and about 20,000:1.
[0016] In other embodiments, the method also includes introducing
an aggregation inhibitor to the liposomes or the cochleates. The
aggregation inhibitor can be any aggregation inhibitor described
herein.
[0017] In another aspect, the instant invention provides
composition which includes one or more cochleates made by any one
of the methods described herein.
[0018] In yet another aspect, the instant invention provides a
composition including an anhydrous cochleate. In one embodiment,
the anhydrous cochleate includes a negatively charged lipid, a
protonized cargo moiety, and a divalent metal cation. In some
preferred embodiments, the protonized cargo moiety is water
soluble. In other preferred embodiments, the protonized cargo
moiety is a protonized weakly basic cargo moiety. In still other
preferred embodiments, the protonized cargo moiety is a multivalent
cation.
[0019] In particularly preferred embodiments, the protonized cargo
moiety is a protonized peptide, a protonized protein, a protonized
nucleotide, including a protonized DNA, a protonized RNA, a
protonized morpholino, a protonized siRNA molecule, a protonized
ribozyme, a protonized antisense molecule, or a protonized plasmid,
an aminoglycoconjugate, such as a protonized aminoglycoside or a
protonized aminoglycopeptide, including protonized vancomycin,
teicoplanin, bleomycin, peptidolglycan, ristocetin,
sialoglycoproteins, orienticin, avaporcin, helevecardin,
galacardin, actinoidin, gentamycin, netilmicin, tobramycin,
amikacin, kanamycin A, kanamycin B, neomycin, paromomycin, neamine,
streptomycin, dihydrostreptomycin, apramycin, ribostamycin,
spectinomycin, or a protonized echinocandin, including protonized
caspofungin, echinocandin B, aculeacin A, micafungin,
anidulafungin, cilofungin, pneumocandin and any combinations
thereof.
[0020] In some embodiments, the ratio of protonized cargo moiety to
lipid is about 2:1 by weight. In other embodiments, the ratio of
protonized cargo moiety to lipid is between about 4:1 and about
10:1 by weight. In yet other embodiments, the composition can
additionally include a second protonized cargo moiety or a cargo
moiety. The cargo moiety may be any of the cargo moieties discussed
herein. In a preferred embodiment, the cargo moiety is a nutrient.
In a particularly preferred embodiment, the nutrient is Vitamin E.
In other preferred embodiments, the divalent metal cation is barium
or calcium.
[0021] In some embodiments, the composition may include an
aggregation inhibitor. Any of the aggregation inhibitors discussed
herein may be used.
[0022] In some embodiments, the lipid may include a phospholipid.
In preferred embodiments, the phospholipid is a
dioleoylphospbatidylserine (DOPS) and/or a phosphatidylserine
(PS).
[0023] In still other embodiments, the present invention provides a
method for forming an anhydrous cochleate which includes the step
of contacting a negatively charged lipid, a protonized cargo moiety
and a divalent metal cation, such that a cochleate is formed.
[0024] In some preferred embodiments, the method includes the step
of acidifying a cargo moiety to form a protonized cargo moiety. In
other preferred embodiments, the method includes the step of
adjusting the pH of a solution of the cochleate to maintain a
protonized cargo moiety.
[0025] In yet other embodiments, the cochleate further comprises a
second protonized cargo moiety. In still other embodiments, the
divalent metal cation is barium or calcium.
[0026] In still other embodiments, an aggregation inhibitor can be
introduced to the cochleate. In preferred embodiments, the
aggregation inhibitor is introduced to the cochleate before and
after the cochleate is formed. In particularly preferred
embodiments, the aggregation inhibitor comprises casein and
methylcellulose, and the casein is introduced before the cochleate
is formed and the methylcellulose is introduced after the cochleate
is formed.
[0027] In another aspect, the present invention is directed to a
cochleate which includes an aggregation inhibitor. In certain
embodiments, the present invention is directed to a cochleate
composition including a plurality of cochleates and an aggregation
inhibitor. In some preferred embodiments, the aggregation inhibitor
coats the cochleate. The cochleate composition can further include
a cargo moiety, and the cargo moiety can be any of the cargo
moieties discussed herein, including protonized cargo moieties. In
preferred embodiments, the cochleate includes an antifungal drug.
Preferred aggregation inhibitors include proteins, peptides,
polysaccharides, milk or milk products, polymers, gums, waxes
and/or resins. Particularly preferred aggregation inhibitors
include casein, .kappa.-casein, milk, albumin, serum albumin,
bovine serum albumin, rabbit serum albumin, methylcellulose,
ethylcellulose, propylcellulose, hydroxycellulose, hydroxymethyl
cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose,
hydroxypropylmethyl cellulose, polyvinyl pyrrolidone, carboxymethyl
cellulose, carboxyethyl cellulose, pullulan, polyvinyl alcohol,
sodium alginate, polyethylene glycol, polyethylene oxide, xanthan
gum, tragacanth gum, guar gum, acacia gum, arabic gum, polyacrylic
acid, methylmethacrylate copolymer, carboxyvinyl polymer, amylose,
high amylose starch, hydroxypropylated high amylose starch,
dextrin, pectin, chitin, chitosan, levan, elsinan, collagen,
gelatin, zein, gluten, carrageenan, carnauba wax, shellac, latex
polymers, milk protein isolate, soy protein isolate, and/or whey
protein isolate. In particularly preferred embodiments, the
aggregation inhibitor is casein, methylcellulose, albumin, serum
albumin, bovine serum albumin and/or rabbit serum albumin.
[0028] In preferred embodiments, the plurality of cochleates has a
mean diameter of less than about 600 nm. In particularly preferred
embodiments, the plurality of cochleates has a mean diameter of
less than about 500 nm. In other preferred embodiments, the size
distribution of the plurality of cochleates is less than about 700
nm. In particularly preferred embodiments, the size distribution of
the plurality of cochleates is less than about 550 nm.
[0029] In other embodiments, the cochleate further includes an
antifungal drug. In preferred embodiments, the antifungal drug is
Amphotericin B, miconazole nitrate, ketoconazole, itraconazole,
fluconazole, griseofulvin, clotrimazole, econazole, terconazole,
butoconazole, oxiconazole, sulconazole, saperconazole,
voriconazole, ciclopirox olamine, haloprogin, tolnaftate,
naftifine, terbinafine hydrochloride, morpholines, flucytosine,
natamycin, butenafine, undecylenic acid, Whitefield's ointment,
propionic acid, caprylic acid, clioquinol, nystatin, selenium
sulfide, caspofungin, echinocandin B, aculeacin A, micafungin,
anidulafungin, cilofungin, and/or pneumocandin. In particularly
preferred embodiments, the antifungal drug is Amphotericin B and
the aggregation inhibitor comprises methylcellulose. In other
particularly preferred embodiments, the composition is a nasal
spray.
[0030] In yet another aspect, the present invention provides a
cochleate composition which includes a first plurality of
cochleates with a first mean particle size and a second plurality
of cochleates with a second mean particle size, wherein the second
mean particle size is different from the first mean particle size.
In preferred embodiments, the composition includes at least one
cargo moiety. In some particularly preferred embodiments, the first
plurality of cochleates and the second plurality of cochleates
include the same cargo moiety. In other particularly preferred
embodiments, the first plurality of cochleates contains a different
cargo moiety than the second plurality of cochleates.
[0031] In another embodiment, the cochleate composition can include
a third plurality of cochleates with a third mean particle size,
wherein the third mean particle size is different from both the
first and the second mean particle sizes. In a preferred
embodiment, the third plurality of cochleates includes a cargo
moiety.
[0032] In still another aspect, the present invention provides a
method of making a cochleate composition including introducing an
aggregation inhibitor to a cochleate composition. In some
embodiments, the method includes introducing the aggregation
inhibitor to a composition of cochleates. In other embodiments, the
method includes introducing the aggregation inhibitor to a
composition of aggregated cochleates. In still other embodiments,
the method includes introducing the aggregation inhibitor to a
composition of liposomes and inducing formation of the cochleate
composition. In yet other embodiments, the method includes
introducing the aggregation inhibitor to a solution of lipids,
forming a liposomes, and inducing formation of the cochleate
composition. In preferred embodiments, the aggregation inhibitor is
added in an aggregation inhibitor to lipid ratio of between about
4:1 and about 0.1:1 by weight. In particularly preferred
embodiments, the aggregation inhibitor is added in an aggregation
inhibitor to lipid ratio of about 1:1 by weight. In other
particularly preferred embodiments, the aggregation inhibitor is
added in an amount suitable for modulating the resulting cochleate
to the desired size range.
[0033] In yet other embodiments, the present invention includes
pharmaceutical compositions including any of the cochleates or
cochleate compositions discussed herein.
[0034] In still other aspects, the present invention provides
methods for treating a subject that can benefit from the
administration of a cargo moiety, including protonized cargo
moieties, by administering cochleates or cochleate compositions
such that the cargo moiety is administered to the subject and such
that the subject is treated. Cochleates or cochleate compositions
of the invention can be made using any of the methods described
herein, including introducing a cargo moiety to a liposome in the
presence of a solvent such that the cargo moiety associates with
the liposome and precipitating the liposome to form a cargo
moiety-cochleate. Any of the cargo moieties and protonized cargo
moieties described herein can be administered in the cochleates of
the present invention. In another preferred embodiment, the
cochleate compositions include an aggregation inhibitor. In
particularly preferred embodiments, the aggregation inhibitor is
casein, methylcellulose, albumin, serum albumin, bovine serum
albumin and/or rabbit serum albumin.
[0035] In a preferred embodiment, the cochleates are used for
treating a bacterial infection in a host. In other preferred
embodiments, the cochleates are used for treating a fungal
infection in a host. In particularly preferred embodiments, the
host of the bacterial infection or the fungal infection is a cell,
a tissue or an organ. In other preferred embodiments, the subject
can benefit from administration of a nutrient and the cargo moiety
is a nutrient. Administration of cochleates can be used to treat
any of the diseases or disorders described herein. In preferred
embodiments, the compositions of the invention are used to treat
rhinosinusitis.
[0036] Administration of cochleates can be by a mucosal route,
including oral, intranasal, intraocular, intrarectally,
intravaginal, topical, buccal and intrapulmonary, or by a systemic
route, including intravenous, intramuscular, intrathecal,
subcutaneous, transdermal and intradermal. In a preferred
embodiment the administration is intranasal. In another embodiment,
the cochleate composition is delivered in the form of a solid, a
capsule, a cachet, a pill, a tablet, a gelcap, a crystalline
substance, a lozenge, a powder, a granule, a dragee, an electuary,
a pastille, a pessary, a tampon, a suppository, a patch, a gel, a
paste, an ointment, a salve, a cream, a foam, a lotion, a partial
liquid, an elixir, a mouth wash, a syrup, a spray, a nebulae, a
mist, an atomized vapor, an irrigant, an aerosol, a tincture, a
wash, an inhalant, a solution or a suspension in an aqueous or
non-aqueous liquid, and an oil-in-water and/or water-in-oil liquid
emulsion. In a preferred embodiment, the cochleate composition is
delivered in a form of a spray, a nebulae, a mist, an atomized
vapor, an irrigant, an aerosol, a wash, and and/or inhalant. In a
preferred embodiment, the cochleate composition includes an
antifungal drug. The antifungal drug can include any of the
antifungal drugs discussed herein
[0037] In yet another aspect, the present invention involves an
article of manufacture which includes packaging material and a
lipid contained within the packaging material, wherein the
packaging material comprises a label or package insert indicating
the use of the lipid for forming cochleates or cochleate
compositions of the invention. In preferred embodiments, the
article of manufacture can additionally include instructions or
guidelines for the formation of cochleates or cochleate
compositions, a solvent, a phospholipid, a cargo moiety, a
protonized cargo moiety, a multivalent cation, a divalent metal
cation, a control cargo moiety, a chelating agent, and/or an
aggregation inhibitor. In particularly preferred embodiments, one
of the instructions involves mixing a cargo moiety with a solvent
and dripping it into a solution of the lipids.
DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 is two fluorescent images of Rhodamine-labeled
cochleates incubated with splenocytes. These images demonstrate a
transfer of lipid to the cell membrane, and indicate that a fusion
event occurred between the outer layer of the cochleate and the
splenocyte cell membrane.
[0039] FIG. 2 illustrates an exemplary method of the present
invention, wherein drug-liposomes are obtained by addition of a
hydrophobic drug in solvent, optionally with an antioxidant, to a
liposomal suspension.
[0040] FIG. 3 illustrates another aspect of the invention, wherein
hydrosoluble drugs are encochleated.
[0041] FIG. 4 is a series of images of an Amphotericin B
formulation having a lipid to drug ratio of 1:1 at different stages
in the formulation: liposomes, liposomes with AmB, cochleates, and
cochleates after addition of EDTA.
[0042] FIGS. 5, 6 and 7 are each a series of images, before and
after addition of EDTA, of AmB-cochleate formulations having a
lipid to drug ratio of 10:1, 2:1, and 1:1 ratio, respectively.
[0043] FIG. 8 is a graph of the size distribution of the liposomes
after vortexing and prior to filtration, after filtration with 0.45
.mu.m filter, and after introducing DMSO/Amphotericin.
[0044] FIG. 9 is a graph of the size distribution of cochleate
formulations having lipid to AmB ratios of 10:1, 2:1 and 1:1.
[0045] FIG. 10 is a graph of the survival data for C.
albicans-infected mice untreated (control), or dosed daily for 14
days with AmB/deoxycholate, or AmB-cochleates with a lipid to drug
ratio of 2:1, 3:1, 4:1, or 5:1.
[0046] FIG. 11 is a chart of the average number of C. albicans
cells/gram of tissue in the liver, kidney, and lungs of C.
albicans-infected mice untreated and dosed daily for 14 days with
AmB/deoxycholate, or AmB-cochleates with a lipid to drug ratio of
2:1, 3:1, 4:1, or 5:1.
[0047] FIG. 12 is a graph of the number of colony forming units
(CFU) for the C. albicans-infected macrophages dosed with varying
concentrations of AmB-cochleates with lipid to drug ratios of 2:1,
3:1, 4:1, and 5:1.
[0048] FIG. 13 is a series of images of the 5:1 AmB cochleates (top
two panels) and the cochleates after addition of EDTA (bottom two
panels).
[0049] FIG. 14 is a graph of the survival data for the C.
albicans-infected mice untreated or dosed daily for 14 days with
AmB/deoxycholate (AmB/D), or AmB-cochleates with a lipid to drug
ratio of 5:1 (dialysis), 2:1 (dialysis), 1:1 (dialvsis), or 2:1
(wash).
[0050] FIG. 15 is a chart of the average number of C. albicans
cells/gram of tissue in the liver, kidney, and lungs of C.
albicans-infected mice untreated (control), or dosed daily for 14
days with AmB/deoxycholate (AmB/D), or AmB-cochleates with lipid to
drug ratios of 5:1 (dialysis), 2:1 (dialysis), 1:1 (dialysis), or
2:1 (washing).
[0051] FIG. 16 is a graph depicting the number of colony forming
units (CFUs) for C. albicans-infected macrophages dosed with
varying concentrations (0.1, 0.01 and 0.001 .mu.l/mg) of
AmB-cochleate formulations having lipid to drug ratios of 5:1
(dialysis), 2:1 (dialysis), 1:1 (dialysis), and 2:1 (washing), or
AmB/deoxycholate (AmB/D).
[0052] FIG. 17 is a graph of the survival data for C.
albicans-infected mice untreated (control), or dosed daily for 14
days with AmB/deoxycholate or AmB-cochleates (CAMB) in suspension
or lyophilized and formulated with or without methylcellulose
(MC).
[0053] FIG. 18 is a chart of the average number of C. albicans
cells/gram of tissue in the liver, kidney, and lungs of C.
albicans-infected mice untreated and dosed daily for 14 days with
AmB/deoxycholate or AmB-cochleates (CAMB) in suspension or
lyophilized and formulated with or without methylcellulose
(MC).
[0054] FIG. 19 is a graph of the concentration of TY-cochleate
preparations versus free TY over time.
[0055] FIG. 20 is two graphs of the concentration of each impurity
over time for both the free TY and TY-cochleates studied in FIG.
21.
[0056] FIG. 21 is a graph comparing the cytotoxicity of
TY-cochleates in a SKOV3 cell line.
[0057] FIG. 22 is an image of ZnTPP in solution (100% DMSO), and
ZnTPP-cochleates. The ZnTPP in solution was dark purple, and the
cochleate formulation was only slightly colored (pink), indicating
that the ZnTPP was successfully incorporated into the cochleates,
which are white/yellowish.
[0058] FIG. 23 is a series of phase contrast images (left panels)
and fluorescence images (right panels), of the ZnTPP-cochleates
(top panels) and ZnTPP-liposomes (bottom panels) formed. These
images indicate that the ZnTPP was successfully associated with the
liposomes and successfully encochleated.
[0059] FIG. 24 is a series of phase contrast images (left panels)
and fluorescence images (right panels), of ZnTPP-cochleates (top
panels) and ZnTPP-liposomes (bottom panels) formed without the
presence of solvent.
[0060] FIG. 25 is a series of images of the SKOV3 cell culture with
the ZnTPP cochleates at 1 hour and 24 hours.
[0061] FIG. 26 is a series of images of the SKOV3 cell culture with
the free ZnTPP (in DMSO) at 1 hour and 24 hours.
[0062] FIG. 27 is a series of images of the SKOV3 cell culture with
the empty cochleates (including DOPE-pyrene lipid) at 1 hour and 24
hours.
[0063] FIG. 28 is a series of images of the SKOV3 cell culture with
the ZnTPP-cochleates (including DOPE-pyrene lipid) at 1 hour and 24
hours.
[0064] FIG. 29 is a series of images illustrating the use of an
exemplary kit of the invention, this is a model compound used as a
control.
[0065] FIG. 30 is a schematic diagram of cochleate aggregation in
the absence of an aggregation inhibitor.
[0066] FIG. 31 is a schematic diagram of an exemplary method of
making cochleates of the invention by adding an aggregation
inhibitor prior to cochleate formation.
[0067] FIG. 32 is a schematic diagram of an exemplary method of
making cochleates of the invention by adding an aggregation
inhibitor subsequent to cochleate formation.
[0068] FIG. 33A is two fluorescent images demonstrating the uptake
of standard cochleates by cultured cells. FIG. 33B is two
fluorescent images demonstrating the uptake of cochleates of the
invention by cultured cells.
[0069] FIGS. 34A and 34B are two graphs depicting the size
distribution of cochleates of the invention and standard cochleate
aggregates.
[0070] FIGS. 35A-D are four fluorescent images of Rhodamine-labeled
cochleates demonstrating the effect of formulating cochleates in
the presence of various aggregation inhibitors: half and half (FIG.
35A), whole milk (FIG. 35B), and fat-free milk (FIG. 35C). FIG. 35D
is an image of a control composition of cochleates that do not
include an aggregation inhibitor.
[0071] FIGS. 36A and 36B are two fluorescent images of
Rhodamine-labeled cochleates demonstrating the disaggregation of
cochleates upon addition of an aggregation inhibitor.
[0072] FIG. 37A is two images comparing acetaminophen cochleates
with and without aggregation inhibitor (casein). FIG. 37B is two
images comparing aspirin cochleates with and without an aggregation
inhibitor (casein).
[0073] FIG. 38 is a graph comparing the in vivo efficiency of
coated (small) and standard (large) aspirin cochleates at different
concentrations and with optional additive in reducing edema in rat
paws injected with carrageenan.
[0074] FIG. 39 is a graph showing the extent of ulceration and
bleeding in rats treated with free indomethacin, free aspirin,
standard aspirin cochleates, and aspirin cochleates with an
aggregation inhibitor versus an untreated control.
[0075] FIG. 40 is a graph indicating that empty cochleates are
immunologically inert in that they have no effect on the production
of NO in macrophages.
[0076] FIG. 41 is a graph comparing the efficacy of encochleated
and unencochleated aspirin and acetaminophen cochleates in
inhibiting NO formation.
[0077] FIG. 42 is an image of a cochleate prepared in the absence
of calcium.
[0078] FIG. 43 is an image of a cochleate prepared in the presence
of calcium.
[0079] FIG. 44 is an image of a cochleate prepared in the presence
of calcium and subsequently treated with a molar excess of
chelating agent (EDTA).
[0080] FIGS. 45A-D are images depicting vancomycin-lipid
formulations. FIG. 45A depicts vanco-liposomes. FIG. 45B depicts
vanco-cochleates that include an aggregation inhibitor (casein).
FIG. 45C depicts vanco-cochleates without an aggregation inhibitor.
FIG. 45D depicts the cochleates of FIG. 45C upon addition of a
chelator (EDTA).
[0081] FIG. 46 is a graph summarizing efficacy data for free
vancomycin, and vancomycin cochleates with and without casein on S.
aureus at 3 hours.
[0082] FIG. 47 is a graph summarizing efficacy data for free
vancomycin, and vancomycin cochleates with and without casein on S.
aureus at 6 hours.
[0083] FIGS. 48 are images depicting tobramycin-lipid formulations.
FIG. 48A depicts tobramycin-liposomes. FIG. 48B depicts
tobramycin-cochleates that include an aggregation inhibitor
(casein). FIG. 48C depicts tobramycin-cochleates without an
aggregation inhibitor. FIG. 48D depicts the cochleate of FIG. 48C
upon addition of a chelator (EDTA).
[0084] FIG. 49 is a graph summarizing efficacy data for free
tobramycin and tobramycin cochleates with and without casein on P.
aeruginosa alone and cultured in macrophages at 3 hours.
[0085] FIG. 50 is a graph summarizing efficacy data for free
tobramycin, and tobramycin cochleates with and without casein on P.
aeruginosa alone and cultured in macrophages at 6 hours.
[0086] FIG. 51 is a series of images of 10:1 soy PS:caspofungin
cochleates before (FIG. 51A) and after (FIG. 51B) addition of
EDTA.
[0087] FIG. 52 is a series of images of 5:1 -soy PS:caspofungin
cochleates before (FIG. 52A) and after (FIG. 52B) addition of
EDTA.
[0088] FIGS. 53A and 53B are two graphs demonstrating the size
distribution of 10:1 soy PS:caspofungin (FIG. 53A) and 5:1 soy
PS:caspofungin (FIG. 53B) cochleates.
[0089] FIG. 54 is a graph demonstrating the size distribution of
10:1 soy PS:caspofungin cochleates before and after addition of
bovine serum albumin and homogenization.
[0090] FIGS. 55A and 55B are two HPLC spectra depicting the
contents of opened caspofingin cochleates. In all formulations only
caspofungin is evident.
[0091] FIG. 56 is a graph depicting the stability of caspofungin
cochleates formulated in water, in saline and in saline with
additional calcium.
[0092] FIG. 57 is a series of images depicting caspofungin
cochleates at varying pH. Caspofungin cochleates appear most stable
at a pH range of about 4-6.
[0093] FIG. 58 is a series of images depicting Amphotericin B
cochleates with a 5:1 lipid:drug ratio containing 0.2% parabens and
varying amounts of methylcellulose.
[0094] FIG. 59 is a graph depicting particle size distribution of
Amphotericin B cochleates with a 5:1 lipid:drug ratio containing
varying amounts of methylcellulose or 0.2% parabens.
[0095] FIG. 60 is an HPLC spectrum depicting the contents of opened
Amphotericin B cochleates formed using an exemplary method of the
invention. Only Amphotericin B is present in the cochleate.
[0096] FIG. 61 is a graph depicting the number of colony forming
units (CFUs) for C. albicans-infected macrophages dosed with
varying concentrations (5, 1, 0.1, 0.01 and 0.001 .mu.l/mg) of
AmB-cochleate formulations having lipid to drug ratios of 5:1 with
and without 0.3% methylcellulose in suspension and lyophilized to
form a powder.
[0097] FIG. 62 is a graph depicting the particle size distribution
of amphotericin B cochleates formulated in a large batch (>5 L)
with an inset of the image of the amphotericin B cochleates.
[0098] FIG. 63 is a graph depicting the particle size distribution
of amphotericin B cochleates formulated in a large batch (>5 L)
with additional rabbit serum albumin and passed through a
homogenizer 2 times. The inset is an image of the amphotericin B
cochleates after homogenization.
[0099] FIG. 64 is a graph depicting the particle size distribution
of amphotericin B cochleates formulated in a large batch (>5 L)
with additional rabbit serum albumin and passed through a
homogenizer 7 times. The inset is an image of the amphotericin B
cochleates after homogenization.
DETAILED DESCRIPTION OF THE INVENTION
[0100] The present invention is based, at least in part, on the
discovery of a novel method for the formulation of cochleates and
cochleate compositions. These cochleates and cochleate compositions
provide all the advantages of conventional cochleates, but are more
efficiently made, from a cost and a time perspective. The method
generally includes the step of introducing a cargo moiety to a
liposome in the presence of a solvent such that the cargo moiety
associates with the liposome.
[0101] Without wishing to be bound to any particular theory, it is
believed that the solvent facilitates association of the cargo
moiety with the liposome, e.g., incorporation of a cargo moiety
with the liposomal bilayer. For example, in some embodiments, a
hydrophobic or amphipathic cargo moiety is dissolved in the solvent
prior to addition to an aqueous liposomal suspension. When the
solution is added to the liposomal suspension, the solvent is
miscible with the water which changes the polarity and decreases
the solubility of the cargo moiety in the solution. The cargo
moiety then associates, at least in part, with the more hydrophobic
environment of the lipid bilayer. For example, the hydrophobic
portion of an amphipathic molecule may associate itself with the
lipid bilayer, leaving the remainder of the molecule to reside
outside the liposome.
[0102] In another embodiment, the cargo moiety is hydrosoluble
and/or hydrophilic, and the solvent creates an environment such
that the cargo moiety associates with the lipid bilayer (e.g., by
ionic attraction to the lipid and/or cation and/or total or partial
migration into the bilayer). Additionally or alternatively, the
solvent also may facilitate membrane permeation of the cargo moiety
(e.g., an alcohol may be employed to enhance the permeability of
the liposomal bilayer).
[0103] The invention also provides cochleates and cochleate
compositions (e.g., pharmaceutical compositions), prepared by the
methods of the invention.
[0104] The present invention also provides novel cochleates and
cochleate compositions that include an aggregation inhibitor. These
cochleates and cochleate compositions provide all the advantages of
conventional cochleates, and additionally provide a cochleate or
cochleate composition having a stable mean particle size and
distribution. The cochleates and cochleate compositions can have,
e.g., a small particle size, e.g. a mean particle size of less that
600 nm, and/or a narrow particle size range, e.g. less than about
700 nm. Moreover, the composition is stable and does not aggregate
with the passage of time.
[0105] The invention also provides novel methods of formation of
cochleates that allow the cochleates to be produced in any desired
size range using a variety of methods. These methods do not require
such steps as providing a two-phase system and/or particle size
differentiation to obtain a cochleate composition having a mean
particle size of less than a micrometer. The methods of the
invention can be utilized with any known method of making
cochleates.
[0106] The present invention also provides a composition for the
safe and efficient delivery of cargo moieties in anhydrous
cochleates. The invention is based, at least in part, on the
discovery that protonized cargo moieties can be precipitated with a
negatively charged lipid and a divalent metal cation to form
anhydrous, stable and safe compositions for delivery of the moiety.
Moreover, protonized cargo moieties can be included in the
cochleates at surprisingly high concentrations, if desired.
[0107] The cochleates of the invention not only protect the cargo
moiety from the host (e.g., from decomposition by proteolytic
enzymes in the digestive tract), but also protect the host from the
cargo moiety (e.g., preventing damage to vital organs caused by
toxic levels of certain cargo moieties). In addition, the
cochleates of the invention allow for efficient delivery of the
cargo moiety across the digestive tract and to cells, e.g., by
fusion and/or cellular uptake. Thus, a lower dosage of cargo moiety
can be administered to generate the same beneficial results as
compared to conventional preparations, while minimizing the
incidence of toxic side effects and/or buildup of cargo moiety in
the digestive tract
[0108] The methods of the invention are superior to those employing
conventional liposomal preparations. By way of example,
liposome-encapsulated tobramycin has resulted in a low bactericidal
activity in vitro. In contrast, anhydrous tobramycin cochleate
preparations of the present invention facilitate oral delivery with
lower serum levels of drug, thereby lowering the toxicity. In
addition, the anhydrous tobramycin cochleates of the invention may
be absorbed via the gastrointestinal tract and delivered directly
to the site of infection. Moreover, once anhydrous tobramycin
cochleates are within the systemic circulation, they can be
efficiently accumulated by phagocytes resulting in intracellular
delivery of the drug to infected cells.
[0109] The invention also provides methods for forming cochleates
that include contacting a protonized cargo moiety, a negatively
charged lipid and a divalent metal cation.
[0110] The invention further provides methods of using the
cochleates of the invention, including methods of administration.
Finally, the invention provides methods of use, including
therapeutic use, and kits directed to the manufacture and use of
the cochleates and cochleate compositions of the invention.
[0111] In order to more clearly and concisely describe the subject
matter of the claims, the following definitions are intended to
provide guidance as to the meaning of specific terms used in the
following written description, examples and appended claims.
[0112] The term "cargo moiety," as used herein, refers to a moiety
to be encochleated, and generally does not refer to the lipid and
ion employed to precipitate the cochleate. Cargo moieties include
any compounds having a property of biological interest, e.g., ones
that have a role in the life processes of a living organism. A
cargo moiety 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.
[0113] As used herein, the terms "cochleate," "lipid precipitate"
and "precipitate" are used interchangeably to refer to a lipid
precipitate component that generally includes alternating cationic
and lipid bilayer sheets with little or no internal aqueous space,
typically stacked and/or rolled up, wherein the cationic sheet is
comprised of one or more multivalent cations. Additionally, the
term "encochleated" means associated with the cochleate
structure.
[0114] The term "protonized cargo moiety" refers to a protonizable
cargo moiety that has been protonized. "Protonizable" refers to the
ability to gain one or more protons. The protonizable cargo moiety
can be weakly basic, and can be protonized by acidification or
addition of a proton. Additionally or alternatively, the
protonizable cargo moiety can be neutral or weakly acidic and can
be protonized in the same manner. Thus, the protonizable cargo
moiety can be an anionic or a neutral cargo moiety, which is
rendered cationic by protonization, or the protonizable cargo
moiety can be cationic, and be rendered more cationic upon
protonization. The cargo moiety can also be provided protonized.
Optionally, the protonized state can be induced, e.g., by
acidification or other methods, as described herein. Protonization
renders the cargo moiety cationic or increases the valency of a
cargo moiety that is already cationic, e.g., from monovalent to
divalent or trivalent.
[0115] The term "protonization," as used herein, refers to the
process of increasing the valency of a cargo moiety. "Protonized"
refers to a cargo moiety that has undergone protonization. Thus,
valency can be increased, e.g., from 0 to 1, from 1 to 2, from 2 to
3, from 3 to 4, or any combination thereof, e.g., from 0 to 3. Any
method to increase valency, e.g., increasing pH, can be used to
protonize a cargo moiety.
[0116] The term "weakly basic cargo moiety" refers to cargo
moieties that, at neutral pH, have the ability to accept protons.
That is, weakly basic cargo moieties are capable of being rendered
cationic or more cationic by protonization. As such, weakly basic
cargo moieties can be anionic or neutral, and be rendered cationic
by protonization. Alternatively, weakly basic cargo moieties can be
cationic, and can be rendered more cationic, i.e., polycationic, by
protonization.
[0117] The term "weakly acidic cargo moiety" refers to cargo
moieties that, at neutral pH, have the ability to give up protons.
"Protonizable weakly acidic cargo moieties," due to their weak
acidity, have the ability to accept protons at decreased pH.
[0118] "Aminoglycoconjugates," as used herein, refer to compounds
that include an amino sugar or carbohydrate covalently linked with
another moiety. Exemplary subgroups of aminoglycoconjugates
include, but are not limited to, aminoglycoproteins,
aminoglycosides, glycosaminoglycans, and aminoglycopeptides.
[0119] "Aminoglycopeptides" are compounds that include an amino
sugar or carbohydrate covalently linked to one or more peptides,
including synthetic or chemically modified derivatives.
Aminoglycopeptides include, but are not limited to vancomycin,
teicoplanin, bleomycin, peptidolglycan, ristocetin,
sialoglycoproteins, orienticin, avaporcin, helevecardin,
galacardin, and actinoidin. Derivatives of these compounds also are
included, e.g., those provided by reductive alkylation of reactive
amines. See, Sundram et al., J. Org. Chem. 60:1102-03 (1995). U.S.
Pat. Nos. 4,639,433, 4,643,987, and 4,698,327, teach N-alkyl and
N-acyl derivatives of vancomycin. European Patent Nos. 435 503A1
and 667 353 A1, described reductive alkylations of a variety of
aminoglycopeptides including vancomycin and orienticin A.
[0120] "Aminoglycosides" are compounds that include at least two
amino sugars linked by glycoside bonds to a streptidine or a
2-deoxystreptamine or their analogs. Analogs are meant to include
aminoglycosides modified, e.g., to increase resistance to enzyme
cleavage. Such derivatives (e.g., amikacin, a semisynthetic
derivative of kanamycin), are necessary for treatment of
individuals or populations that have built up resistance to other
aminoglycosides, and all such derivatives developed presently or in
the future are aminoglycosides that fall within the scope of the
present invention. Analogs also are meant to include the
structurally related aminocyclitols (e.g., spectnomycin).
Aminoglycosides include, but are not limited to, gentamicin,
netilmicin, tobramicin, amikacin, kanamycin A, kanamycin B,
neomycin, paromomycin, neamine, streptomycin, dihydrostreptomycin,
apramycin, ribostamycin, and spectinomycin. Aminoglycosides can
optionally be grouped as streptomycins (e.g., streptomycin and
dihydrostreptomycin), kanamycins (e.g., kanamycin, amikacin,
tobramycin), gentamicins (e.g., gentamicin and netilmicin), and
neomycins. Apramycin and specinomycin are aminoglycosides typically
used by veterinarians to treat non-human animals.
[0121] "Echinocandins" are large lipopeptide molecules, which are
active as antifungal agents. Echinocandins act by inhibiting glucan
synthesis via inhibition of 1,3-beta-D-glucan synthase. This
interferes with the synthesis of chitin, an important cell-wall
component, and results in fungal cell lysis. These drugs have
fungicidal activity against a vast species of fungi, e.g., Candida
and Aspergillus. Examples of echinocandins include, but are not
limited to, caspofungin, echinocandin B, aculeacin A, micafungin,
anidulafungin, cilofungin, and pneumocandin. Any antifungal
molecule with an echinocandin core structure is meant to be
included.
[0122] As used herein, the term "peptide" refers to a compound
containing two or more amino acids, such as a protein. The term
"nucleotide" refers to one or more purine or pyrimidine molecules
attached to a backbone. The backbone can be a sugar-phosphate
backbone, or a modified backbone, e.g., a morpholino backbone. The
terms "protonized peptide" and "protonized nucleotide" are meant to
include any peptide or nucleotide that can be rendered divalent or
polyvalent.
[0123] "Carbohydrates" include any carbohydrate including those
that include one or more monosaccharides, disaccharides,
oligosaccharides or polysaccharides, and their derivatives.
[0124] Cochleates
[0125] Cochleate delivery vehicles represent a unique technology
platform suitable for the oral and systemic administration of a
wide variety of molecules with important therapeutic biological
activities, including drugs, genes, and vaccine antigens. Miller et
al., J Exp Med 176:1739-1744 (1992); Gould-Fogerite and Mannino, J.
Liposome Res 6(2):357-79 (1996); Mannino and Gould-Fogerite, New
Generation Vaccines, ch. 18, pp 229-39 (Marcel Dekker, New York,
N.Y., Myron M. Levine, Ed. 2nd ed. 1997); Gould-Fogerite et al.,
Advanced Drug Delivery Reviews 32(3):273-387 (1998); Gould-Fogerite
and Mannino, Methods in Molecular Medicine, Vaccine Adjuvants:
Preparation Methods and Research Protocols pp 179-196 (Humana
Press, Totowa 1999); Gould-Fogerite et al., J Liposome Research
10(4): 339-358 (2000); U.S. Pat. No. 5,834,015; Gould-Fogerite et
al., Gene 84:429-438 (1989); Zarif et al., J. Liposome Research
10(4), 523-538 (2000); Zarif et al., Antimicrobials Agents and
Chemotherapy 44(6):1463-1469 (2000); Santangelo et al.,
Antimicrobials Agents and Chemotherapy 44(9):2356-2360 (2000);
Parker et al., Methods in Enzymology: Antisense Technology, Part B,
314: 411-29 (M. Ian Phillips, Ed., 1999).
[0126] Cochleate formulation technology is particularly applicable
to macromolecules as well as small molecule drugs that are
hydrophobic, positively charged, negatively charged and/or possess
poor oral bioavailability. Proof-of-principle studies for cochleate
mediated oral delivery of macromolecules as well as small molecule
drugs is being carried out in appropriate animal models with well
established, clinically important drugs which currently can only be
effectively delivered by injection (e.g., antifungal agents such as
amphotericin B).
[0127] The cochleate structure provides protection from degradation
for associated "encochleated" moieties. Divalent cation
concentrations in vivo in serum and mucosal secretions are such
that the cochleate structure is maintained. Hence, the majority of
cochleate-associated molecules are present in the inner layers of a
primarily solid, non-aqueous, stable, impermeable structure. Since
the cochleate structure includes a series of solid layers,
components within the interior of the cochleate structure remain
substantially intact, even though the outer layers of the cochleate
may be exposed to harsh environmental conditions or enzymes. In an
exemplary method of cochleate formation, liposomes, which include
negatively charged lipids associated with a cargo moiety, are
exposed to a cation, e.g., calcium, that interacts with the
liposomes to displace water and condense the lipid. The
cation/lipid sheets "roll-up" and/or stack against each other to
minimize contact with water, which provides an environment for the
encochleated molecule that is substantially free of water. This
structure provides protection to encochleated molecules from
digestion in the stomach.
[0128] The cochleate interior is primarily free of water and
resistant to penetration by oxygen. Oxygen and water are primarily
responsible for the decomposition and degradation of cargo moieties
(e.g., drugs and nutrients), which leads to reduced shelf-life.
Accordingly, encochleation also imparts extensive shelf-life
stability. For example, for DNA vaccine-cochleates, the
encochleation efficiency, the percentage of supercoiled versus
relaxed plasmid, and immunogenicity are equivalent to fresh
preparations for more than one year.
[0129] With respect to storage, cochleates can be stored in
cation-containing buffer, or lyophilized or otherwise converted to
a powder, and stored at room temperature. If desired, the
cochleates also can be reconstituted with liquid prior to
administration. Cochleate preparations have been shown to be stable
for more than two years at 4.degree. C. in a cation-containing
buffer, and at least one year as a lyophilized powder at room
temperature.
[0130] As used herein, the term "multivalent cation" refers to a
divalent cation or higher valency cation, or any compound that has
at least two positive charges, including mineral cations such as
calcium, barium, zinc, iron and magnesium and other elements
capable of forming ions or other structures having multiple
positive charges capable of chelating and bridging negatively
charged lipids. Additionally or alternatively, the multivalent
cation can include other multivalent cationic compounds, e.g.,
cationic or protonized cargo moieties. The term "divalent metal
cation," as used herein, refers to a metal having two positive
charges.
[0131] The lipid employed in the present invention preferably
includes one or more negatively charged lipids. As used herein, the
term "negatively charged lipid" includes lipids having a head group
bearing a formal negative charge in aqueous solution at an acidic,
basic or physiological pH, and also includes lipids having a
zwitterionic head group.
[0132] The cochleates of the invention also can include
non-negatively charged lipids (e.g., positive and/or neutral
lipids). Preferably, a majority of the lipid is negatively charged.
In one embodiment, the lipid is a mixture of lipids, comprising at
least 75% negatively charged lipid. In another embodiment, the
lipid includes at least 85% negatively charged lipid. In other
embodiments, the lipid includes at least 90%, 95% or even 99%
negatively charged lipid. All ranges and values between 60% and
100% negatively charged lipid are meant to be encompassed
herein.
[0133] The negatively charged lipid can include soy-based lipids.
Preferably, the lipid includes phospholipids, such as soy-based
phospholipids. The negatively charged lipid can include
phosphotidyl serine (PS), dioleoylphosphatidylserine (DOPS),
phosphatidic acid (PA), phosphatidylinositol (PI), and/or
phosphatidyl glycerol (PG) and or a mixture of one or more of these
lipids with other lipids. Additionally or alternatively, the lipid
can include phosphatidylcholine (PC), phosphatidylethanolamine
(PE), diphosphotidylglycerol (DPG), dioleoyl phosphatidic acid
(DOPA), distearoyl phosphatidylserine (DSPS), dimyristoyl
phosphatidylserine (DMPS), dipalmitoyl phosphatidylgycerol (DPPG)
and the like.
[0134] The lipids can be natural or synthetic. For example, the
lipid can include esterified fatty acid acyl chains, or organic
chains attached by non-ester linkages such as ether linkages (as
described in U.S. Pat. No. 5,956,159), disulfide linkages, and
their analogs.
[0135] In one embodiment the lipid chains are from about 6 to about
26 carbon atoms, and the lipid chains can be saturated or
unsaturated. Fatty acyl lipid chains useful in the present
invention include, but are not limited to, n-tetradecanoic,
n-hexadecanoic acid, n-octadecanoic acid, n-eicosanoic acid,
n-docosanoic acid, n-tetracosanoic acid, n-hexacosanoic acid,
cis-9-hexadecenoic acid, cis-9-octadecenoic acid,
cis,cis-9,12-octadecedienoic acid, all-cis-9,12,15-octadecetrienoic
acid, all-cis-5,8,11,14-eicosatetraenoic acid,
all-cis-4,7,10,13,16,19-docosahe- xaenoic acid, 2,4,6,8-tetramethyl
decanoic acid, and lactobacillic acid, and the like.
[0136] The cochleates of the invention can further include
additional compounds known to be used in lipid preparations, e.g.,
cholesterol and/or pegylated lipid. Pegylated lipid includes lipids
covalently linked to polymers of polyethylene glycol (PEG). PEG's
are conventionally classified by their molecular weight, thus PEG
6,000 MW, e.g., has a molecular weight of about 6000. Adding
pegylated lipid generally will result in an increase of the amount
of compound (e.g., peptide, nucleotide, and nutrient) that can be
incorporated into the cochleate. An exemplary pegylated lipid is
dipalmitoylphosphatidylehtanolamine (DPPE) bearing PEG 5,000
MW.
[0137] Methods of Forming Cochleates
[0138] In one aspect, the invention provides methods for forming
cochleates. Any known method can be used to form cochleates,
including but not limited to those described in U.S. Pat. Nos.
5,994,318 and 6,153,217, the entire disclosures of which are
incorporated herein by this reference.
[0139] In one embodiment, the method generally includes introducing
a cargo moiety to a lipid in the presence of a solvent, adding an
aqueous solution to form liposomes, and precipitating to form a
cochleate.
[0140] In a preferred embodiment, the method generally includes
introducing a cargo moiety to a liposome in the presence of a
solvent such that the cargo moiety associates with the liposome,
and precipitating the liposome to form a cargo
moiety-cochleate.
[0141] The step of introducing a cargo moiety to a liposome in the
presence of a solvent can be achieved in a variety of ways, all of
which are encompassed within the scope of the present invention. In
one embodiment, the cargo moiety is introduced by introducing a
solution of the solvent and the cargo moiety to the liposome.
Preferably, the liposome is in a liposomal suspension, preferably,
an aqueous liposomal suspension. In a preferred embodiment, the
solution is introduced to the liposome by dropwise addition of the
solution. In other embodiments, the solution can be added by
continuous flow or as a bolus. In addition the solution may be
introduced to dried lipid, with water added before, after or with
the solution.
[0142] In another embodiment, the cargo moiety is introduced to the
liposome prior to or after the solvent. For example, the cargo
moiety may be introduced to a liposomal suspension that includes
the solvent. The mixture can then be agitated, mixed, vortexed or
the like to facilitate association of the cargo moiety with the
liposome. The cargo moiety introduced may be in a powder or a
liquid form.
[0143] An antioxidant (e.g., Vitamin E) may also be employed in the
methods of the present invention. It can be introduced with the
cargo moiety or with the liposome. Preferably, it is incorporated
into the liposomal suspension or a solution of the cargo moiety and
solvent.
[0144] The liposome may be prepared by any known method of
preparing liposomes. Thus, the liposomes may be prepared for
example by solvent injection, lipid hydration, reverse evaporation,
freeze drying by repeated freezing and thawing. The liposomes may
be multilamellar (MLV) or unilamellar (ULV), including small
unilamellar vesicles (SUV). The concentration of lipid in these
liposomal solutions can be from about 0.1 mg/ml to 500 mg/ml.
Preferably, the concentration of lipid is from about 0.5 mg/ml to
about 50 mg/ml, more preferably from about 1 mg/ml to about 25
mg/ml.
[0145] The liposomes may be large unilamellar vesicles (LUV),
stable plurilamellar vesicles (SPLV) or oligolamellar vesicles
(OLV) prepared, e.g., by detergent removal using dialysis, column
chromatography, bio beads SM-2, by reverse phase evaporation (REV),
or by formation of intermediate size unilamellar vesicles by high
pressure extrusion. Methods in Biochemical Analysis, 33:337 (1988).
Liposomes made by all these and other methods known in the art can
be used in practicing this invention.
[0146] In a preferred embodiment at least majority of the liposomes
are unilamellar. The method can further include the step of
filtering a liposomal suspension and/or mechanically extruding the
suspension through a small aperture that includes both MLV and ULV
liposomes, such that a majority of the liposomes are ULV. In
preferred embodiments, at least 70%, 80%, 90% or 95% of the
liposomes are ULV.
[0147] The method is not limited by the method of forming
cochleates. Any known method can be used to form cochleates from
the liposomes of the invention (i.e., the liposomes associated with
the cargo moiety). In a preferred embodiment, the cochleate is
formed by precipitation. The liposome can be precipitated with a
multivalent cation to form a-cargo moiety-cochleate. The
multivalent cation can consist entirely or consist essentially of a
cationic metal, including, but not limited to calcium, magnesium,
barium, zinc, and/or iron. Additionally or alternatively, the
multivalent cation can include other multivalent cationic
compounds. As used herein, the term "multivalent" refers to ions
having a valency of at least 2, e.g., divalent, trivalent, etc.
[0148] Any suitable solvent can be employed in connection with the
present invention. Solvents suitable for a given application can be
readily identified by a person of skill in the art. Preferably, the
solvent is an FDA acceptable solvent.
[0149] The solvent can be an organic solvent or an inorganic
solvent. In one embodiment, the solvent is a water miscible
solvent. Suitable solvents include but are not limited to
dimethylsulfoxide (DMSO), a methylpyrrolidone, N-methylpyrrolidone
(NMP), acetonitrile, alcohols, e.g., ethanol (EtOH),
dimethylformamide (DMF), tetrahydrofuran (THF), and combinations
thereof. In general, the cargo moiety concentration within the
solvent is between about 0.01 mg/ml and 200 mg/ml. Preferably, the
cargo moiety concentration is between about 0.05 mg/ml and about
100 mg/ml, more preferably between about 0.1 mg/ml and 20
mg/ml.
[0150] The solvent can optionally be removed, e.g., before the
formation of liposomes, at the liposome stage and/or after the
cochleates are formed. Any known solvent removal method can be
employed. For example, solvent may be removed from the liposomal
suspension by tangential flow and/or filtration and/or dialysis, or
from the cochleates by washing, filtration, centrifugation, and/or
dialysis. The cochleates can be washed, e.g., with buffer or water,
optimally with calcium or another cation.
[0151] Utilizing the methods of the invention a wide range of lipid
to cargo moiety ratios can be achieved. Different ratios can have
varying biological activity. The amount of cargo moiety
incorporated into the cochleates can be varied as desired. The
optimal lipid:cargo moiety ratio for a desired purpose can readily
be determined without undue experimentation. The cochleates can be
administered to the targeted host to ascertain the nature and tenor
of the biologic response to the administered cochleates. It is
evident that the optimized ratio for any one use may range from a
high ratio to a low ratio to obtain maximal amount of cargo moiety
in the cochleates. All ratios disclosed herein are w/w, unless
otherwise indicated. In one embodiment, the ratio of lipid to cargo
moiety is between about 10,000:1 and 1000:1. Ratios in this range
may be suitable when it is desired to administer small amounts of
the moiety, (e.g., in the case of administration of radioactive
agents or highly active, rare or expensive molecules). In another
embodiment, the ratio is between about 8,000:1 and 4,000:1, e.g.,
about 6,000:1. Such a ratio may be suitable, e.g., in delivering
porphyrins. In yet another embodiment, the ratio is between about
5,000:1 and 50:1. In yet another embodiment, the ratio of the lipid
to the cargo moiety is between about 20:1 and about 0.5:1. In
another embodiment, the ratio of the lipid to the cargo moiety is
between about 1:1 and about 10:1. Such a ratio may be suitable,
e.g., for delivery of an antifungal agent. In yet another
embodiment, the ratio of lipid to the cargo moiety is about 2:1,
about 3:1, or between about 1.5:1 and 3.5:1. All individual values
and ranges between about 0.25:1 and about 40,000:1 are within the
scope of the invention. Further values also are within the scope of
the invention. The cochleate formulations also can be prepared both
with and without targeting molecules (e.g., fusogenic molecules,
such as Sendai virus envelope polypeptides), to target specific
cells and/or tissues.
[0152] In some embodiments, the cargo moiety is hydrophobic. In
others, it is amphipathic. In still others, it is hydrophilic
and/or hydrosoluble. Exemplary cargo moieties are disclosed
below.
[0153] In preferred embodiments, hydrophobic cargo moiety
cochleates (e.g., beta-carotene cochleates) are formed by
introducing a hydrophobic cargo moiety to a liposome in the
presence of a solvent such that the hydrophobic cargo moiety
associates with the liposome, and precipitating the liposome to
form a hydrophobic cargo moiety-cochleate. In particularly
preferred embodiments, the loading of hydrophobic cargo moiety in
the cochleate is considerably higher than the loading observed when
cochleates are formed using conventional methods, ie., those
described in U.S. Pat. No. 5,994,318.
[0154] Formation of the cochleates of the invention in the above
methods involves crystallization of multivalent cation with
negatively charged lipids. It is evident, therefore, that all of
the parameters that govern crystallization, e.g., temperature,
lipid concentration, multivalent cation concentrations, rate of
cation addition, pH and rate of mixing, can be utilized to regulate
cochleate formation. In certain embodiments, ionic conditions can
be created or adjusted to affect the efficiency of the association
and/or the encochleation of the cargo moiety. For example,
increasing the salt concentration in a liposomal suspension can
render the environment less hospitable to a hydrophobic or
amphipathic cargo moiety, thereby increasing liposome and cochleate
loading efficiency. Ionic conditions can also affect the ultimate
structure of the cochleate generated. High loads of a cargo moiety
can also affect the highly ordered structure observed in cochleates
formed, e.g., exclusively from calcium and PS. Additionally or
alternatively, pH conditions can be created or adjusted to affect
the loading and structure of the resulting cochleates. Such
variations can readily be manipulated by the skilled practitioner
using no more than the instant specification and routine
experimentation. In addition, because a cochleate is highly
thermodynamically stable, once a cochleate formulation method is
developed for a given product, the end product can be made
predictably and reliably.
[0155] Accordingly, in another aspect, the present invention
provides methods of making anhydrous cochleates with protonized
cargo moieties. The method generally includes the step of
contacting a negatively charged lipid, a protonized cargo moiety
and a divalent metal cation. Without wishing to be bound by any
particular theory, it is believed that the negatively charged lipid
forms an ionic interaction with the cationic protonized cargo
moiety. The divalent metal cation then precipitates the lipid and
protonized cargo moiety to form an anhydrous cochleate.
[0156] In a preferred embodiment, the protonized cargo moiety is
introduced to the negatively charged lipid. A divalent metal cation
is then added to the lipid-protonized cargo moiety mixture in order
to form anhydrous cochleates. The divalent metal cation can be,
e.g., calcium, barium, etc. In particularly preferred embodiments,
the divalent metal cation is capable of inducing the formation of
an anhydrous cochleate. In other particularly preferred
embodiments, the divalent metal cation is calcium.
[0157] In one embodiment, liposomes are formed that include
negatively charged lipid using known methods, and the protonized
cargo moiety is added prior to, during or after formation of the
liposomal suspension. Alternatively, the protonized cargo moiety is
introduced to a preformed liposomal suspension, e.g., as a solid or
in an aqueous or organic solution.
[0158] The method can further include the step of protonizing the
cargo moiety prior to or during the formation of the cochleate,
e.g., by acidification. Any known method of acidification can be
employed. For example, a weakly basic cargo moiety can be
protonized with acidic aqueous buffer. A buffer is chosen based
upon the pK.sub.a of the cargo moiety. A cargo moiety with a lower
pK.sub.a would necessitate a buffer. with a lower pH range than
that of a cargo moiety with a higher pK.sub.a. Thus, for
caspofingin, with pK.sub.a values of 5.1, 8.7, 9.7 and 10.7, a
buffer with a pH range of between 4.5 and 5.5 would be sufficient
to maintain its multivalency. Buffers with a pH range suitable for
acidification can readily be identified by the skilled practitioner
based upon the cargo moiety being protonized. Suitable buffers
include low molecular weight buffers having an acidic pK.sub.a,
e.g., amino acids and TES. Acidic buffers are known in the art, and
identification of a variety of acidic buffers would require no more
than routine experimentation by one of ordinary skill in the art.
Alternatively, the weakly basic cargo moiety can be protonized by
slow addition of an acid, e.g., hydrochloric acid, to an aqueous
solution of lipid and weakly basic cargo moiety.
[0159] In still other embodiments, the protonizable cargo moiety
has more than one pK.sub.a value. In preferred embodiments, the pH
is lowered to below the highest pK.sub.a value. In other preferred
embodiments, pH is lowered to below the second highest pK.sub.a
value. In other preferred embodiments, the pH is lowered to below
the third, fourth, fifth or even sixth highest pK.sub.a value. In
yet other preferred embodiments, the pH is lowered to below the
lowest pK.sub.a value.
[0160] Optionally, the cargo moiety can be protonized in the
lipid-cargo moiety mixture, e.g., by lowering the pH or introducing
the cargo moiety to a suspension of lipids at a low pH. Because of
its cationic nature, the protonized cargo moiety tends to associate
with the negatively charged surface of the liposome bilayers.
[0161] In yet other embodiments, the cargo moieties can be
protonized prior to incorporation into the cochleates. For example,
they can be obtained or purchased protonized from the manufacturer.
Additionally or alternatively, they can be protonized, isolated as
a protonized cargo moiety, and subsequently incorporated into a
cochleate at a suitable pH. A suitable pH is a pH that allows the
cargo moiety to remain protonized, and can be readily determined by
the skilled artisan.
[0162] In other embodiments, the pH of the resultant anhydrous
cochleates in solution can be adjusted using, e.g., acid. Without
wishing to be bound by any particular theory, it is believed that
this would help to maintain the protonized cargo moiety within the
cochleate structure.
[0163] In another aspect, the present invention generally is
directed to methods of making cochleates that include an
aggregation inhibitor. The aggregation inhibitor can be introduced
prior to, during or after formation of cochleates. That is, the
aggregation inhibitor can be added to the lipid-cargo moiety
solution, to the liposomal solution or to the precipitated
cochleate. For example, in one embodiment, the aggregation
inhibitor is introduced to a liposomal suspension from which
cochleates will subsequently be formed (e.g., by addition of cation
or dialysis). That is, the aggregation inhibitor may be introduced
prior to formation of liposomes, e.g., it may be added to dried
lipid prior to suspension or added directly to a liposomal
suspension, before of after addition of a cargo moiety. In such
embodiments, the cochleates may be initially formed in the desired
size range and aggregation thereafter prevented by the presence of
the aggregation inhibitor.
[0164] In other embodiments, the methods of the invention can
include the step of introducing an aggregation inhibitor to a
cochleate composition. For example, the method can further include
forming cochleates (prior to introducing the aggregation
inhibitor). The method can include providing cochleates already
formed, e.g., cochleates obtained from a supplier. The method can
further include the step of disaggregating cochleates by adding an
aggregation inhibitor to aggregated cochleates.
[0165] In still other embodiments, aggregated cochleates may be
disaggregated using alternative disaggregation methods, e.g.,
homogenization, and an aggregation inhibitor can be introduced in
order to prevent reaggregation.
[0166] In yet another embodiment, the aggregation inhibitor can be
introduced during the formation of the cochleate, e.g., it can be
added with the cation or during dialysis.
[0167] In a preferred embodiment, the aggregation inhibitor is
added in an amount suitable for modulating the resulting cochleate
to the desired size.
[0168] The method can include forming cochleates with any or all of
the optional ingredients disclosed herein. For example, the
cochleates can include additional cationic compounds, protonized
cargo moieties, non-negative lipids, and/or aggregation
inhibitors.
[0169] Any of the methods described herein can be utilized to
produce anywhere from about 1 mg to about 500 g of cochleates in
one batch. A smaller batch may be preferred in a laboratory setting
where characterization of cochleates is desired. On the other hand,
larger batches may be preferred in a manufacturing setting where
mass production is desired. Preferably, larger batches are at least
50 g, and more preferably at least 75 g.
[0170] FIG. 2 illustrates an exemplary method of the present
invention, wherein drug-liposomal are obtained by addition of
a-hydrophobic drug in solvent (e.g., DMSO, DMF, THF, EtOH),
optionally with an antioxidant (e.g., Vitamin E), to a liposomal
suspension. A liposomal suspension is prepared by vortexing lipid
(e.g., soyPS) and water and filtered, however, other methods of
obtaining liposomal suspensions can be employed in the methods of
the present invention. Cochleates are precipitated out by the
addition of calcium (e.g., calcium chloride), and subsequently can
be washed (e.g., with calcium containing buffers) to remove any
residual solvent, if desired. Alternatively, residual solvent can
be removed by other methods, e.g., dialysis.
[0171] FIG. 3 illustrates another aspect of the invention, wherein
hydrosoluble drugs are encochleated. In this method, a hydrosoluble
drug is added directly to liposomes and subsequently precipitated.
The liposomes are prepared by adding lipid (e.g., dry Soy PS
powder) to water, but could be prepared or provided by any other
known means.
[0172] Aggregation Inhibitors
[0173] In some preferred embodiments, the cochleates of the present
invention can optionally include one or more aggregation
inhibitors. The term "aggregation inhibitor," as used herein,
refers to an agent that inhibits aggregation of cochleates. The
aggregation inhibitor typically is present at least on the surface
of the cochleate, and may only be present on the surface of the
cochleate (e.g., when the aggregation inhibitor is introduced after
cochleate formation). Aggregation inhibitors can be added before,
after, or during cochleate formation. The type and/or amount of
aggregation inhibitor can be adjusted to obtain a desired cochleate
size and/or distribution. Additionally or alternatively,
aggregation inhibitor(s) can be used to stabilize cochleate size
and/or size distribution such that aggregation of cochleates is
minimized or eliminated.
[0174] Such compositions are advantageous for several reasons
including that smaller cochleates can allow for greater uptake by
cells and rapid efficacy. Such a composition is suitable, e.g.,
when it is desired to rapidly and effectively deliver a cargo
moiety (e.g., an antifungal or antibacterial agent against a fungal
or bacterial infection). Moreover, particle size can have a
targeting affect in that some cells may take up particles of a
certain size more or less effectively. Size may also affect the
manner in which cochleates interact with a cell (e.g., fusion
events or uptake).
[0175] Aggregation inhibitors work in part by modifying the surface
characteristics of the cochleates such that aggregation is
inhibited. Aggregation can be inhibited, for example, by steric
bulk around the cochleate, which inhibits aggregation and/or
changes the nature of the cochleate structure, e.g., a change in
the surface hydrophobicity and/or surface charge.
[0176] The terms "coat," "coated," "coating," and the like, unless
otherwise indicated, refer to an agent (e.g. an aggregation
inhibitor) present at least on the outer surfaces of a cochleate.
Such agents may be associated with the bilayer by incorporation of
at least part of the agent into the bilayer, and/or may be
otherwise associated, e.g., by ionic attraction to the cation or
hydrophobic or ionic attraction to the lipid.
[0177] As discussed herein, cochleates can be formed by the calcium
induced restructuring and fusion of lipid, e.g., phospholipid such
as phosphatidylserine (PS). Due to the hydrophobic nature of the
surfaces of cochleates in aqueous, calcium containing solutions,
cochleates formed without the aggregation inhibitors of the
invention can aggregate and form larger masses, e.g., needle-like
structures in aspirin cochleates (FIG. 37A, right panel). It has
been discovered that restricting and/or inhibiting the interaction
of liposomes that can coalesce into cochleates at the time of
cation addition limits the size of the resultant cochleate crystal,
and prevents aggregation into larger particles. The addition of an
aggregation inhibitor (e.g., casein) to liposomes prior to the
addition of calcium results in stable non-aggregated nanocochleate
structures (FIG. 37A, left panel).
[0178] The type and/or amount of aggregation inhibitor used can
also determine the size of resulting cochleate. The presence of an
aggregation inhibitor in differing concentrations also allows
regulation of cochleate size distribution.
[0179] It also has been discovered that addition of one or more
aggregation inhibitors after formation of cochleates also inhibits
and even reverses aggregation. For example, it is shown in FIG. 35
that the addition of half and half, whole milk, and fat free milk
to Rhodamine-PE cochleates inhibits aggregation. It can also be
noted that the milk products with more fat content (milk and half
and half) inhibited aggregation more than the fat free milk, which
has less fat content. Additionally, the addition of an aggregation
inhibitor (milk) to aggregated cochleates has been demonstrated to
disaggregate the cochleates as depicted in FIG. 36.
[0180] Suitable aggregation inhibitors that can be employed in
accordance with the present invention include but are not limited
to at least one of the following: casein, .kappa.-casein, milk,
albumin, serum albumin, bovine serum albumin, rabbit serum albumin,
methylcellulose, ethylcellulose, propylcellulose, hydroxycellulose,
hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl
cellulose, hydroxypropylmethyl cellulose, polyvinyl pyrrolidone,
carboxymethyl cellulose, carboxyethyl cellulose, pullulan,
polyvinyl alcohol, sodium alginate, polyethylene glycol,
polyethylene oxide, xanthan gum, tragacanth gum, guar gum, acacia
gum, arabic gum, polyacrylic acid, methylmethacrylate copolymer,
carboxyvinyl polymer, amylose, high amylose starch,
hydroxypropylated high amylose starch, dextrin, pectin, chitin,
chitosan, levan, elsinan, collagen, gelatin, zein, gluten,
carrageenan, carnauba wax, shellac, latex polymers, milk protein
isolate, soy protein isolate, whey protein isolate and mixtures
thereof
[0181] A preferred aggregation inhibitor is casein. Casein is a
highly phosphorylated, calcium binding protein. Without wishing to
be bound to any particular theory, it is believed that calcium
mediates an interaction between negatively charged lipid (e.g., PS)
and casein, thereby changing the surface properties of cochleates
such that aggregation is inhibited. Another preferred aggregation
inhibitor is milk and other milk products such as Half and Half,
cream etc. Preferred milk products also contain casein. Another
preferred aggregation inhibitor is an excipient, e.g.,
methylcellulose. Other preferred aggregation inhibitors include
albumin, serum albumin, bovine serum albumin and rabbit serum
albumin.
[0182] More than one aggregation inhibitor may be employed in the
compositions of the invention. For example, both milk and
methylcellulose may be used as an aggregation inhibitor.
[0183] In one embodiment, the cochleate compositions of the
invention include between about 10% and about 0.1% aggregation
inhibitor. Preferably, the aggregation inhibitor comprises about 1%
of the cochleate composition.
[0184] In another embodiment, the cochleate compositions of the
invention include an aggregation inhibitor to lipid ratio of
between about 0.1:1 to about 4:1 by weight.
[0185] Preferably, the aggregation inhibitor to lipid ratio is
about 1:1. A person of ordinary skill in the art will readily be
able to determine the amount of aggregation inhibitor needed to
form cochleates of the desired size with no more than routine
experimentation.
[0186] Cochleate Size and Distribution
[0187] The formation of cochleates can be envisioned as a
crystallization event that spontaneously occurs upon the
interaction of charged lipids and oppositely charged multivalent
cations. Modulating of the size of cochleate crystals formed,
however has prior to the present invention proved difficult.
[0188] In aqueous suspension, plain cochleates generally aggregate
and upon long term storage form larger masses which can be several
microns in size. Because of the association of the calcium with the
lipid head group, the surfaces of cochleates have a hydrophobic
character. When suspended in aqueous buffer, cochleate aggregation
is a consequence of hydrophobic interactions, minimizing the amount
of surface area exposed to water. FIG. 30 is a schematic model of
cochleate aggregation in aqueous solution.
[0189] It has been discovered that aggregation can be inhibited and
even reversed, and individual cochleate particles can be stabilized
by changing the surface properties of the cochleates and thereby
inhibiting cochleate-cochleate interaction. Aggregation can be
inhibited by including in the liposome suspension a material that
prevents liposome-liposome interaction at the time of calcium
addition and thereafter. Alternatively, the aggregation inhibitor
can be added after formation of cochleates. Additionally, the
amount of aggregation inhibitor can be varied, thus allowing
modulation of the size of the cochleates.
[0190] FIG. 31 is a schematic model of cochleates coated in
proteins to reduce the amount of cochleate aggregation to near
zero. As demonstrated in greater detail below, the resulting
cochleates are surprisingly small. Particle size analysis
demonstrates that these formulations are stable nanocochleates.
Additional experiments, presented below in the Examples, have
extended these observations providing the conceptual basis for the
development of protocols for the preparation of stabilized
nanocochleate formulations of defined size.
[0191] FIG. 32 is a schematic diagram of an exemplary method of
making cochleates of the invention by adding an aggregation
inhibitor subsequent to cochleate formation. Accordingly, in one
aspect, the invention provides a cochleate composition comprising a
plurality of cochleates and an aggregation inhibitor. In a
preferred embodiment, the aggregation inhibitor comprises a coating
on the cochleates. Such a "coating" can be formed by addition of
the aggregation inhibitor after formation of cochleates. The amount
of aggregation inhibitor employed and the point at which the
aggregation inhibitor is added can be used to control the particle
sizes of the cochleates.
[0192] Accordingly, the present invention provides a cochleate
composition comprising a plurality of cochleates and an aggregation
inhibitor having a desired particle size distribution, and methods
of making the same. As demonstrated herein, the amount of
aggregation inhibitor and/or time of addition can be varied to
modulate and/or stabilize the size and/or size distribution of a
cochleate composition.
[0193] In one embodiment, the aggregation inhibitor can be employed
to achieve cochleates that are significantly smaller and have
narrower particle size distributions than compositions without
aggregation inhibitors as demonstrated, e.g., in FIG. 34. Such
compositions are advantageous for several reasons including that
they can allow for greater uptake by cells (see e.g., FIG. 33), and
rapid efficacy (see e.g., FIGS. 46, 47, 49 and 50). Such a
composition is suitable, e.g., when it is desired to rapidly and
effectively deliver a cargo moiety (e.g., an antifungal or
antibacterial agent against a fungal or bacterial infection).
Moreover, cochleate size can have a targeting affect in that some
cells may take up particles of a certain size more or less
effectively. Size may also affect the manner in which cochleates
interact with a cell (e.g., fusion events or uptake).
[0194] In another embodiment, the aggregation inhibitor can be
employed in an amount to achieve cochleate compositions having a
particle size relatively larger than that which can be achieved
without or with other aggregation inhibitors (e.g., if more and/or
a different aggregation inhibitor used). Such a composition can be
useful, e.g., when delayed uptake and/or release of the cargo
molecule is desired, or when targeted cells or organs more
effectively take up cochleates in the relatively larger size range.
Such compositions also may have sustained activity (relative to
smaller cochleate compositions) because it can take longer for the
cargo moiety to be released from a larger cochleate, e.g., if
multiple fusion events are required.
[0195] In yet another embodiment, the amount and/or types of
aggregation inhibitor can be chosen to manufacture a cochleate
composition that has a wide particle size distribution such that
the cargo moiety is released over a period of time because smaller
cochleates are rapidly taken up initially followed by take up or
fusion events with increasingly larger cochleates. In addition,
size may not only affect what type of cells take up the cochleate,
but also how the cochleates interact with certain cells, e.g., size
may effect whether a cochleate is taken up by a cell or undergoes
one or more fusion events with a cell.
[0196] Moreover, in yet further embodiments, several compositions
can be combined for desired release profiles, e.g., a pulsed
released, or combined release. For example, a rapid release
nanocochleate composition can be mixed with a delayed-release
larger size or even standard cochleate composition, such that an
immediate and a delayed release are both realized. In an exemplary
case, both small and large antibiotic cochleates are administered
in order to treat a subject with a high initial dose (small
cochleates) and to maintain enough antibiotic in the serum to be
effective against remaining bacteria (large cochleates). In
addition, the cochleate compositions may have different cargo
moieties, e.g., a stomach protecting medication can be formulated
with nanocochleates for initial release (or a large distribution
for long term release), and one or more non-steroidal
anti-inflammatory drugs can be formulated with larger cochleates
(NSAID) for release after the stomach protecting medication is
released.
[0197] An aggregation inhibitor also can be employed to stabilize
particle size and particle size distribution. For example, it can
be used to "lock-in" the cochleate size and distribution of
standard cochleates and/or cochleates having an aggregation
inhibitor. While the cochleates of the invention typically are
stable over long periods of time, standard cochleates (cochleates
formed without aggregation inhibitors) can tend to aggregate over
time. Thus, standard cochleates can be reduced in size and/or
stabilized by addition to such aggregation inhibitors, e.g.,
addition of methylcellulose after cochleate formation. FIG. 54
shows the decrease in size of caspofungin cochleates which have
been homogenized and treated with bovine serum albumin.
[0198] Cochleates formed in the presence of aggregation inhibitors
do not aggregate. Accordingly, such compositions are advantageous
for several reasons including, e.g., greater uptake by cells, and
increased efficacy. Cochleate compositions of the invention
preferably have a mean diameter less than about 5, 4, 3, 2, or 1
micrometer. Preferably, the cochleate compositions have a mean
diameter less than about 900 nm, 800 nm, 700 nm, 600 nm, 500 nm,
400 nm, 300 nm, 200 nm, or 100 nm. All individual values between
these values (880, 435, 350, etc.), are meant to be included and
are within the scope of this invention. In another embodiment,
cochleate compositions of the invention include cochleate
populations having a mean diameter about equal to or greater than
about 1 micrometer, e.g., 2, 3, 4, 5, 10, 50, or 100 micrometers.
All individual values and ranges within these ranges are meant to
be included and are within the scope of this invention.
[0199] Preferably, the size distribution is narrow relative to that
observed in standard cochleates (cochleates formed without
aggregation inhibitors). As demonstrated, e.g. in FIG. 34, the size
distribution of cochleate compositions with aggregation inhibitors
is significantly improved relative to that observed in standard
cochleate compositions. Preferably, the cochleates have a size
distribution of less than about 30, 20, 10, 5, 3 or 1 .mu.m, 900
nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100
nm. All individual values between these values (550 nm, 420 nm, 475
nm, etc.), are meant to be included and are within the scope of
this invention. Such compositions are particularly desirable where
uptake by macrophages is desired. It can readily be appreciated
that particle size can be adjusted to a size suitable for uptake by
desired organs or cells and/or unsuitable for uptake by organs or
cells. In another embodiment, a wider size distribution of
cochleates is employed, e.g., about 10, 20, 50, 100, 200 . . . 500
micrometers. All individual values within these ranges are meant to
be included and are within the scope of this invention. Such
compositions can be useful for long term release of cargo
moieties.
[0200] Additionally, as discussed above, the invention contemplates
combination of cochleate populations with one or more cargo
moieties, one or more size distributions, and one or more mean
diameter, to achieve a desired release pattern, e.g., pulsed
release, delayed release and/or timed release of different cargo
moieties.
[0201] Cargo Moieties
[0202] The cochleates of the present invention are preferably
associated or "loaded" with a cargo moiety. A "cargo moiety" is a
moiety to be encochleated, and generally does not refer to the
lipid and ion employed to precipitate the cochleate. Cargo moieties
include any compounds having a property of biological interest,
e.g., ones that have a role in the life processes of a living
organism. A cargo moiety 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.
[0203] Thus, examples include vitamins, minerals, nutrients,
micronutrients, amino acids, toxins, microbicides, microbistats,
co-factors, enzymes, polypeptides, polypeptide aggregates,
polynucleotides, lipids, carbohydrates, nucleotides, starches,
pigments, fatty acids, saturated fatty acids, monounsaturated fatty
acids, polyunsaturated fatty acids, flavorings, essential oils,
extracts, hormones, cytokines, viruses, organelles, steroids and
other multi-ring structures, saccharides, metals, metabolic
poisons, antigens, imaging agents, porphyrins, tetrapyrrolic
pigments, drugs and the like.
[0204] The cargo moiety can be a diagnostic agent, such as an
imaging agent. Imaging agents include nuclear agents and
fluorescent probes, e.g., porphyrins. Porphyrins include
tetrapyrrolic agents or pigments. One such tetrapyrrolic agent is
Zinc Tetra-phenyl Porphyrin (ZnTPP), which is a hydrophobic,
fluorescent molecule that has high absorption in the visible
spectrum (dark purple).
[0205] The polynucleotide can be one that is expressed to yield a
biologically active polypeptide or polynucleotide. Thus, the
polypeptide may serve as an immunogen or, for example, have
enzymatic activity. The polynucleotide may have catalytic activity,
for example, be a ribosome, or may serve as an inhibitor of
transcription or translation, e.g., a small interfering RNA (siRNA)
or an antisense molecule. The polynucleotide can be an antisense
molecule including modified antisense molecule, such as a
morpholino antisense molecule. The polynucleotide can be modified,
e.g., it can be synthesized to have a morpholino backbone. If
expressed, the polynucleotide preferably includes the necessary
regulatory elements, such as a promoter, as known in the art. A
specific example of a polypeptide is insulin.
[0206] The cargo moiety can be an organic molecule that is
hydrophobic in aqueous media. The cargo moiety can also be a
water-soluble monovalent or polyvalent cationic molecule, anionic,
or net neutral at physiological pH.
[0207] The drug can be, but is not limited to, a protein, a small
peptide, a bioactive polynucleotide, an antibiotic, an antiviral,
an anesthetic, antipsychotic, an anti-infectious, an antifungal, an
anticancer, an immunosuppressant, an immunostimulant, a steroidal
anti-inflammatory, a non-steroidal anti-inflammatory, an
antioxidant, an antidepressant which can be synthetically or
naturally derived, a substance which supports or enhances mental
function or inhibits mental deterioration, an anticonvulsant, an
HIV protease inhibitor, a non-nucleophilic reverse transcriptase
inhibitor, a cytokine, a tranquilizer, a mucolytic agent, a
dilator, a vasoconstrictor, a decongestant, a leukotriene
inhibitor, an anti-cholinergic, an anti-histamine, a cholesterol
lipid metablolism modulating agent or a vasodilatory agent. The
drug can also be any over the counter (non-prescription)
medication.
[0208] An antifungal drug can be a polyene macrolide, tetraene
macrolide, pentaenic macrolide, fluorinated pyrimidine, imidazole,
azole, triazole, halogenated phenolic ether, thiocarbamate,
allylamine, sterol inhibitor, and an agent that interpolates fungal
cell wall components.
[0209] Nonsteroidal anti-inflammatory drugs (NSAIDS) are typically
used to treat inflammation, muscle strains, and high fever. NSAIDS
function by inhibiting cyclooxygenase-1 (COX1) and cyclooxygenase-2
(COX2). COX1 enzymes are responsible for protecting the lining of
the stomach and COX2 enzymes are responsible for the production of
prostaglandins, which are important in the inflammatory process.
Unfortunately, commercially available preparations of NSAIDS are
active against both COX1 and COX2, and therefore have unwanted side
effects such as ulcers, upset stomach or nausea.
[0210] Examples of suitable drugs include Amphotericin B,
acyclovir, adriamycin, carbamazepine, ivermectin, melphalen,
nifedipine, indomethacin, curcumin, aspirin, ibuprofen, naproxen,
acetaminophen, rofecoxib, diclofenac, ketoprofin, meloxicam,
nabumetone, estrogens, testosterones, steroids, phenytoin,
ergotamines, cannabinoids, rapamycin, propanadid, propofol,
alphadione, echinomycin, miconazole, miconazole nitrate,
ketoconazole, itraconazole, fluconazole, griseofulvin,
clotrimazole, econazole, terconazole, butoconazole, oxiconazole,
sulconazole, saperconazole, voriconazole, ciclopirox olamine,
haloprogin, tolnaftate, naftifine, terbinafine hydrochloride,
morpholines, flucytosine, natamycin, butenafine, undecylenic acid,
Whitefield's ointment, propionic acid, and caprylic acid,
clioquinol, selenium sulfide, teniposide, hexamethylmelamine,
taxol, taxotere, 18-hydroxydeoxycorticosterone, prednisolone,
dexamethazone, cortisone, hydrocortisone, piroxicam, diazepam,
verapamil, vancomycin, tobramycin, teicoplanin, bleomycin,
peptidolglycan, ristocetin, sialoglycoproteins, orienticin,
avaporcin, helevecardin, galacardin, actinoidin, gentamycin,
netilmicin, amikacin, kanamycin A, kanamycin B, neomycin,
paromomycin, neamine, streptomycin, dihydrostreptomycin, apramycin,
ribostamycin, spectinomycin, caspofungin, echinocandin B, aculeacin
A, micafungin, anidulafungin, cilofungin, pneumocandin,
geldanamycin, nystatin, rifampin, tyrphostin, a glucan synthesis
inhibitor, vitamin A acid, mesalamine, risedronate, nitrofurantoin,
dantrolene, etidronate, nicotine, amitriptyline, clomipramine,
citalopram, dothepin, doxepin, fluoxetine, imipramine, lofepramine,
mirtazapine, nortriptyline, paroxetine, reboxitine, sertraline,
trazodone, venlafaxine, dopamine, St. John's wort,
phosphatidylserine, phosphatidic acid, amastatin, antipain,
bestatin, benzamidine, chymostatin, 3,4-dichloroisocoumarin,
elastatinal, leupeptin, pepstatin, 1,10-phenanthroline,
phosphoramidon, ethosuximide, ethotoin, felbamate,. fosphenytoin,
lamotrigine, levitiracetam, mephenytoin, methsuximide,
oxcarbazepine, phenobarbital, phensuximide, primidone, topirimate,
trimethadione, zonisamide, saquinavir, ritonavir, indinavir,
nelfinavir, and amprenavir.
[0211] Tyrphostin and geldanamycin (GA) target the
oncoprotein/oncogene erb B2, which is overexpressed on a variety of
tumor cells, and this high level of expression is functionally
related to transformation.
[0212] GA is a hydrophobic small molecule drug that has been shown
to have activity in vitro against cancer cell lines. It inhibits
ErbB2 expression by destabilizing chaperone proteins. GA has been
traditionally dissolved in DMSO for in vitro and in vivo testing.
In vivo, it has anti-tumor activity, but has significant
hepatotoxicity.
[0213] Tyrphostin AG-825 is a tyrosine kinase inhibitor that has
activity against cancer cell lines over-expressing erbB2. It
inhibits its activity, and therefore cellular proliferation, but
not erb B2 expression.
[0214] The drug can be a polypeptide such as cyclosporin,
Angiotensin I, II and III, enkephalins and their analogs, ACTH,
anti-inflammatory peptides I, II, III, bradykinin, calcitonin,
b-endorphin, dinorphin, leucokinin, leutinizing hormone releasing
hormone (LHRH), insulin, neurokinins, somatostatin, substance P,
thyroid releasing hormone (TRH) and vasopressin.
[0215] The drug can be an antigen, but is not limited to a protein
antigen. The antigen can also be a carbohydrate or DNA. Examples of
antigenic proteins include membrane proteins, carbohydrates,
envelope glycoproteins from viruses, animal cell proteins, plant
cell proteins, bacterial proteins, and parasitic proteins.
[0216] The antigen can be extracted from the source particle, cell,
tissue, or organism by known methods. Biological activity of the
antigen 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
employed. Suitable detergents include ionic detergents such as
cholate salts, deoxycholate salts and the like or heterogeneous
polyoxyethylene detergents such as Tween, BRIG or Triton.
[0217] Utilization of this method allows reconstitution of antigens
into the liposomes with retention of biological activities, and
efficient association with the cochleates. The method may also be
employed without sonication, extreme pH, temperature, or pressure
all of which may have an adverse effect upon efficient
reconstitution of the antigen in a biologically active form.
[0218] Suitable nutrients include, but are not limited to lycopene,
micronutrients such as phytochemicals or zoochemicals, vitamins,
minerals, fatty acids, amino acids, fish oils, fish oil extracts,
saccharides, herbal products and essential oils and flavor agents.
Specific examples include Vitamins A, B, B1, B2, B3, B12, B6,
B-complex, C, D, E, and K, vitamin precursors, caroteniods, and
beta-carotene, resveratrol, biotin, choline, inositol, ginko,
lutein, zeaxanthine, quercetin, silibinin, perillyl alcohol,
genistein, sulfurophane, and essential fatty acids, including
eicosapentanoic acid (EPA), gamma-3, omega-3, gamma-6 and omega-6
fatty acids, herbs, spices, and iron. Minerals include, but are not
limited to boron, chromium, colloidal minerals, colloidal silver,
copper, manganese, potassium, selenium, vanadium, vanadyl sulfate,
calcium, magnesium, barium, iron and zinc.
[0219] As used herein, "micronutrient" is a nutrient that the body
must obtain from outside sources. Generally micronutrients are
essential to the body in small amounts.
[0220] The cargo moiety can be a saccharide or sweetener, e.g.,
saccharine, isomalt, maltodextrine, aspartame, glucose, maltose,
dextrose, fructose and sucrose. Flavor agents include oils,
essential oils, or extracts, including but not limited to oils and
extracts of cinnamon, vanilla, almond, peppermint, spearmint,
chamomile, geranium, ginger, grapefruit, hyssop, jasmine, lavender,
lemon, lemongrass, marjoram, lime, nutmeg, orange, rosemary, sage,
rose, thyme, anise, basil, black pepper, tea or tea extracts, an
herb, a citrus, a spice or a seed.
[0221] In some preferred embodiments, the cargo moiety can be a
protonized cargo moiety. In one embodiment, the cargo moiety is a
protonized weakly basic cargo moiety. The pharmacokinetics of
weakly basic cargo moieties (e.g., vancomycin and tobramycin),
conventionally has been dominated by their poor solubility in
lipids such as milk. Because of this poor solubility and the lack
of water in the cochleates, it was surprising that weakly basic
cargo moieties could be incorporated into cochleates at the
concentrations achieved in the present invention. It has been
discovered, however, that protonized weakly basic cargo moieties
can be incorporated into anhydrous cochleates. Protonized neutral
cargo moieties can similarly be precipitated with negatively
charged lipid, provided that acidification renders them cationic.
Additionally, cargo moieties suitable for use in accordance with
the present invention can include protonized weakly acidic cargo
moieties or protonized amphoteric cargo moieties. Weakly acidic
cargo moieties or amphoteric cargo moieties may or may not include
an initial positive charge. Such cargo moieties would also be
rendered cationic by protonization. Protonizable cargo moieties can
be negatively charged, positively charged, uncharged or
zwitterionic. The invention is particularly advantageous in the
preparation of protonized water-soluble cargo moieties.
[0222] In one embodiment, the protonized cargo moiety is
monovalent. In other embodiments, the protonized cargo moiety is
multivalent, e.g., divalent, trivalent, etc. In certain
embodiments, a higher valency may be preferable due to the size
and/or conformation of the cargo moiety.
[0223] Moreover, because the protonized cargo moieties are
cationic, hydrous cochleates can be a made without additional
cation (e.g., a metal cation, such as calcium). For example,
vancomycin-cochleates have been made without cation, as described
below. Anhydrous cochleates made with divalent metal cation, e.g.,
Ca.sup.2+, are preferred and are active against Staph. A. infection
in vitro.
[0224] In one embodiment, the protonized cargo moiety is a
multivalent cation (i.e., polycationic). The protonization or
acidification can render a non-cationic moiety cationic or increase
the valency of a cationic moiety. The protonized cargo moiety can
optionally be isolated and characterized prior to formulation into
a cochleate. Alternatively, the cargo moiety can be obtained or
purchased protonized (e.g., vancomycin hydrochloride or caspofungin
acetate).
[0225] In one embodiment, the protonized cargo moiety is a
protonized peptide, such as a protonized protein.
[0226] In another embodiment, the protonized cargo moiety is a
protonized nucleotide. The protonized nucleotide can be, but is not
limited to a protonized DNA, a protonized RNA, a protonized
morpholino, a protonized siRNA molecule, a protonized ribozyme, a
protonized antisense molecule, or a protonized plasmid.
[0227] In a preferred embodiment, the cargo moiety is a drug,
including, but not limited to, an aminoglycoconjugate, e.g., an
aminoglycoside or an aminoglycopeptide. Preferably the
aminoglyconjugate is weakly basic.
[0228] In a particularly preferred embodiment, the
aminoglycoconjugate is one or more of the following: vancomycin,
teicoplanin, bleomycin, peptidolglycan, ristocetin,
sialoglycoproteins, orienticin, avaporcin, helevecardin,
galacardin, actinoidin, gentamycin, netilmicin, tobramycin,
amikacin, kanamycin A, kanamycin B, neomycin, paromomycin, neamine,
streptomycin, dihydrostreptomycin, apramycin, ribostamycin, and
spectinomycin.
[0229] In another preferred embodiment, the cargo moiety is an
echinocandin. In a particularly preferred embodiment, the
echinocandin is one or more of the following: caspofungin,
echinocandin B, aculeacin A, micafungin, anidulafungin, cilofungin,
and pneumocandin.
[0230] The cochleates of the invention can be prepared with a wide
range of cargo moiety to lipid ratios. By way of example, the ratio
of cargo moiety to lipid can be between about 20,000:1 and about
0.5:1 by weight. In one embodiment the ratio is about 1:1 by
weight. In others the ratio is about 2:1, 3:1, 4:1, 5:1, 10:1,
20:1, 50:1, 100:1, 200:1, or 400:1 by weight. All individual ranges
and values between 20,000:1 and 0.5:1 are encompassed by the
invention.
[0231] The cochleates of the present invention can optionally
include one or more additional cargo moieties. The additional cargo
moiety can be a second protonized cargo moiety or any other cargo
moiety.
[0232] Additional pharmacologically active agents may be delivered
in combination with the primary active agents, e.g., the cochleates
of this invention. In one embodiment, such agents include, but are
not limited to agents that reduce the risk of atherosclerotic
events and/or complications thereof. Such agents include, but are
not limited to beta blockers, beta blockers and thiazide diuretic
combinations, HMG CoA reductase inhibitors, statins, aspirin, ace
inhibitors, ace receptor inhibitors (ARBs), and the like.
[0233] Suitable beta blockers include, but are not limited to
cardioselective (selective beta 1 blockers), e.g., acebutolol
(e.g., Sectral.TM.), atenolol (e.g., Tenormin.TM.), betaxolol
(e.g., Kerlone.TM.), bisoprolol (e.g., Zebeta.TM.), metoprolol
(e.g., Lopressor.TM.), and the like. Suitable non-selective
blockers (block beta 1 and beta 2 equally) include, but are not
limited to carteolol (e.g., Cartrol.TM.), nadolol (e.g.,
Corgard.TM.), penbutolol (e.g., Levatol.TM.), pindolol (e.g.,
Visken.TM.), propranolol (e.g., Inderal.TM.), timolol (e.g.,
Blockadren.TM.), labetalol (e.g., Normodyne.TM., Trandate.TM.), and
the like.
[0234] Suitable beta blocker thiazide diuretic combinations
include, but are not limited to Lopressor HCT, ZIAC, Tenoretic,
Corzide, Timolide, Inderal LA 40/25, Inderide, Normozide, and the
like.
[0235] Suitable statins include, but are not limited to pravastatin
(e.g., Pravachol.TM.), simvastatin (e.g., Zocor.TM.), lovastatin
(e.g., Mevacor.TM.), and the like.
[0236] Suitable ace inhibitors include, but are not limited to
captopril (e.g., Capoten.TM.), benazepril (e.g., Lotensin.TM.),
enalapril (e.g., Vasotec.TM.), fosinopril (e.g., Monopril.TM.),
lisinopril (e.g., Prinivil.TM. or Zestril.TM.), quinapril (e.g.,
Accupril.TM.), ramipril (e.g., Altace.TM.), imidapril, perindopril
erbumine (e.g., Aceon.TM.), trandolapril (e.g., Mavik.TM.), and the
like. Suitable ARBS (Ace Receptor Blockers) include but are not
limited to losartan (e.g., Cozaar.TM.), irbesartan (e.g.,
Avapro.TM.), candesartan (e.g., Atacand.TM.), valsartan (e.g.,
Diovan.TM.), and the like.
[0237] Suitable HMG CoA reductase inhibitors that are useful in
accordance with the methods and compositions of the invention are
statin molecules. These include: Lovastatin (e.g., Mevacor.TM.),
Pravastatin (e.g., Pravachol.TM.), Simvastatin (e.g., Zocor.TM.),
Fluvastatin (e.g., Lescol.TM.), Atorvastatin (e.g., Lipitor.TM.),
or Cerivastatin (e.g., Baycol.TM.).
[0238] Other agents that may be administered in conjuction with the
cochleates of the invention for treatment of atherosclerotic events
and/or complications thereof are phytosterols, phytostanols and
their derivatives and isomers; soy protein; soluble fibers, e.g.
beta-glucan from, for example, oat and psyllium, nuts, rice bran
oil, each of which is particularly suitable for use in food,
dietary supplements and food additive compositions. Phytosterols
may be solid (e.g., powder, granules) or liquid (e.g., oil)
form.
[0239] It will be obvious to a person of skill in the art that the
choice of the agent for treatment of atherosclerotic events and/or
complications thereof depends on the intended delivery vehicle
(e.g., food, supplement, pharmaceutical) and the mode of
administration.
[0240] The cargo moiety can additionally be bound to a cochleate
component or to a hydrophobic tail. In one embodiment, the cargo
moiety is bound to the lipid cochleate component or the hydrophobic
tail with a digestible, reducible, or otherwise reversible linker.
The cargo moiety can be bound in a reversible manner (e.g., with a
reducible or digestible linker) or a linker susceptible to target
conditions (e.g., pH, temperature, ultrasonic energy and the like).
This is particularly useful as the linker can be chosen such that
it is readily digestible, e.g., by an enzyme, in the body generally
or even in a target structure. Thus, e.g., a linker can be chosen
such that it is degraded by an enzyme in the plasma, interstitial
fluids, in a cell (e.g. a macrophage) or in an endosome, such that
the protonized cargo moiety becomes detached and available in
unbound form in these structures. In another embodiment, the
reversible linker can be an electrostatic or other bond that is
broken by a change in pH, e.g., in an organ or other structure in
which the cochleate experiences a pH gradient. In another
embodiment, the linker is reversed by a change in temperature,
e.g., by exposure to body temperature.
[0241] In one embodiment, the cargo moiety is bound by an
electrostatic, hydrophobic, covalent, or ionic interaction with a
lipid component such as a hydrophobic tail. In a preferred
embodiment, the cargo moiety is bound to a component of the bilayer
of the cochleate, e.g., a phospholipid or other lipid. Covalently
binding the cargo moiety to the lipid by cross-linking can be
accomplished by known methods. In one embodiment, the covalent bond
is reversible so that the cargo moiety can be detached from the
lipid component or hydrophobic tail under suitable conditions. For
example, a cargo moiety can be attached to a phospholipid via a
linker that can be cleaved by an enzyme endogenous to a target
tissue, organ, or structure (e.g., a plasma protein, interstitial
protein, an endosome or the intracellular milieu), such that the
cargo moiety is delivered to the target tissue, organ or other
structure. In alternative embodiments the cargo moiety can be
attached by any other means, for example, by electrostatic
interactions and/or hydrophobic interactions.
[0242] The cargo moiety can be associated with the lipid component
or hydrophobic tail in any of the methods described herein. For
example, in one embodiment, the cargo moiety is associated with the
lipid component, such that the cargo moiety dissociates with the
lipid component upon contact with a target environment. The cargo
moiety can be bound to a component of the cochleate with any of the
linkers described herein, e.g., a linker that is reducible, or
otherwise reversible or digestible by an enzyme, protein, or
molecule endogenous to the target environment. The enzyme can be an
extracellular, intracellular or endosomal enzyme endogenous to the
subject. In another embodiment, the cargo moiety component is
electrostatically associated with the lipid component and
dissociates with the cochleate upon contact with a pH gradient in a
cell or organ of the subject.
[0243] Delivery of Cargo Moieties
[0244] Many naturally occurring membrane fusion events involve the
interaction of calcium with negatively charged phospholipids (e.g.,
PS and phosphatidylglycerol). Calcium-induced perturbations of
membranes containing negatively charged lipids, and the subsequent
membrane fusion events, are important mechanisms in many natural
membrane fusion processes. Therefore, cochleates can be envisioned
as membrane fusion intermediates.
[0245] Phase/fluorescent and fluorescent images of
Rhodamine-labeled cochleates incubated with splenocytes were
captured and are shown in FIG. 1. These images indicate that a
fusion event occurs between the outer layer of the cochleate and
the cell membrane, resulting in the delivery of encochleated
material into the cytoplasm of the target cell. As the calcium
rich, highly ordered membrane of a cochleate first comes into close
approximation to a natural membrane, a perturbation and reordering
of the cell membrane is induced, resulting in a fusion event
between the outer layer of the cochleate and the cell membrane.
This fusion results in the delivery of a small amount of the
encochleated material into the cytoplasm of the target cell. The
cochleate can then break free of the cell and be available for
another fusion event, either with the same or another cell.
[0246] Additionally or alternatively, particularly with active
phagocytic cells, cochleates may be taken up by endocytosis and
fuse from within the endocytic vesicle. Cochleates made with trace
amounts of fluorescent lipids have been shown to bind and gradually
transfer lipids to the plasma membrane and interior membranes of
white blood cells in vitro. FIG. 33, for example, demonstrates the
uptake of cochleates by macrophages.
[0247] Cochleates are useful for the delivery of a cargo moiety to
cultured cells, tissues or organisms by a variety of administration
routes. The term "delivery," as used herein, refers to any means of
bringing or transporting a cargo moiety to a host, a food item, a
formulation, a pharmaceutical composition, or any other system,
wherein the cargo moiety maintains at least a portion of its
activity. For example, the use of cochleates to deliver protein or
peptide molecules as vaccines has been disclosed in U.S. Pat. No.
5,840,707, issued Nov. 24, 1998. Similarly, polypeptide-cochleates
are effective immunogens when administered to animals by
intraperitoneal and intramuscular routes of immunization (G.
Goodman-Snitkoff, et al., J. Immunol., Vol. 147, p.410 (1991); M.
D. Miller, et al., J. Exp. Med., Vol. 176, p. 1739 (1992)).
Further, cochleates are effective delivery vehicles for
encapsulated proteins and/or DNA to animals and to cells in
culture. For example, reconstituted Sendai or influenza virus
glycoproteins are efficiently delivered in encochleated form
(Mannino and Gould-Fogerite, Biotechniques 6(1):682-90 (1988);
Gould-Fogerite et al., Gene 84:429 (1989); Miller et al., J. Exp.
Med. 176:1739 (1992)).
[0248] The cochleates can be coadministered with a further agent.
The second agent can be delivered in the same cochleate
preparation, in a separate cochleate preparation mixed with the
cochleate preparation of the invention, separately in another form
(e.g., capsules or pills), or in a carrier with the cochleate
preparation. The cochleates can further include one or more
additional cargo moieties, such as other drugs, peptides,
nucleotides (e.g., DNA and RNA), antigens, nutrients, flavors
and/or proteins.
[0249] The cochleates of the invention also can include a reporter
molecule for use in in vitro diagnostic assays, which can be a
fluorophore, radiolabel or imaging agent. The cochleates can
include molecules that direct binding of the cochleate to a
specific cellular target, or promotes selective entry into a
particular cell type.
[0250] One advantage of the cochleates of the present invention is
the stability of the composition. Cochleates can be administered by
any route, e.g., mucosal or systemic, without concern. Cochleates
can be administered orally or by instillation without concern, as
well as by the more traditional routes, such as oral, intranasal,
intraoculate, intrarectal, intravaginal, intrapulmonary, topical,
subcutaneous, intradermal, intramuscular, intravenous, transdermal,
systemic, intrathecal (into CSF), and the like. Direct application
to mucosal surfaces is an attractive delivery means made possible
with cochleates. Delivery can be effected by, e.g., a nasal spray
or nasal bath or irrigation.
[0251] Another advantage of the present invention is the ability to
modulate cochleate size. Modulation of the size of cochleates and
cochleate compositions changes the manner in which the cargo moiety
is taken up by cells. For example, in general, small cochleates are
taken up quickly and efficiently into cells, whereas larger
cochleates are taken up more slowly, but tend to retain efficacy
for a longer period of time. Also, in some cases small cochleates
are more effective than large cochleates in certain cells, while in
other cells large cochleates are more effective than small
cochleates.
[0252] Cochleates and cochleate compositions can also be
administered to humans and non-human animals, such as dog, cats,
and farm animals, in food or beverage preparations. Such
compositions can be introduced to the food or beverage compositions
by the manufacturer (e.g., to supplement food with nutrients), or
by the consumer (e.g., where the cochleate composition is sold
separately as a food additive). For example, nutrients and/or
flavorings may be incorporated into dog or cat food, particularly
where such nutrient and/or flavoring is fragile and normally
decomposes or loses, activity when exposed to oxygen and or water.
Cochleates may be added at any stage into the preparation of dog or
cat food, as the cochleates are stable under extreme pressure and
temperature conditions.
[0253] Another advantage of cochleates and cochleate compositions
of the present invention is their ability to reduce a number of
unwanted side effects. A number of drugs currently on the market
cause gastrointestinal distress and often high circulating blood
levels lead to toxicity in a number of vital organs. The ingestion
of, e.g., aspirin may result in epigastric distress, nausea, and
vomiting. Aspirin may also cause gastric ulceration; exacerbation
of peptic ulcer symptoms, gastrointestinal hemorrhage, and erosive
gastritis have all been reported in patients on high-dose therapy
but also may occur even when low doses are administered. In high
doses, aspirin can also cause hepatic injury. Aspirin can cause
retention of salt and water as well as an acute reduction of renal
function in patients with congestive heart failure or renal
disease. Although long-term use of aspirin alone rarely is
associated with nephrotoxicity, the prolonged and excessive
ingestion of aspirin in combination with other compounds can
produce papillary necrosis and interstitial nephritis. Although
acetaminophen is usually well tolerated, skin rash (generally
erythematous or urticarial) `and other allergic reactions occur
occasionally. Occasionally, the rash can be more serious and may be
accompanied by drug fever and mucosal lesions. In other examples,
the use of acetaminophen has been associated with neutropenia,
thrombocytopenia, and pancyotpenia. The most serious adverse effect
of acute overdosage of acetaminophen is a dose-dependent,
potentially fatal hepatic necrosis. Renal tubular necrosis and
hypoglycemic coma also may occur.
[0254] Another advantage of the present invention is that the
cochleates can be formulated for uptake by particular cells or
organs. Conventionally, high levels of drugs are often administered
intravenously to obtain moderate levels at the sites of infection
in order to combat opportunistic infections. This can cause
undesirable side effects, for example, in the case of vancomycin,
macular skin rashes, anaphylaxis, phlebitis and pain at the site of
intravenous injection, chills, rash, and fever may occur. Also,
rapid intravenous infusion may cause a variety of symptoms,
including erythematous or urticarial reactions, flushing,
tachycardia, and hypotension, generally non-permanent auditory
impairment, ototoxicity associated with excessively high
concentrations of the drug in plasma and less commonly,
nephrotoxicity. By employing the cochleates of the present
invention, toxicity levels can be lowered by decreasing the free
drug in the circulating blood. Additionally, the cargo moiety can
be delivered directly to the site of infection, which can lower or
eliminate the incidence of gastrointestinal distress.
[0255] Aminoglycosides are very poorly absorbed from the
gastrointestinal tract. Less than 1% of the dose typically is
absorbed following either oral or rectal administration. Also,
inadequate concentrations of aminoglycosides are found in
cerebrospinal fluid. Additionally, the drugs are not inactivated in
the intestine, and are excreted relatively rapidly by the normal
kidney, i.e., they are eliminated quantitatively in the feces.
Long-term oral or rectal administration, however, may result in
accumulation of aminoglycosides to toxic concentrations in patients
with renal impairment. Instillation of these drugs into body
cavities with serosal surfaces may result in rapid absorption and
unexpected toxicity, i.e., neuromuscular blockade. Similarly,
intoxication may occur when aminoglycosides are applied topically
for long periods to large wounds, burns, or cutaneous ulcers,
particularly if there is renal insufficiency.
[0256] Moreover, due to their polar nature, aminoglycosides largely
are excluded from most cells, from the central nervous system, and
from the eye. Concentrations of conventionally administered
aminoglycosides in secretions and tissues are low. High
concentrations, however, are found in the renal cortex and in the
endolymph and perilymph of the inner ear; this is thought to
contribute to the nephrotoxicity and ototoxicity caused by these
drugs. Although they are widely used agents, serious toxicity is a
major limitation to the usefulness of the aminoglycosides.
[0257] Both vestibular and auditory dysfunction can follow the
administration of any of the aminoglycosides. Studies of both
animals and human beings have documented progressive accumulation
of these drugs in the perilymph and endolymph of the inner ear.
Accumulation occurs predominantly when plasma concentrations are
high. Diffusion back into the bloodstream is slow; the half-lives
of the aminoglycosides are five to six times longer in the otic
fluids than in plasma. Ototoxicity is more likely to occur in
patients with persistently elevated concentrations of drug in
plasma. However, even a single dose of tobramycin has been reported
to produce slight temporary cochlear dysfunction during periods
when the concentration in plasma is at its peak. The relationship
of this observation to permanent loss of hearing is not known.
[0258] Approximately 8% to 26% of patients who receive an
aminoglycoside for more than several days develop renal impairment,
which is almost always reversible. The toxicity results from
accumulation and retention of aminoglycoside in the proximal
tubular cells. The initial manifestation of damage at this site is
excretion of the enzymes of the renal tubular brush border. Several
variables have been found to influence nephrotoxicity from
aminoglycosides, including total amount of drug administered and
duration of therapy. Constant concentrations of drug in plasma
above a critical level, which is manifested by elevated trough
serum concentrations, correlate with toxicity in human beings.
Aminoglycosides have the potential to produce reversible and
irreversible vestibular, cochlear, and renal toxicity. These side
effects complicate the use of these compounds and make their proper
administration difficult.
[0259] Accordingly, the cochleates of the present invention can be
employed to avoid harmful side effects of drugs caused by their
high concentration or presence in organs such as the kidneys,
stomach or liver.
[0260] Echinocandins are a relatively new class of antifungal
drugs. Although the most widely known echinocandin, caspofungin, is
considered less toxic than other antifungal drugs, (e.g.,
Amphotericin B), this is not true of the entire class. Caspofungin
is especially effective against Candida species, however, other
members of the echinocandin class have activity against other
species, (e.g., Cryptococcus). Additionally, echinocandins are
generally administered intravenously due to their poor oral
absorption. Cochleates of the present invention can be used not
only to facilitate oral absorption, but also to avoid potential
side effects from this class of compounds.
[0261] Safety/Biocompatibility
[0262] Cochleates readily can be prepared from safe, simple,
well-defined, naturally occurring substances, e.g., PS and calcium.
Mixtures of naturally occurring (e.g., soy lipids), synthetic
lipids, and/or modified lipids can also be utilized.
Phosphatidylserine is a natural component of all biological
membranes, and is most concentrated in the brain. The phospholipids
used can be produced synthetically, or prepared from natural
sources. Soy PS is inexpensive, available in large quantities and
suitable for use in humans. Clinical studies indicate that PS is
safe and may play a role in the support of mental functions in the
aging brain. Unlike many cationic lipids, cochleates (which are
composed of anionic lipids) are non-inflammatory and biodegradable.
The tolerance in vivo of mice to multiple administrations of
cochleates by various routes, including intravenous,
intraperitoneal, intranasal and oral, has been evaluated. Multiple
administrations of high doses of cochleate formulations to the same
animal show no toxicity, and do not result in either the
development of an immune response to the cochleate matrix, or any
side effects relating to the cochleate vehicle.
[0263] The cochleates and cochleate compositions of the present
invention can be administered to animals, including both human and
non-human animals. It can be administered to animals, e.g., in
animal feed or water. For example, antibiotic-cochleates of the
present invention can be administered to poultry and other farm
animals, including the ruminants and pigs, to control infection or
to promote growth or milk production. Among a number of conditions
which can be treated with these agents is enteritis, a disease
which can cause severe economic losses to livestock producers.
Enteritis occurs in chickens, swine, cattle and sheep and is
attributed mainly to anaerobic bacteria, particularly Clostridium
perfringens. Enterotoxemia in ruminants, an example of which is
"overeating disease" in sheep, is a condition caused by C.
perfringens infection. The treatment of such conditions is
therefore also encompassed within the methods of the present
invention.
[0264] Methods of Treatment
[0265] In yet another aspect, the present invention provides for
both prophylactic and therapeutic methods of treating a subject at
risk of (or susceptible to) a disorder or having a disorder which
can be treated with one or more cargo moiety.
[0266] "Treatment", or "treating" as used herein, is defined as the
application or administration of a therapeutic agent (e.g.,
antibiotics encochleated by cochleates of the invention) to a
patient, or application or administration of a therapeutic agent to
an isolated tissue or cell line from a patient, who has a disease
or disorder, a symptom of disease or disorder or a predisposition
toward a disease or disorder, with the purpose to cure, heal,
alleviate, relieve, alter, remedy, ameliorate, improve or affect
the disease or disorder, the symptoms of the disease or disorder,
or the predisposition toward disease or disorder. "Treated," as
used herein, refers to the disease or disorder being cured, healed,
alleviated, relieved, altered, remedied, ameliorated improved or
affected. For example, certain methods of treatment of the instant
invention provide for administration of anti-inflammatory
cochleates, such that inflammation is lessened or alleviated. Other
methods of treatment of the instant invention include the
administration of antifungal cochleates, such that fungal infection
is relieved or remedied.
[0267] The terms "cure," "heal," "alleviate," "relieve," "alter,"
"remedy," "ameliorate," "improve" and "affect" are evaluated in
terms of a suitable or appropriate control. A "suitable control" or
"appropriate control" is any control or standard familiar to one of
ordinary skill in the art useful for comparison purposes. In one
embodiment, a "suitable control" or "appropriate control" is a
value, level, feature, characteristic, property, etc. determined
prior to administration of a cargo moiety cochleate, as described
herein. For example, the number of colony forming units can be
determined prior to administering an echinocandin cochleate of the
invention to a host. In another embodiment, a "suitable control" or
"appropriate control" is a value, level, feature, characteristic,
property, etc. determined in a subject, e.g., a control or normal
subject exhibiting, for example, normal traits. In yet another
embodiment, a "suitable control" or "appropriate control" is a
predefined value, level, feature, characteristic, property,
etc.
[0268] The methods of the present invention include methods of
administering a cargo moiety to a host, wherein the cargo moiety is
associated with a cochleate or cochleate composition of the
invention. The cochleates and cochleate compositions of the present
invention may be administered orally, nasally, topically,
intravenously, transdermally, buccally, sublingually, rectally,
vaginally or parenterally.
[0269] The present invention provides a method for treating a
subject that would benefit from administration of a composition of
the present invention. Any therapeutic indication that would
benefit from a cargo moiety, e.g., a drug or nutrient, can be
treated by the methods of the invention. Accordingly, the present
invention provides methods of treating a subject at risk for or
having a disease or disorder which can be treated with, for
example, a protein, a small peptide, a bioactive polynucleotide, an
antibiotic, an antiviral, an anesthetic, antipsychotic, an
anti-infectious, an antifungal, an anticancer, an
immunosuppressant, an immunostimulant, a steroidal
anti-inflammatory, a non-steroidal anti-inflammatory, an
antioxidant, an antidepressant which can be synthetically or
naturally derived, a substance which supports or enhances mental
function or inhibits mental deterioration, an anticonvulsant, an
HIV protease inhibitor, a non-nucleophilic reverse transcriptase
inhibitor, a cytokine, a tranquilizer, a mucolytic agent, a
dilator, a vasoconstrictor, a decongestant, a leukotriene
inhibitor, an anti-cholinergic, an anti-histamine, a cholesterol
lipid metablolism modulating agent or a vasodilatory agent. The
method includes the step of administering to the subject a
composition of the invention, such that the disease or disorder is
treated. The disease or disorder can be, e.g., inflammation, pain,
infection, fungal infection, bacterial infection, viral infection,
parasitic disorders, an immune disorder, genetic disorders,
degenerative disorders, cancer, proliferative disorders, obesity,
depression, hair loss, impotence, hypertension, hypotension,
dementia, senile dementia, or malnutrition, acute and chronic
leukemia and lymphoma, sarcoma, adenoma, carcinomas, epithelial
cancers, small cell lung cancer, non-small cell lung cancer,
prostate cancer, breast cancer, pancreatic cancer, hepatocellular
carcinoma, renal cell carcinoma, biliary cancer, colorectal cancer,
ovarian cancer, uterine cancer, melanoma, cervical cancer,
testicular cancer, esophageal cancer, gastric cancer, mesothelioma,
glioma, glioblastoma, pituitary adenomas, schizophrenia, obsessive
compulsive disorder (OCD), bipolar disorder, Alzheimer's disease,
Parkinson's disease, cell proliferative disorders, blood
coagulation disorders, Dysfibrinogenaemia and hemophilia (A and B),
autoimmune disorders, e.g., systemic lupus erythematosis, multiple
sclerosis, myasthenia gravis, autoimmune hemolytic anemia,
autoimmune thrombocytopenia, Grave's disease, allogenic transplant
rejection, ankylosing spondylitis, psoriasis, scleroderma, uveitis,
eczema, dermatological disorders, hyperlipidemia, hyperglycemia,
and hypercholesterolemia.
[0270] Cochleates of the instant invention can also be used to
promote greater health or quality of life, for example limit
cholesterol uptake or regulate lipid metabolism, weight gain,
hunger, aging, or growth. Cosmetic effects such as wrinkle
reduction, hair growth, pigmentation, or dermatologic disorders may
also be treated. Cochleates may also treat hereditary disease such
as cystic fibrosis or muscular dystrophy.
[0271] The cochleates of the instant invention can be used to treat
a variety of inflammations, including headache, arthritis,
rheumatoid arthritis, osteoarthritis, atherosclerosis, acute gout,
acute or chronic soft tissue damage associated with, e.g., a sports
injury, tennis elbow, bursitis, tendonitis, acute or chronic back
pain, such as a herniated disc, carpal tunnel syndrome,
glomerulonephritis, carditis, ulcerative colitis, asthma, sepsis,
and plantar fasciitis. The cochleates of the invention can also be
used to relieve pain resulting from surgery or other medical
procedure. The cochleates of the instant invention can further be
used to treat a variety of fungal infections, including candida,
e.g., yeast infection, tinea, e.g., Athlete's foot, pityriasis,
thrush, cryptococcal meningitis, histoplasmosis, and
blastomycosis.
[0272] The cochleates of the instant invention can also be used to
treat a variety of bacterial infections, including but not limited
to moderate to severe lower respiratory tract infections, skin
infections, biliary tract infections, bone infections, antibiotic
prophylaxis, pseudomembraneous enterocolitis, central nervous
system infections (e.g., meningitis and ventriculitis),
intra-abdominal infections (e.g., peritonitis), pneumonia,
septicemia, soft tissue infections, neutropaenic sepsis, joint
infections, infective endocartidis, and urinary tract
infections.
[0273] Exemplary bacteria that can be treated with the antibiotic
preparation of the present invention include, but are not limited
to, Staphylococcus aureus, Staphylococcus epidermidis,
Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus
Group D, Clostridium perfringens, Haemophilus influenzae,
Escherichia coli, Pseudomonas aeruginosa, and Klebsiella
pneumoniae.
[0274] The cochleate compositions of the invention are demonstrated
herein to effectively mediate the presence of bacteria such as
Pseudomona and Staphylococcus. One species, S. aureus, one of the
leading causes of hospital acquired infections, causes a wide
variety of suppurative diseases, including superficial and deep
abscesses, empyema, meningitis, purulent arthritis, and septicemia
and endocarditis. In addition, it causes two toxinoses: food
poisoning and exfolative skin disease.
[0275] Staphyloccoci found in infected tissues are mainly located
extracellularly. However, virulent staphylococci can survive within
leukocytes after phagocytosis and may protect themselves against
the bactericidal action of antibiotics by means of their
intracellular location. Intraphagocytic survival of S. aureus has
also been observed in patients with various disorders of phagocytic
functions. Certain infections caused by S. aureus have the tendency
to become recurrent, which is attributable to the intraphagocytic
survival of small numbers of the organism. Antibiotics that are
able to penetrate leukocytes have been shown to have superior
clinical efficacy in such recurrent and persistent staphylococcal
infections.
[0276] Because intracellular residence of infectious agents can
complicate treatment, a complete cure can require the eradication
of all intracellular bacteria. Therefore, a therapeutic approach
that increases the intracellular antibiotic concentration may
enhance the bactericidal killing and ensure complete elimination of
infection.
[0277] Pseudomonas aeruginosa infections occur in individuals with
altered host defenses, including burn patients, persons with
malignant or metabolic disease, or those who have had prior
instrumentation or manipulation. Prolonged treatment with
immunosuppressive or antimicrobial drugs and radiation therapy also
predispose individuals to Pseudomonas infections. P. aeruginosa is
a frequent cause of life-threatening infection, and is the most
common cause of nosocomial gram-negative pneumonia, with an
associated mortality rate of less than 60%. Among immunocompromised
patients, P. aeruginosa is a frequent cause of nosocomial
bacteremia. In cystic fibrosis, P. aeruginosa chronically colonizes
the lung, eventually causing respiratory failure and death.
[0278] In a preferred embodiment, antibacterial cochleates of the
present invention have the ability to reduce the number of
bacterial colonies by at least 10%. More preferably, antibacterial
cochleates can reduce the number of bacterial colonies by at least
25% and even more preferably by 50%, 75%, 85%, 95%, . . . 100%. All
individual values and ranges falling between these ranges and
values are within the scope of the present invention.
[0279] The present invention also provides a means for treating a
variety of fungal infections, including, but not limited to,
asthma, chronic rhinosinusitis, allergic fungal sinusitis, sinus
mycetoma, non-invasive fungus induced mucositis, non-invasive
fungus induced intestinal mucositis, chronic otitis media, chronic
colitis, inflammatory bowel diseases, ulcerative colitis, Crohn's
disease, candidemia, intraabdominal abscesses, peritonitis, pleural
space infections, esophageal candidiasis and invasive
aspergillosis. Exemplary fungi that can be treated using antifungal
cochleates of the invention include, without limitation, Absidia,
Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus,
Aspergillus nidulans, Aspergillus terreus, Aspergillus versicolor,
Alternaria, Basidiobolus, Bipolaris, Candida albicans, Candida
glabrata, Candida guilliermondii, Candida krusei, Candida
lypolytica, Candida parapsilosis, Candida tropicalis, Cladosporium,
Conidiobolus, Cunninahamella, Curvularia, Dreschlera, Exserohilum,
Fusarium, Malbranchia, Paecilonvces, Penicillium, Pseudallescheria,
Rhizopus, Schizophylum, Sporothrix, Acremonium, Arachniotus
citrinus, Aurobasidioum, Beauveria, Chaetomium, Chryosporium,
Epicoccum, Exophilia jeanselmei, Geotrichum, Oidiodendron, Phoma,
Pithomyces, Rhinocladiella, Rhodoturula, Sagrahamala,
Scolebasidium, Scopulariopsis, Ustilago, Trichodermia, and
Zygomycete.
[0280] Candida albicans is part of the normal microbial flora that
colonizes mucocutaneous surfaces of the oral cavity,
gastrointestinal tract, and vagina of many mammals and birds.
Because both antibody- and cell-mediated immune responses to
Candida antigens are evoked in healthy individuals, C. albicans
colonies are generally infectious for the host. C. albicans,
however, does not normally cause disease in immunocompetent
colonized hosts. It is the setting of congenital, induced, or
disease-related immune dysfunction that C. albicans causes
cutaneous, mucocutaneous, and life-threatening systemic
disease.
[0281] C. albicans is able to not only compete with other microbes
but also adhere to and survive on mucosal surfaces of a host with
Candida-specific antibody and cell-mediated immunity. Numerous
putative C. albicans virulence factors exist that may enable this
opportunistic fungus to survive and thrive in the adverse
conditions of host tissues. Among these putative virulence factors,
the cell wall of C. albicans is one of the most important. The cell
wall provides rigidity as well as protection against osmotic lysis,
and it promotes infection by supporting the interaction of C.
albicans adhesins and host-cell receptors. Also, the C. albicans
cell wall contains mannoproteins which have immunosuppressive
properties that can enhance the persistence of the fungus in
lesions. Echinocandins, unlike other antifungals, function by
interfering with the synthesis of the fungal cell wall.
[0282] In a preferred embodiment, echinocandin cochleates of the
present invention have the ability to reduce fungal colony forming
units (CFU's) by at least 10%. More preferably, echinocandin
cochleates can reduce CFU's by at least 25% and even more
preferably by 50%, 75%, 85%, 95%, . . . 100%. All individual values
and ranges falling between these ranges and values are within the
scope of the present invention. Reduction in colony forming units
may be in vivo or in vitro. The host of the fungal infection can be
a human or non-human animal.
[0283] Macrophages are important in the uptake of bacteria, fungi
and parasites, and also play an important role in the inflammatory
response. In addition to performing phagocytosis, macrophages have
the potential of being activated, a process that results in
increased cell size, increased levels of lysosomal enzymes, more
active metabolism, and greater ability to phagocytose and kill
ingested microbes. After activation, macrophages secrete a wide
variety of biologically active products that, if unchecked, result
in tissue injury and chronic inflammation. One of the secreted
products, nitric oxide (NO) has come into the forefront as a
mediator of inflammation.
[0284] Nitric oxide (NO) produced by inducible NOS plays an
important role in inflammation, killing of bacterial pathogens, and
tissue repair. NO formation increases during inflammation (i.e., in
rheumatoid arthritis, ulcerative colitis, and Crohns disease), and
several classic inflammatory symptoms, (i.e., erythema and vascular
weakness) are reversed by NOS inhibitors. Nitric oxide has also
been recognized as playing a versatile role in the immune system.
It is involved in the pathogenesis and control of infectious
diseases, tumors, autoimmune processes and chronic degenerative
diseases.
[0285] Aspirin and acetaminophen are used as anti-inflammatory
drugs to relieve pain and fever. The mechanism of action and side
effects of these drugs are explained in part by the generation of
NO from iNOS. Inhibition of iNOS expression and NO production,
therefore, could be a way to therapeutically decrease the
inflammatory actions of these drugs.
[0286] The above methods can be employed in the absence of other
treatment, or in combination with other treatments. Such treatments
can be started prior to, concurrent with, or after the
administration of the compositions of the instant invention.
Accordingly, the methods of the invention can further include the
step of administering a second treatment, such as for example, a
second treatment for the disease or disorder or to ameliorate side
effects of other treatments. Such second treatment can include,
e.g., radiation, chemotherapy, transfusion, operations (e.g.,
excision to remove tumors), and gene therapy. Additionally or
alternatively, further treatment can include administration of
drugs to further treat the disease or to treat a side effect of the
disease or other treatments (e.g., anti-nausea drugs).
[0287] With regard to both prophylactic and therapeutic methods of
treatment, such treatments may be specifically tailored or
modified, based on knowledge obtained from the field of
pharmacogenomics. "Pharmacogenomics", as used herein, refers to the
application of genomics technologies such as gene sequencing,
statistical genetics, and gene expression analysis to drugs in
clinical development and on the market.
[0288] More specifically, the term refers the study of how a
patient's genes determine his or her response to a drug (e.g., a
patient's "drug response phenotype", or "drug response genotype").
Thus, another aspect of the invention provides methods for
tailoring an individual's prophylactic or therapeutic treatment
according to that individual's drug response genotype.
Pharmacogenomics allows a clinician or physician to target
prophylactic or therapeutic treatments to patients who will most
benefit from the treatment and to avoid treatment of patients who
will experience toxic drug-related side effects.
[0289] The language "therapeutically effective amount" is that
amount necessary or sufficient to produce the desired physiologic
response. The effective amount may vary depending on such factors,
as the size and weight of the subject, or the particular compound.
The effective amount may be determined through consideration of the
toxicity and therapeutic efficacy of the compounds by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it may be
expressed as the ratio LD.sub.50/ED.sub.50. Compounds which exhibit
large therapeutic indices are preferred. While compounds that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that targets such compounds to the site of
affected tissue in order to minimize potential damage to unaffected
cells and, thereby, reduce side effects.
[0290] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any composition used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
EC50 (i.e., the concentration of the test composition that achieves
a half-maximal response) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
[0291] 1. Prophylactic Methods
[0292] In one aspect, the invention provides a method for
preventing in a subject, a disease or disorder which can be treated
with at least one cargo moiety, e.g., a protein, a small peptide,
an antiviral, an anesthetic, an anti-infectious, an antifungal, an
anticancer, an immunosuppressant, a steroidal anti-inflammatory, a
non-steroidal anti-inflammatory, a tranquilizer, a mucolytic agent,
a dilator, a vasoconstrictor, a decongestant, a leukotriene
inhibitor, an anti-cholinergic, an anti-histamine or a vasodilatory
agent. Subjects at risk for a disease or condition which can be
treated with the agents mentioned herein can be identified by, for
example, any or a combination of diagnostic or prognostic assays
known to those skilled in the art. Administration of a prophylactic
agent can occur prior to the manifestation of symptoms
characteristic of the disease or disorder, such that the disease or
disorder is prevented or, alternatively, delayed in its
progression. Amphotericin B cochleates, for example, have been
administered prophylactically in mice, and were at least as
efficacious, if not more efficacious, than Amphotericin B
deoxycholate.
[0293] 2. Therapeutic Methods
[0294] Another aspect of the invention pertains to methods of
administering a cochleate composition for therapeutic purposes. In
one embodiment, the present invention provides a method for
treating a subject that would benefit from administration of a
composition of the present invention. Any therapeutic indication
that would benefit from a cochleate composition of the invention
can be treated by the methods of the invention. The present
invention provides methods of treating a subject at risk for or
having a disease or disorder that can be treated with one ore more
cargo moiety. The method includes the step of administering to the
subject a composition of the invention, such that the disease or
disorder is prevented, ameliorated, terminated or delayed in its
progression. The disease or disorder can be any of the diseases or
disorders discussed herein.
[0295] The compositions of the invention can be administered to a
subject alone or in combination with a second therapy as described
above. The compositions of the invention can be administered to a
subject prior to, at the same time, or after a second therapy is
administered.
[0296] Therapeutic agents can be tested in an appropriate animal
model. For example, cochleate compositions of the present invention
can be used in an animal model to determine the efficacy, toxicity,
or side effects of treatment with said agent. Alternatively, a
therapeutic agent can be used in an animal model to determine the
mechanism of action of such an agent. For example, an agent can be
used in an animal model to determine the efficacy, toxicity, or
side effects of treatment with such an agent. Alternatively, an
agent can be used in an animal model to determine the mechanism of
action of such an agent.
[0297] Pharmaceutical Compositions
[0298] The invention pertains to uses of the cochleate compositions
of the invention for prophylactic and therapeutic treatments as
described infra. Accordingly, the compounds of the present
invention can be incorporated into pharmaceutical compositions
suitable for administration. Such compositions typically comprise
the compositions of the invention and a pharmaceutically acceptable
carrier. As used herein the language "pharmaceutically acceptable
carrier" is intended to include any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances is well known in the art.
[0299] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, sweetening, flavoring and perfuming agents,
preservatives and antioxidants may also be present in the
compositions.
[0300] Examples of pharmaceutically acceptable antioxidants, which
may also be present in formulations of therapeutic compounds of the
invention, include water soluble antioxidants, such as ascorbic
acid, cysteine hydrochloride, sodium bisulfate, sodium
metabisulfite, sodium sulfite and the like; oil-soluble
antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole
(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,
alpha-tocopherol, and the like; and metal chelating agents, such as
citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,
tartaric acid, phosphoric acid, and the like.
[0301] Furthermore, the present invention can further include one
or more additional agents, including water, antimicrobial agents,
plasticizing agents, flavoring agents, surfactants, stabilizing
agents, emulsifying agents, thickening agents, binding agents,
coloring agents, sweeteners, fragrances, and the like.
[0302] Suitable antimicrobial agents include triclosan, cetyl
pyridium chloride, domiphen bromide, quaternary ammonium salts,
zinc compounds, sanguinarine, fluorides, alexidine, octonidine,
EDTA, and essential oils such as thymol, methyl salicylate, menthol
and eucalyptol.
[0303] Suitable plasticizing agents include, for example, polyols
such as sugars, sugar alcohols, or polyethylene glycols (PEGs),
urea; glycol, propylene glycol, triethyl citrate, dibutyl or
dimethyl phthalate, monoacetin, diacetin or triacetin.
[0304] Suitable surfactants include pluronic acid, sodium lauryl
sulfate, mono and diglycerides of fatty acids and polyoxyethylene
sorbitol esters, such as, Atmos 300 and polysorbate 80. Suitable
stabilizing agents include xanthan gum, locust bean gum, guar gum,
and carrageenan. Suitable emulsifying agents include
triethanolamine stearate, quaternary ammonium compounds, acacia,
gelatin, lecithin, bentonite, veegum, and the like. Suitable
thickening agents include methylcellulose, carboxyl
methylcellulose, and the like. Suitable binding agents include
starch.
[0305] Suitable sweeteners that can be included are those well
known in the art, including both natural and artificial sweeteners.
Suitable sweeteners include water-soluble sweetening agents such as
monosaccharides, disaccharides and polysaccharides; water-soluble
artificial sweeteners such as soluble saccharin salts, cyclamate
salts, or the free acid form of saccharin, and the like; dipeptide
based sweeteners, such as L-aspartic acid derived sweeteners;
water-soluble sweeteners derived from naturally occurring
water-soluble sweeteners, such as a chlorinated derivative of
ordinary sugar (sucrose), known, under the product description of
sucralose; and protein based sweeteners such as thaumatoccous
danielli (Thaumatin I and II).
[0306] In general, an effective amount of auxiliary sweetener is
utilized to provide the level of sweetness desired for a particular
composition, and this amount will vary with the sweetener selected.
This amount will normally be 0.01% to about 10% by weight of the
composition when using an easily extractable sweetener.
[0307] The flavorings that can be used include those known to the
skilled artisan, such as natural and artificial flavors. These
flavorings may be chosen from synthetic flavor oils and flavoring
aromatics, and/or oils, oleo resins and extracts derived from
plants, leaves, flowers, fruits and so forth, and combinations
thereof. Representative flavor oils include: spearmint oil,
cinnamon oil, peppermint oil, clove oil, bay oil, thyme oil, cedar
leaf oil, oil of nutmeg, oil of sage, and oil of bitter almonds.
Also useful are artificial, natural or synthetic fruit flavors such
as vanilla, chocolate, coffee, cocoa and citrus oil, and fruit
essences. These flavorings can be used individually or in
admixture. Flavorings such as aldehydes and esters including
cinnamyl acetate, cinnamaldehyde, citral, diethylacetal,
dihydrocarvyl acetate, eugenyl formate, p-methylanisole, and so
forth may also be used. Generally, any flavoring or food additive,
such as those described in Chemicals Used in Food Processing,
publication 1274 by the National Academy of Sciences, pages 63-258,
may be used.
[0308] The amount of flavoring employed is normally a matter of
preference subject to such factors as flavor type, individual
flavor, and strength desired. Thus, the amount may be varied in
order to obtain the result desired in the final product. Such
variations are within the capabilities of those skilled in the art
without the need for undue experimentation.
[0309] The compositions of this invention can also contain coloring
agents or colorants. The coloring agents are used in amounts
effective to produce the desired color. The coloring agents useful
in the present invention include pigments such as titanium dioxide,
which may be incorporated in amounts of up to about 5 wt %, and
preferably less than about 1 wt %. Colorants can also include
natural food colors and dyes suitable for food, drug and cosmetic
applications. These colorants are known as FD&C dyes and lakes.
A full recitation of all FD&C and D&C dyes and their
corresponding chemical structures may be found in the Kirk-Othmer
Encyclopedia of Chemical Technology, Volume 5, Pages 857-884, which
text is accordingly incorporated herein by reference.
[0310] Formulations of the present invention include those suitable
for oral, nasal, topical, transdermal, buccal, sublingual, rectal,
vaginal or parenteral administration. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any methods well known in the art of pharmacy. The amount of
active ingredient which may be combined with a carrier material to
produce a single dosage form will generally be that amount of the
composition which produces a therapeutic effect. Generally, out of
100%, this amount will range from about 1% to about 99% of active
ingredient, preferably from about 5% to about 70%, most preferably
from about 10% to about 30%.
[0311] Methods of preparing these formulations or compositions
include the step of bringing into association a composition of the
present invention with the carrier and, optionally, one or more
accessory ingredients. In general, the formulations are prepared by
uniformly and intimately bringing into association a composition of
the present invention with liquid carriers, or finely divided solid
carriers, or both, and then, if necessary, shaping the product.
[0312] Formulations of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, gelcaps, crystalline substances, lozenges (using a
flavored basis, usually sucrose and acacia or tragacanth), powders,
granules, gel, partial liquid, spray, nebulae, mist, atomized
vapor, aerosol, tincture, or as a solution or a suspension in an
aqueous or non-aqueous liquid, or as an oil-in-water or
water-in-oil liquid emulsion, or as an elixir or syrup, or as
pastilles (using an inert base, such as gelatin and glycerin, or
sucrose and acacia) or as mouth washes and the like, each.
containing a predetermined amount of a composition of the present
invention as an active ingredient. A composition of the present
invention may also be administered as a bolus, electuary, or
paste.
[0313] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules, and the like), the active ingredient is mixed with one or
more pharmaceutically acceptable carriers, such as sodium citrate
or dicalcium phosphate, or any of the following: fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
or silicic acid; binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose or acacia; humectants, such as glycerol; disintegrating
agents, such as agar-agar, calcium carbonate, potato or tapioca
starch, alginic acid, certain silicates, and sodium carbonate;
solution retarding agents, such as paraffin; absorption
accelerators, such as quaternary ammonium compounds; wetting
agents, such as, for example, cetyl alcohol and glycerol
monostearate; absorbents, such as kaolin and bentonite clay;
lubricants, such a talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof; and coloring agents.
[0314] In the case of capsules, tablets and pills, the
pharmaceutical compositions may also comprise buffering agents.
Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugars, as well as high molecular
weight polyethylene glycols and the like.
[0315] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered composition moistened with an inert liquid
diluent.
[0316] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes or
microspheres.
[0317] They may be sterilized by, for example, filtration through a
bacteria-retaining filter, or by incorporating sterilizing agents
in the form of sterile solid compositions which may be dissolved in
sterile water, or some other sterile injectable medium immediately
before use.
[0318] These compositions may also optionally contain opacifying
agents and may be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain portion of the
gastrointestinal tract, optionally, in a delayed manner. Examples
of embedding compositions which may be used include polymeric
substances and waxes. The active ingredient may also be in
micro-encapsulated form, if appropriate, with one or more of the
above-described excipients.
[0319] Liquid dosage forms for oral administration of the compounds
of the invention include pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert dilutents commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof. Besides inert
dilutents, the oral compositions may also include adjuvants such as
wetting agents, emulsifying and suspending agents, sweetening,
flavoring, coloring, perfuming and preservative agents.
[0320] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0321] Formulations of the pharmaceutical compositions of the
invention for rectal or vaginal administration may be presented in
liquid or aerosol form, or as a suppository, which may be prepared
by mixing one or more compounds of the invention with one or more
suitable nonirritating excipients or carriers comprising, for
example, cocoa butter, polyethylene glycol, a suppository wax or a
salicylate, and which is solid at room temperature, but liquid at
body temperature and, therefore, will melt in the rectum or vaginal
cavity and release the active compound. Liquid or aerosol forms
include, but are not limited to, gels, pastes, ointments, salves,
creams, solutions, suspensions, partial liquids, sprays, nebulaes,
mists, atomized vapors, and tinctures. Formulations of the present
invention which are suitable for vaginal administration also
include pessaries, tampons, creams, gels, pastes, foams or spray
formulations containing such carriers as are known in the art to be
appropriate.
[0322] Formulations of the pharmaceutical compositions of the
invention for nasal administration can be in solid, liquid, or
aerosol form (e.g., powder, crystalline substance, gel, paste,
ointment, salve, cream, solution, suspension, partial liquid,
spray, nebulae, irrigant, wash, mist, atomized vapor or
tincture).
[0323] Dosage forms for the topical or transdermal administration
of a composition of this invention include powders, sprays,
ointments, pastes, creams, lotions, gels, solutions, patches and
inhalants. The composition may be mixed under sterile conditions
with a pharmaceutically acceptable carrier, and with any
preservatives, buffers, or propellants which may be required.
[0324] The ointments, pastes, creams and gels may contain, in
addition to an composition of this invention, excipients, such as
animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and zinc oxide, or mixtures
thereof.
[0325] Powders and sprays may contain, in addition to a composition
of this invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates and polyamide powder, or
mixtures of these substances. Sprays may additionally contain
customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0326] Transdermal patches have the added advantage of providing
controlled delivery of a composition of the present invention to
the body. Such dosage forms may be made by dissolving or dispersing
the composition in the proper medium. Absorption enhancers may also
be used to increase the flux of the composition across the skin.
The rate of such flux may be controlled by either providing a rate
controlling membrane or dispersing the composition in a polymer
matrix or gel.
[0327] Ophthalmic formulations, eye ointments, powders, solutions
and the like are also within the scope of this invention.
[0328] Pharmaceutical compositions of this invention suitable for
parenteral administration comprise a cochleate or cochleate
composition of the invention in combination with one or more
pharmaceutically acceptable sterile isotonic aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions, or sterile
powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
antioxidants, buffers, bacteriostats, solutes which render the
formulation isotonic with the blood of the intended recipient or
suspending or thickening agents.
[0329] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity may be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0330] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents which delay
absorption such as aluminum monostearate and gelatin.
[0331] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0332] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, and sodium chloride, in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0333] Sterile injectable solutions can be prepared by
incorporating a composition of the invention in the desired amount
in an appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the
composition into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the cochleate compositions of the invention plus any
additional desired ingredient from a previously sterile-filtered
solution thereof.
[0334] Injectable depot forms are made by forming microencapsule
matrices of the subject compounds in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release may be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions which are
compatible with body tissue.
[0335] Oral compositions generally include an inert diluent or an
edible carrier. Thev can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the composition can be incorporated with excipients
and used in the form of tablets, troches, or capsules. Oral
compositions can also be prepared using a fluid carrier for use as
a mouthwash, wherein the composition in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0336] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0337] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0338] The compositions of the invention also can be prepared in
the form of suppositories (e.g., with conventional suppository
bases such as cocoa butter and other glycerides) or retention
enemas for rectal delivery.
[0339] In one embodiment, the compositions of the invention are
prepared with carriers that will protect the composition against
rapid elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811.
[0340] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of a composition calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the composition and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such a composition for the treatment of
individuals.
[0341] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0342] The pharmaceutical compositions can be included in a
container along with one or more additional compounds or
compositions and instructions for use. For example, the invention
also provides for packaged pharmaceutical products containing two
agents, each of which exerts a therapeutic effect when administered
to a subject in need thereof. A pharmaceutical composition may also
comprise a third agent, or even more agents yet, wherein the third
(and fourth, etc.) agent can be another agent against the disorder,
such as a cancer treatment (e.g., an anticancer drug and/or
chemotherapy) or an HIV cocktail. In some cases, the individual
agents may be packaged in separate containers for sale or delivery
to the consumer. The agents of the invention may be supplied in a
solution with an appropriate solvent or in a solvent-free form
(e.g., lyophilized). Additional components may include acids,
bases, buffering agents, inorganic salts, solvents, antioxidants,
preservatives, or metal chelators. The additional kit components
are present as pure compositions, or as aqueous or organic
solutions that incorporate one or more additional kit components.
Any or all of the kit components optionally further comprise
buffers.
[0343] The present invention also includes packaged pharmaceutical
products containing a first agent in combination with (e.g.,
intermixed with) a second agent. The invention also includes a
pharmaceutical product comprising a first agent packaged with
instructions for using the first agent in the presence of a second
agent of instructions for use of the first agent in a method of the
invention. The invention also includes a pharmaceutical product
comprising a second or additional agents packaged with instructions
for using the second or additional agents in the presence of a
first agent or instructions for use of the second or additional
agents in a method of the invention. Alternatively, the packaged
pharmaceutical product may contain at least one of the agents and
the product may be promoted for use with a second agent.
[0344] In yet another aspect, the invention provides an article of
manufacture of cochleates and/or cochleate compositions of the
invention (FIG. 29). The article of manufacture includes packaging
material and a lipid contained within the packaging material. The
packaging material includes a label or package insert indicating
the use of the lipid for forming cochleates or cochleate
compositions of the invention. The article of manufacture can
further include instructions or guidelines for the formation of
cochleates or cochleate compositions of the invention, e.g., mixing
a cargo moiety with a solvent and dripping it into a solution of
the lipids. Optionally, the article of manufacture can include a
solvent, a cargo moiety, a multivalent cation (e.g., calcium and/or
magnesium), a control cargo moiety, and/or a chelating agent (e.g.,
EDTA).
[0345] The article of manufacture may further include other
ingredients or apparatus that can be employed to manufacture the
compositions of the present invention. One non-limiting example of
an article of manufacture would include 5g of powdered Soy PS, a
solution of a model hydrophobic compound in DMSO as a positive
control, a solution of calcium chloride to induce cochleate
formation, and a solution of EDTA to visualize the opening of the
cochleates into liposomes.
[0346] The instructions and/or guidelines may generally include one
or more of the following statements:
[0347] 1. Prepare a liposomal suspension by vigorously mixing lipid
in water or buffer.
[0348] 2. Monitor lipid concentrations: low concentration would
require a large volume of buffer in order to formulate an adequate
amount of end product and high concentration may produce large
cochleate aggregates upon the addition of calcium.
[0349] 3. Experimentally determine whether to use water or buffered
solution: the presence of salts and the pH of the suspension may
affect the formation of the cargo moiety-liposome intermediate
depending on the properties of the cargo moiety.
[0350] 4. Optionally filter or perform other standard procedures to
prepare liposomes of a defined size and/or to sterilize the
suspension.
[0351] 5. Prepare a cargo moiety solution with an appropriate
solvent: many solvents may potentially be used in this process,
e.g., DMSO.
[0352] 6. Add the cargo moiety-solvent solution, preferably
dropwise, to the liposome suspension with vigorous mixing.
[0353] 7. Experimentally determine the optimal rate of addition and
speed of mixing: a suspension of cargo moiety-liposomes essentially
free of unencochleated cargo moiety when viewed by light microscopy
should be produced.
[0354] 8. Calculate the amount of calcium to be added by assuming
one mole of calcium for every two moles of lipid, and adding extra
calcium to bring the buffer to between 2 and 6 mM.
[0355] 9. Induce cochleate formation through addition of a calcium
salt. The salt may be added as a solution, e.g., 0.1 M calcium
chloride, or may be slowly added as a solid calcium salt, e.g.,
calcium chloride, with vigorous mixing.
[0356] 10. If the presence of solvent in the buffer is unwanted,
optionally harvest the cargo moiety-cochleates, e.g., by
centrifugation or filtration, and resuspending them in an
appropriate medium. The association of calcium ions with PS is
easily reversible, therefore, in order to remain intact and in
their crystalline state, cochleate formulations can be resuspended
in a medium containing at least 1 to 2 mM calcium ions.
[0357] 11. Optionally evaluate the quality of the cochleate
formulation. The presence of sufficient calcium ions initiates and
maintains the cochleate structure. One method of evaluating the
quality of a cochleate formulation is visualization of the
liposomes that are produced upon removal of the calcium ions from a
cochleate crystal. This may be accomplished using light microscopy.
An aliquot of a cargo moiety-cochleate suspension may be visualized
by phase contrast microscopy at 1000.times. magnification. A small
amount of a concentrated solution of a chelating agent, e.g., EDTA,
may be added to the edge of the cover slip, thus reaching the
sample through capillary action. A high-quality cochleate product
will open into intact liposomes upon contact with the
calcium-chelating agent. When using EDTA as the chelating agent,
the pH of the EDTA solution should be about pH 9.5. Cochleates will
not convert to liposomes at a pH below 6.5. If EDTA solutions at pH
7.4 are used, the release of hydrogen ions upon the binding of
calcium to the acetate groups of the chelating agent lowers the pH
of the solution and inhibits cochleate conversion to liposomes.
[0358] Choice of solvent and other materials, optimal rate of
dropwise addition, speed of mixing the amount of calcium, etc., can
readily be determined by the skilled practitioner employing the
teachings provided herein.
[0359] In addition, a skilled practitioner can introduce, modify
and/or eliminate elements and/or steps to the above without
departing from the scope of the invention. For example, a liposome
suspension might be provided already prepared, a combination of
solvents might be used, excess calcium might be used to obviate the
calculation of calcium, alternative or additional cations might be
employed, etc.
[0360] Practice of the invention will be still more fully
understood from the following examples, which are presented herein
for illustration only and should not be construed as limiting in
any way.
Exemplification
[0361] Materials and Methods
[0362] Materials
[0363] The following materials were used, unless otherwise
indicated: powdered Amphotericin B (AmB) U.S. Pharmacopela grade
was obtained from USP (Rockville, Md.) and Alpharma (Copenhagen,
Denmark), and stored at 4.degree. C. powdered Soy PS was obtained
from American Lethicin Corporation (Oxford, Conn.) and Degussa
(Champaign, Ill.) and stored at room temperature; Vitamin E (V-E)
was obtained from Roche (Parsippany, N.J.); sterile water was
obtained from Baxter (Canada); Dioleoylphosphatidyl serine (DOPS)
was obtained from Avanti Polar Lipid (Alabaster, Ala.), methyl
sulfoxide (DMSO) HPLC grade was obtained from Aldrich (Milwaukee,
Wis.), and micellar AmB/deoxycholate suspension sold under the
trademark FUNGIZONE was obtained from Sigma (St. Louis, Mo.).
[0364] General Method for Forming Cochleates with a Cargo
Moiety
[0365] Lipid powder (soy PS or synthetic PS) is dispersed in water
(pure water or saline) by vortexing, resulting mixture of
unilamellar and multilamellar liposomes. The liposomal suspension
is filtered to obtain a suspension having a majority of unilamellar
liposomes. To this liposome suspension, a water miscible organic
solvent (e.g., DMSO) including a cargo moiety (and optional
antioxidant) is introduced. The liposomal suspension is
precipitated with cation. The solvent may be removed from the
liposomal suspension by tangential flow and/or filtration and/or
dialysis, or from the cochleates by washing, filtration,
centrifugation, tangential flow, and/or dialysis.
[0366] Cell Lines and Culture Conditions
[0367] Mouse macrophage J774A.1 cell line and ovarian cancer cell
line SKOV3 cell line were obtained from ATCC and PPD, respectively.
The cells were grown in monolayers in humidified air with
5%CO.sub.2 at 37.degree. C. in 60 mm.sup.2 Petri dishes (Corning)
containing 5 mL of DMEM supplemented with 10 % FBS. For
experiments, cells were harvested by scraping (J774A.1) or
trypsinization (SKOV3), and were seeded into 24 or 96-well plates
at a density of 5.times.10.sup.5 cells.
[0368] Staphylococcal aureus (ATCC 29213) and Pseudomonas
aeruginosa (ATCC 700289) were maintained weekly on Nutrient agar
plates and slants. Fresh cultures were grown up to 24 hours prior
to experiment.
[0369] Imaging
[0370] Phase contrast light microscopy and confocal microscopy
(Olympus) was used to image liposomal suspensions, cochleates and
cells, with and without the aid of fluorescence, which can be used,
e.g., to study cellular uptake and intracellular distribution of
fluorescently labeled cochleates and cargo moieties. Confocal
microscopy is particularly advantageous as it is a 3-dimensional
digital imaging device that can be used to effectively view slices
of cell culture. This allows verification of the presence of
cochleate and other agents within a cell.
[0371] Particle Size Analysis
[0372] Two different devices were used to examine particle size.
The N4 plus (Coulter) measures particles in the range 10 nm to 3000
nm. The LS230 (Beckman/Coulter) measures particles in the range 40
nm to 1 mm. Using the two devices provides the flexibility and
capability to evaluate formulations ranging from nanocochleates to
large aggregates of cochleates.
[0373] Fraunhofer was used as the optical model for all the
experiments. The optical models used to calculate absolute particle
size were for spherical particles. Since cochleates are not
spherical, the numbers given are relative, not absolute values, but
nonetheless allow batch to batch sample comparisons. The results
obtained by the two different devices give a qualitative comparison
of the size differences between the formulations. However, light
and electron microscopy have confirmed that the "nanocochleates"
obtained are submicron in size.
EXAMPLE 1
Amphotericin B Cochleate (CAMB) Prepared with DMSO and Lipid:AmB
Ratios of 10:1, 2:1 and 1:1 w/w
[0374] Amphotericin B cochleates (CAMB) were prepared with Soy PS
and DMSO with Vitamin E, and a Lipid to AmB ratio of 10:1 as
follows.
[0375] Preparation of Liposomes
[0376] 20 ml of water was added to 200 mg of Soy-PS in a 50 ml
plastic tube, vortexed for about 15 minutes to form a liposomal
suspension, and filtered using a 0.45 .mu.m filter. The suspension
was sonicated for about 4 minutes and filtered again with a 0.22
.mu.m filter.
[0377] Addition of Cargo Moiety and Antioxidant in Solvent
[0378] 1.90 ml of DMSO solvent was added to 20 mg of Amphotericin B
in a 15 ml plastic tube. To the AmB/DMSO mixture, 0.1 ml of Vitamin
E (20 mg/ml in DMSO), vortexed for about 10 minutes. This solution
was then added to the liposomal suspension by drop wise addition
using a 1 ml pipette while vortexing. The final mixture was
vortexed for about 2 minutes.
[0379] Precipitation of Cochleates
[0380] 2 ml of calcium (0.1 N) was added to the liposomal
suspension at a rate of 10 .mu.l/10s while vortexing to form
cochleates.
[0381] Solvent Removal Washing
[0382] The mixture was vortexed for about 1-2 minutes, centrifuged
for about 1 hour at 9000 rpm, and the supernatant was removed and
replaced with fresh supernatant (water with 2 mM calcium). This
washing step was repeated once.
[0383] Employing the above method, cochleates having a lipid to
drug ratio 2:1 and 1:1 also were prepared by varying the
ingredients to conform to following formulations.
1TABLE 1 Cochleate Formulations CAMB 1:1 CAMB 2:1 Soy PS (mg) 100
200 AmB (mg) 100 100 Vitamin E (mg) 1 2 DMSO (ml) 2 2 Water (ml) 10
20 Calcium (ml) 1 2 Washings with sterile water w/ 2 2 calcium (1
mM) Final Volume (ml) 10 20
[0384] Summary of Results
[0385] For each cochleate formulation, a yellowish suspension with
some of the cochleates floating on the top and/or residing on the
bottom of the suspension were observed macroscopically.
[0386] FIG. 4 is a series of images of the formulation having a 1:1
ratio at different stages in the formulation: liposomes, liposomes
with AmB, cochleates, and cochleates after addition of EDTA. FIGS.
5, 6 and 7 are each a series of images, before and after addition
of EDTA, of the cochleate formulations having a 10:1, 2:1, and 1:1
ratio, respectively.
[0387] FIG. 8 is a graph of the size distribution of the liposomes
after vortexing and prior to filtration, after filtration with 0.45
.mu.m filter, and after introducing DMSO/Amphotericin. FIG. 9 is a
graph of the size distribution of cochleate formulations having
lipid to AmB ratios of 10:1, 2:1 and 1:1.
EXAMPLE 2
Amphotericin B Cochleates Prepared with NMP
[0388] Cochleates were prepared as described in Example 1, except
N-methylpyrrolidone (NMP) solvent was used instead of DMSO, and the
formulation was adjusted as indicated in the following table.
2TABLE 2 NMP 10:1 Formulation CAMB/NMP 10:1 Soy PS (mg) 200 AmB
(mg) 20 Vitamin E (mg) 2 DMSO (Ml) 2 Water (ml) 20 Calcium (ml) 2
Washings with sterile water w/ 2 calcium (1 mM) Final Volume 20
ml
[0389] Cochleates in the final formulations were observed as a
yellowish suspension. Mice infected with a lethal dose of
Aspergillus fumigatus were dosed with 2 mg/kg of the AmB-cochleate
formulation for 14 days. The cochleates were efficacious against
the A. fumigatus.
Example 3
Amphotericin B Cochleates Prepared with DMSO and Lipid:AmB Ratios
of 5:1, 4:1, 3:1 and 2:1 w/w
[0390] Amphotericin B cochleates (CAMB) were prepared with soy PS
and DMSO with tocopherol (Vitamin E) with the following
protocol:
[0391] 1. Weighing and placing 300 mg of soy PS into a 50 ml pp
sterile tube with 10 ml sterile water.
[0392] 2. Vortexing the suspension for 2 minutes.
[0393] 3. Sonicating the suspension for 3 minutes.
[0394] 4. Filtering the suspension with a 0.22 .mu.m filter and
pooling liposomes into a 50 ml tube.
[0395] 5. Weighing and placing 10 mg (5:1), 12.5 mg (4:1) 16.6 mg
(3:1) and 25 mg (2:1), of AmB (individually) into four 15 ml pp
sterile tubes with 0.5 ml DMSO and vortexing.
[0396] 6. Adding 6.0 .mu.l (5:1), 6.2 .mu.l (4:1), 6.6 .mu.l (3:1),
and 7.5 .mu.l (2:1) of tocopherol at 10 mg/ml in DMSO to the 15 ml
tubes with AmB. (The concentration of the AmB was 20 mg/ml (5:1),
25 mg/ml (4:1), 33.2 mg/ml (3:1), and 50 mg/ml (2:1), at this
time)
[0397] 7. Vortexing the solution for a few minutes until the AmB
completely dissolved.
[0398] 8. Adding 5 ml of liposomes to each AmB/Vitamin E/DMSO
solution, and vortexing the sample for a few minutes.
[0399] 9. Adding 0.5 ml of 0.1M calcium solution into the
suspension with vortexing, using an eppendorf repeater pipette with
a 500 .mu.l tip and adding 10 .mu.l aliquots to the tube per every
10 sec.
[0400] 10. Centrifuging the suspension for 30 minutes at 9000 rpm
at 4.degree. C.
[0401] 11. Removing the supernatant from the tube and re-suspending
the pellet with the same volume of wash buffer (sterile water with
2 mM calcium).
[0402] 12. Repeating steps 10 and 11 two more times. Adjusting the
final volume of the suspension to 6 ml with wash buffer.
[0403] 13. Examining the final preparation under a microscope and
confirming the pH of 5.5.
[0404] 14. Streaking the sample on a chocolate plate to check the
sterilization of the final preparation and incubating plates at
37.degree. C., 4.degree. C., and room temperature for 24 hrs, 48
hrs, and 72 hrs.
[0405] 15. Storing the sample, treated with nitrogen and covered
with parafilm, at 4.degree. C.
[0406] The following table summarizes the above formulations.
3TABLE 3 CAMB Preparations Name of PS:AmB AmB 0.1M Final AmB Sample
(w/w) AmB Soy PS V-E liposome Calcium (mg/ml) CAMB 5:1 5:1 10 mg 50
mg 60 .mu.g 5.5 ml 0.5 ml 1.66 mg/ml CAMB 4:1 4:1 12.5 mg 50 mg
62.5 .mu.g 5.5 ml 0.5 ml 2.08 mg/ml CAMB 3:1 3:1 16.6 mg 50 mg 66.6
.mu.g 5.5 ml 0.5 ml 2.76 mg/ml CAMB 2:1 2:1 25 mg 50 mg 75 .mu.g
5.5 ml 0.5 ml 4.16 mg/ml
[0407] About 0.5 ml DMSO was used in each preparation. HPLC and
LC/MS were used to measure the AmB and DMSO concentrations.
[0408] Summary of Results
[0409] 1. Macroscopic observations: yellowish suspension.
[0410] 2. Microscopic observations: cochleates with different size
of aggregates.
[0411] 3. Addition of EDTA: liposomes formed after addition of EDTA
(chelator).
[0412] 4. Recovery: HPLC analysis indicated that approximately 100%
of the AMB was successfully encochleated.
[0413] 5. For the mouse study described in Example 4, the following
amounts of each formulation were set aside:
[0414] 5:1=>0.4 mg/ml.times.1.2 ml, 14 bottles
[0415] 4:1=>0.4 mg/ml.times.1.2 ml, 14 bottles
[0416] 3:1=>0.4 mg/ml.times.1.2 ml, 14 bottles
[0417] 2:1=>0.4 mg/ml.times.1.2 ml 14 bottles
[0418] For the in vitro study described in Example 5, the following
amounts of each formulation were set aside:
[0419] 5:1=>0.34 mg/ml.times.100 .mu.l (Conc: PS=1.7 mg/ml)
[0420] 4:1=>0.42 mg/ml.times.100 .mu.l (Conc: PS=1.7 mg/ml)
[0421] 3:1=>0.56 mg/ml.times.100 .mu.l (Conc: PS=1.7 mg/ml)
[0422] 2:1=>0.85 mg/ml.times.100 .mu.l (Conc: PS=1.7 mg/ml)
[0423] 6. Sterility: No bacteria observed in the formulations after
72 hrs.
EXAMPLE 4
Efficacy Studies in Mice
[0424] The formulations of Example 3 were administered to mice to
study the efficacy of the formulations to protect mice from a
lethal dose of Candida albicans, and to clear the organs of C.
albicans in the surviving mice.
[0425] Six groups of 10 mice were studied. The mice were
administered 5.times.10.sup.5 cells C. albicans intravenously
through the tail vein. Starting 24 hours post-infection, the
following compositions were administered to each group orally once
daily at 2 mg AmB/kg body weight for 14 days, except for the
control group which remained untreated.
[0426] a. Control (untreated)
[0427] b. AmB/deoxycholate (FUNGIZONE)
[0428] c. CAMB 2:1 AmB
[0429] d. CAMB 3:1 AmB
[0430] e. CAMB 4:1 AmB
[0431] f. CAMB 5:1 AmB
[0432] Appearance and behavior was monitored each day of the study.
Tissue burden of C. Albicans was determined in kidney, liver and
lungs for each animal, and colony counts were taken. Organs were
obtained and weighed, homogenized, dilutions in buffer made, and
aliquots plated onto plates; colony counts of fungus were taken
several days later. FIG. 10 is a graph of the survival data for C.
albicans-infected mice untreated (control), or dosed daily for 14
day with AmB/deoxycholate, or AmB-cocbleates with a lipid to drug
ratio of 2:1, 3:1, 4:1, or 5:1.
[0433] FIG. 11 is a chart of the average number of C. albicans
cells/gram of tissue in the liver, kidney, and lungs of C.
albicans-infected mice untreated and dosed daily for 14 days with
AmB/deoxycholate, or AmB-cochleates with a lipid to drug ratio of
2:1, 3:1, 4:1, or 5:1.
[0434] One hundred percent of the control (untreated) group did not
survive the study and showed high tissue burdens. All four
AmB-cochleate (CAMB) formulations were effective in preventing
mortality and reducing fungal cell burdens in target organs
(kidneys, lungs and liver). The CAMB 5:1 and CAMB 3:1 formulation
appeared somewhat better than the others clearing the liver and
lungs completely (the principal target organ for C. albicans
(kidney) was not completely cleared). The CAMB 5:1 formulation
appeared to be the most effective versus the others in reducing
fungal cell burdens in the kidneys. In general, the CAMB
formulations were more effective than the AmB/deoxycholate
formulation.
EXAMPLE 5
Efficacy Studies in Cells
[0435] The relative efficacy of the compositions of Example 3 were
studied in J774A.1 macrophages to compare the relative efficacy of
the cochleate compositions (5:1, 4:1, 3:1 and 2:1) against Candida
albicans.
[0436] Macrophages were seeded into a 96-well plate and incubated
overnight as described above. Following incubation, the macrophages
were infected with C. albicans at a ratio of 1:200 with respect to
the macrophages. The AmB-cochleates were then added at the
concentrations of 0.1, 0.01 and 0.001 .mu.g. Twenty-four hours
later, the cell cultures were lysed, samples were plated onto agar
plates, and colonies were counted the following day.
[0437] FIG. 12 is a graph of the number of colony forming units
(CFU) for the C. albicans-infected macrophages dosed with varying
concentrations of AmB-cochleates with lipid to drug ratios of 2:1,
3:1, 4:1, and 5:1. All cochleate formulations were efficacious at
killing C. albicans.
EXAMPLE 6
Amphotericin B Cochleates Prepared with DMSO and Lipid:AmB Ratios
of 5:1, 2:1 and 1:1 w/w
[0438] Multiple batches of Amphotericin B cochleates (CAMB) were
prepared with DMSO and tocopherol (Vitamin E) by the following
steps. Two methods for the removal of solvent were employed:
removal of solvent by washing the cochleates and removal of solvent
by dialysis of the liposomal suspension.
[0439] 1. Weighing and placing 100 mg of soy PS into a 50 ml pp
sterile tube with 10 ml sterile water.
[0440] 2. Vortexing the suspension for 2 minutes.
[0441] 3. Sonicating the suspension for 3 minutes.
[0442] 4. Filtering the suspension with a 0.22 .mu.m filter and
pooling the liposomes into a 50 ml tube.
[0443] 5. Weighing and placing 10 mg (5:1), 25 mg (2:1), and 50 mg
(1:1) of AmB into 4, 15 ml pp sterile tubes with 0.5 ml DMSO (1 ml
DMSO for 1:1).
[0444] 6. Adding 7.5 .mu.l (2:1), 6.0 .mu.l (5:1), and 10 .mu.l
(1:1) of tocopherol at 10 mg/ml to the DMSO (the concentration of
the AmB will be 20 mg/ml (5:1), 50 mg/ml (2:1), and 50 mg/ml (1:1)
at this time),
[0445] 7. Vortexing the solution for a few minutes until the AmB
dissolved completely.
[0446] Remainder of Method when Removing Solvent by Washing
Cochleates
[0447] 1. Mixing 5 ml of liposome with 0.507 ml of the AmB/DMSO
suspension, and vortexing the sample for a few minutes.
[0448] 2. Adding 0.5 ml of 0.1 M calcium solution into the
suspension with vortexing, using an eppendorf repeater pipette with
a 500 .mu.l tip and adding 10 .mu.l aliquots to the tube per every
10 sec.
[0449] 3. Centrifuging the suspension for 30 minutes at 9000 rpm at
4.degree. C.
[0450] 4. Removing the supernatant from the tube and re-suspending
the pellet with wash buffer of same volume (2 mM calcium with
sterile water).
[0451] 5. Repeating steps 3 and 4. Adjusting the final volume of
the suspension to 5 ml with 2 mM calcium wash buffer.
[0452] Remainder of Method when Removing Solvent by Dialysis of
Liposomes
[0453] 1. Transferring 6 ml (1:1), and 5.5 ml (2:1 and 5:1), of
AmB/DMSO/liposomes into dialysis tubes individually.
[0454] 2. Starting the removal the DMSO using dialysis by changing
the sterile water several times and leaving overnight.
[0455] 3. On the next day, transferring the AmB/liposomes into the
50 ml sterile tubes, and saving 0.5 ml of AmB/liposomes for the
HPLC analysis.
[0456] 4. Precipitating the liposomes by adding 0.5 ml of 0.1 M
calcium to each 50 ml tube of AmB/liposomes. About 6.0 ml were
precipitated for the 5:1 and 2:1 samples, and 6.5 ml were
precipitated for the 1:1 sample. 500 .mu.l of liposomes were saved
for the HPLC assay from each sample. The pH was about 4.0 at this
point after dialysis, and was readjusted to a pH of 5.5 to 6.0 with
IN NaOH in final preparation.
[0457] Sterilization/Stability/Storage of Preparations
[0458] 1. Stability: The final preparations from both methods were
examined under a microscope, and the pH (about 5.5) was
confirmed.
[0459] 2. Sterility: Samples of each preparation were streaked on a
chocolate plate to check the sterilization of the final
preparation, and incubated at 37.degree. C., 4.degree. C., and room
temperature for 24 hrs, 48 hrs, and 72 hrs.
[0460] 3. Storage: The samples, treated with nitrogen and covered
with parafilm, were stored a 4.degree. C.
[0461] The above formulations can be summarized as follows.
4TABLE 4 CAMB Formulations Ratio of PS: Amount of Amount of 0.1M
Final Conc. Of Name of Sample AmB(w/w) AmB soy PS Tocopherol AmB
liposome Calcium AmB (mg/ml) AmB/DMSO 2:1 25 mg 50 mg 75 .mu.g 5.5
ml 0.5 ml .apprxeq.5 mg/ml (washing) AmB/DMSO 5:1 10 mg 50 mg 60
.mu.g 5.5 ml 0.5 ml 1.52 mg/ml (dialysis) AmB/DMSO 2:1 25 mg 50 mg
75 .mu.g 5.5 ml 0.5 ml .apprxeq.3.8 mg/ml (dialysis) AmB/DMSO 1:1
50 mg 50 mg 100 .mu.g 6.0 ml 0.5 ml .apprxeq.7.1 mg/ml
(dialysis)
[0462] The recovery of AmB was determined using HPLC assay.
[0463] Results
[0464] 1. Macroscopic observations: yellowish suspension with some
settles on the bottom of the tubes.
[0465] 2. Microscopic observations: aggregated and individual
cochleates were observed.
[0466] 3. Addition of EDTA: liposomes formed upon addition of
EDTA
[0467] 4. Images of cochleate: FIG. 13 is a series of images of the
5:1 AmB cochleates (top two panels) and the cochleates after
addition of EDTA (bottom two panels).
[0468] 6. Recovery: HPLC analysis indicated that following amounts
of AmB encochleated for each formulation indicated.
[0469] 2:1 (Washing)=>81%
[0470] 5:1 (Dialysis)=>91%
[0471] 2:1 (Dialysis)=>92%
[0472] 1:1 (Dialysis)=>92%
[0473] 7. Outcome: For the mouse study described in Example 7, the
following amounts of each formulation were set aside:
[0474] 2:1 (Washing)=>0.2 mg/ml.times.2.5 ml, 14 bottles
[0475] 5:1 (Dialysis)=>0.2 mg/ml.times.2.2 ml, 14 bottles (using
2.sup.nd batch)
[0476] 2:1 (Dialysis)=>0.2 mg/ml.times.2.5, 14 bottles
[0477] 1:1 (Dialysis)=>0.2 mg/ml.times.2.5 ml 14 bottles
EXAMPLE 7
Efficacy Studies in Mice
[0478] The formulations of Example 6 were administered to mice to
study the efficacy of the formulations to protect mice from a
lethal dose of Candida albicans, and to clear the organs of C.
albicans in the surviving mice.
[0479] Six groups of 10 mice were studied. The mice were
administered 10.sup.6 cells C. albicans intravenously through the
tail vein. Starting 24 hours post-infection, the following
compositions were administered to each mouse once daily at 2 mg/kg
orally for 14 days, except for the control group, which remained
untreated.
[0480] a. Control Group (untreated)
[0481] b. AmB/deoxycholate
[0482] c. CAMB 2:1 (Washed)
[0483] d. CAMB 5:1 (Dialysis)
[0484] e. CAMB 2:1 (Dialysis)
[0485] f. CAMB 1:1 (Dialysis)
[0486] Appearance and behavior was monitored each day of the study.
Tissue burdens of C. albicans were determined in kidney, liver and
lungs for each animal at the end of the study, and colony counts
were taken. Organs were obtained and weighed, homogenized,
dilutions in buffer made, and aliquots plated onto plates and
colony counts of fungus taken several days later.
[0487] Summary of Results
[0488] FIG. 14 is a graph of the survival data for the C.
albicans-infected mice untreated or dosed daily for 14 days with
AmB/deoxycholate (AmB/D), or AmB-cochleates with a lipid to drug
ratio of 5:1 (dialysis), 2:1 (dialysis), 1:1 (dialysis), or 2:1
(wash).
[0489] FIG. 15 is a chart of the average number of C. albicans
cells/gram of tissue in the liver, kidney, and lungs of C.
albicans-infected mice untreated (control), or dosed daily for 14
days with AmB/deoxycholate (AmB/D), or AmB-cochleates with lipid to
drug ratios of 5:1 (dialysis), 2:1 (dialysis), 1:1 (dialysis), or
2:1 (washing).
[0490] Seventy percent of control (untreated) animals died and
showed high tissue burdens, while all four cochleate formulations
were effective in preventing mortality and reducing fungal cell
burdens in target organs (kidneys, lungs and liver). The 5:1
(dialysis) formulation appeared more effective than the others in
clearing the liver completely. The 2:1 (washed) and 2:1 (dialysis)
formulations had nearly the same efficacy. All formulations
(excepted for 1:1 (dialysis) formulation) reduced the fungal cell
burden as well as or better than the AmB/deoxycholate formulation.
Overall, the data are consistent with effective oral delivery of
AmB from cochleates.
EXAMPLE 8
Efficacy of Cochleates in Cells
[0491] The relative efficacy of the compositions of Example 6 were
studied in J774A.1 macrophages to compare the relative efficacy of
the cochleate compositions against Candida albicans.
[0492] Macrophages were seeded into a 96-well plate and incubated
overnight as described above. Following incubation, the macrophages
were infected with C. Albicans at a ratio of 1:200 with respect to
the macrophages. The AmB-cochleate formulations were then added at
the concentrations of 0.1, 0.01 and 0.001 .mu.g/ml. Twenty-four
hours later, the cell cultures were lysed, samples plated onto agar
plates, and counted the following day.
[0493] FIG. 16 is a graph depicting the number of colony forming
units (CFUs) for C albicans-infected macrophages dosed with varying
concentrations (0.1, 0.01 and 0.001 .mu.g/ml) of AmB-cochleate
formulations having lipid to drug ratios of 5:1 (dialysis), 2:1
(dialysis), 1:1 (dialysis), and 2:1 (washing), or AmB/deoxycholate
(AmB/D). The 2:1 (washing) and the 5:1 (dialysis) formulation were
the most efficacious at killing the C. albicans at 0.001 .mu.l/ml.
In contrast, the AmB/deoxycholate CFU's were too numerous to count
at this concentration.
EXAMPLE 9
Amphotericin B Cochleates Prepared with DMSO and Lipid:AmB Ratio of
5:1 with and without Methylcellulose
[0494] Amphotericin B cochleates were prepared using Soy PS and
DMSO with Vitamin E, and a Lipid to AmB ratio of 5:1 as
follows.
[0495] Preparation of Liposomes
[0496] 20 ml of water was added to 200 mg of Soy-PS, vortexed for
about 15 minutes to form a liposomal suspension, and filtered using
a 0.45 .mu.m filter. The suspension was sonicated for about 4
minutes and filtered again with a 0.22 .mu.m filter.
[0497] Addition of Cargo Moiety and Antioxidant in Solvent
[0498] 2 ml of DMSO solvent was added to 40 mg of Amphotericin B.
To the AmB/DMSO mixture was added 2 mg of Vitamin E and the
solution was vortexed for about 10 minutes. This solution was then
added to the liposomal suspension by drop wise addition while
vortexing. The final mixture was vortexed for about 2 minutes.
[0499] Precipitation of Cochleates
[0500] 2 ml of calcium (0.1 M) was added to the liposomal
suspension at a rate of 10 .mu.l/10 while vortexing to form
cochleates.
[0501] Solvent Removal/Washing
[0502] The mixture was vortexed for about 1-2 minutes, centrifuged
for about 1 hour at 9000 rpm, and the supernatant.was removed and
replaced with fresh supernatant (water with 2 mM calcium). This
washing step was repeated twice.
[0503] Inhibition of Aggregation
[0504] 0.1%, 0.2%, 0.3%, 0.4%, or 0.5% (w/w) methylcellulose (MC)
to inhibit aggregation and 0.2% (w/w) parabens to maintain
sterility were added to the suspension, and it was lyophilized to
form a powder.
[0505] Images of cochleates containing 0.2%, 0.3% and 0.5%
methylcellulose are given in FIG. 58. Particle size distributions
in FIG. 59 show that the addition of methylcellulose decreases
aggregation, and that the addition of paraben slightly increases
aggregation. FIG. 60 is an HPLC analysis of the cochleate showing
that amphotericin B is the only compound within the cochleate
structure.
EXAMPLE 10
Efficacy Studies in Mice
[0506] AmB/deoxycholate and 5:1 AmB cochleates (CAMB) formulated as
described in Example 9 with and without 0.3% methylcellulose (MC)
were administered to mice to study the efficacy of the formulations
to protect mice from a lethal dose of Candida albicans, and to
clear the organs of C. albicans in the surviving mice.
[0507] Six groups of 10 mice were studied. The mice were
administered 5.times.10.sup.5 cells C. albicans intravenously
through the tail vein. Starting 24 hours post-infection, the
following compositions were administered to each group once daily
for 14 days in the dosage indicated, except for the control group
which remained untreated.
[0508] a. Control
[0509] b. AmB/deoxycholate 2 mg/kg ip
[0510] c. 5:1 CAMB (suspension) with 0.3% MC, 1 mg/kg AmB oral
dosing
[0511] d. 5:1 Lyophilized CAMB with 0.3% MC, 1 mg/kg AmB oral
dosing
[0512] e. 5:1 CAMB (suspension), 1 mg/kg AmB oral dosing
[0513] f. 5:1 Lyophilized CAMB, 1 mg/kg AmB oral dosing
[0514] Appearance and behavior was monitored each day of the study.
On day 15, mice were sacrificed and tissue burden of C. Albicans
was determined in kidney, liver and lungs for each animal. Organs
were obtained and weighed, homogenized, diluted in buffer, and
aliquots were plated onto plates; colony counts of fungus were
taken several days later.
[0515] FIG. 17 is a graph of the survival data for the C.
albicans-infected mice untreated or dosed daily for 14 days with
AmB/deoxycholate (AmB/D), or AmB-cochleates (CAMB) in suspension or
lyophilized and formulated with or without methylcellulose
(MC).
[0516] FIG. 18 is a chart of the average number of C. albicans
cells/gram of tissue in the liver, kidney, and lungs of C.
albicans-infected mice untreated (control), or dosed daily for 14
days with AmB/deoxycholate, or AmB-cochleates (CAMB) in suspension
or lyophilized and formulated with or without methylcellulose
(MC).
[0517] One hundred percent of control (untreated) animals died by
day 10 and showed high tissue burdens. AmB/deoxycholate at 2 mg/kg
resulted in 100% survival and completely cleared Candida from the
liver and lungs and decreased the tissue burden in the kidney by
2-3 log units. Forty percent of the mice treated with CAMB without
methylcellulose (both in suspension and lyophilized) died and both
groups also showed substantial tissue burdens in target organs.
However, CAMB with methylcellulose in suspension and lyophilized
CAMB with methylcellulose afforded 100% and 80% survival,
respectively, and showed several log order reductions in tissue
burden relative to the other CAMB formulation. The antifungal
properties of CAMB with methylcellulose in suspension at 1 mg/kg
administered PO mimicked the behavior of AmB/deoxycholate at 2
mg/kg administered IP. Overall, cochleates with methylcellulose
showed stronger antifungal properties than cochleates without
methylcellulose.
EXAMPLE 11
Efficacy Studies in Cells
[0518] The relative efficacy of the compositions of Example 9 were
studied in J774A.1 macrophages to compare the relative efficacy of
the cochleate compositions (with and without methylcellulose)
against Candida albicans.
[0519] Macrophages were seeded into a 96-well plate and incubated
overnight as described above. Following incubation, the macrophages
were infected with C. albicans at a ratio of 1:200 with respect to
the macrophages. The AmB-cochleates were then added at the
concentrations of 5, 1, 0.1, 0.01 and 0.001 .mu.g/ml. Twenty-four
hours later, the cell cultures were lysed, samples were plated onto
agar plates, and colonies were counted the following day.
[0520] FIG. 61 is a graph of the number of colony forming units
(CFU) for the C. albicans-infected macrophages dosed with
AmB-cochleates in suspension and lyophilized with lipid to drug
ratio 5:1, with and without methylcellulose. All cochleate
formulations were efficacious at killing C. albicans.
EXAMPLE 12
Scale Up
[0521] Amphotericin B cochleate preparation was scaled up to 5
liters using Soy PS and DMSO with Vitamin E, and a Lipid to AmB
ratio of 5:1 as follows.
[0522] Preparation of Liposomes
[0523] 5.4 L of water was added to 50 g of Soy-PS, vortexed for
about 15 minutes to form a liposomal suspension, and filtered using
a 10 .mu.m filter.
[0524] Addition of Cargo Moiety and Antioxidant in Solvent
[0525] 500 ml of DMSO solvent was added to 10 g of Amphotericin B.
To the AmB/DMSO mixture was added 60 mg of Vitamin E and the
solution was vortexed for about 10 minutes. This solution was then
added to the liposomal suspension by drop wise addition using a
separatory funnel while vortexing. The final mixture was vortexed
for about 2 minutes.
[0526] Precipitation of Cochleates
[0527] 100 ml of calcium (0.5 M) was added to the liposomal
suspension at a rate of 10 .mu.l/10 s while vortexing to form
cochleates.
[0528] Solvent Removal/Washing
[0529] The mixture was vortexed for about 1-2 minutes, centrifuged
for about 1 hour at 9000 rpm, and the supernatant was removed and
replaced with fresh supernatant (water with 2 mM calcium). This
washing step was repeated twice.
[0530] Optional Inhibition of Aggregation
[0531] 0.3% (w/w) methylcellulose (MC) to inhibit aggregation and
0.2% (w/w) parabens to maintain sterility were added to the
suspension, and it was lyophilized to, form a powder. Subsequently,
the cochleates were treated with rabbit serum albumin and forced
multiple times through a high pressure homogenizer such as the
Avestin EmulsiFex-C5. Homogenization pressure was maintained around
15K to 20K psi. Particle size distributions of cochleates before
treatment with albumin, after two passes through a homogenizer and
after seven passes through a homogenizer are shown in FIGS. 62, 63
and 64, respectively.
EXAMPLE 13
Geldanamycin Cochleates
[0532] Geldanamycin (GA)-cochleates were prepared as described in
Example 1. The cochleates were observed macroscopically to have
successfully encochleated GA, and also included crystals, possibly
including unencochleated GA. When cochleates were centrifuged,
about one third of the GA was present in the supernatant. Overall,
the GA was successfully encochleated.
EXAMPLE 14
Tyrphostin Cochleates
[0533] Tyrphostin AG-825 (TY)-cochleates were prepared using the
solvent drip method described in Example 1. Good morphology of
TY-cochleates was observed, in that it appeared that TY was
successfully encochleated.
[0534] HPLC Analysis and Stability of TY-Cochleate Formulation
[0535] HPLC was used to study the stability of the TY in the
cochleates, by measuring the concentration of TY in TY-cochleates
as compared to free, i e., unencochleated TY in solution.
[0536] FIG. 19 is a graph of the concentration of TY-cochleate
preparations versus free TY over time. As can the seen in FIG. 19,
the free TY concentration decreased to zero over time. In contrast,
the TY concentration initially dropped for the TY-cochleates
(possibly due to the degradation of free TY in the cochleate
formulation), and stabilized thereafter.
[0537] TY degrades into two products (identified as impurity 1 and
impurity 2 in FIG. 20). FIG. 20 is two graphs of the concentration
of each impurity over time for both the free TY and TY-cochleates
studied in FIG. 19. FIG. 20 confirms that the free TY degraded over
time, whereas, after an initial degradation was observed, the
concentration of degradation products remained fairly stable for
the TY-cochleates.
[0538] Biological Evaluation of Tyrphostin AG-825 Cochleates
[0539] Cytotoxicity of TY-cochleates was studied in a SKOV3 cell
line (FIG. 21). The TY-cochleates showed slightly higher
cytotoxicity against the cancer cell line than that. from free TY.
The data for empty (drug free) cochleates also is shown.
[0540] Results
[0541] Tyrphostin AG-825 was successfully formulated into
cochleates using the method of the present invention. Stability
tests demonstrated that TY-cochleates have a superior stability as
compared to free TY in solution, and biological analysis of
TY-cochleates indicated that it delivered similar cytotoxic effects
on SKOV3 cell line to that of its free form in solution.
[0542] Porphyrin-cochleates also have been successfully made with
ethanol, DMG and THF solvents.
Example 15
Porphyrin Cochleates
[0543] Porphyrin cochleates were prepared with Zinc Tetra-Phenyl
Porphyrin (ZnTPP) and DMSO as described in Example 1, adjusted for
a lipid to ZnTPP ratio of 20,000:1 w/w.
[0544] The particle size and fluorescence of: plain liposomes;
liposomes with ZnTPP; and cochleates with ZnTPP, were evaluated and
the following results were obtained.
5TABLE 5 Particle Size and Fluorescence Particle Size Particle Size
Fluorescence Intensity Mean (nm) StD (nm) Max (nm) (a.u.) Liposomes
300.1 132.4 Liposomes + 280.1 122.9 595.5 45338 ZnTPP Cochleates +
<10 .mu.m 596 43589 ZnTpp
[0545] FIG. 22 is an image of ZnTPP in solution (100% DMSO), and
the ZnTPP-cochleates. The ZnTPP in solution was dark purple, and
the cochleate formulation was only slightly colored (pink),
indicating that the ZnTPP was successfully incorporated into the
cochleates. which are white.
[0546] FIG. 23 is a series of phase contrast images (left panels)
and fluorescence images (right panels), of the ZnTPP-cochleates
(top panels) and ZnTPP-liposomes (bottom panels) formed. These
images indicate that the ZnTPP was successfully associated with the
liposomes and successfully encochleated.
[0547] Comparison to Cochleates Formed without Solvent
[0548] FIG. 24 is a series of phase contrast images (left panels)
and fluorescence images (right panels), of ZnTPP-cochleates (top
panels) and ZnTPP-liposomes (bottom panels) formed without the
presence of solvent. FIG. 24 indicates that the ZnTPP did not
successfully associate with the liposomes or cochleates in the
absence of solvent.
[0549] Interaction of ZnTPP-Cochleates with SKOV3 Cells
[0550] In order to study any interaction of the cochleates with
cells, ZnTPP-cochleates and free ZnTPP (in solution with DMSO) were
introduced to SKOV3 cell cultures, and imaged with fluorescence
under a confocal microscope.
[0551] FIG. 25 is a series of images of the SKOV3 cell culture with
the ZnTPP cochleates at 1 hour and 24 hours. The images demonstrate
uptake of the ZnTPP-cochleates into the perinuclear region, and
that the ZnTPP had not significantly degraded at 24 hours.
[0552] FIG. 26 is a series of images of the SKOV3 cell culture with
the free ZnTPP (in DMSO) at 1 hour and 24 hours. The images
indicate high uptake of the ZnTPP solution at one hour, but
significant degradation at 24 hours. The appearance and
distribution of ZnTPP is different than that observed with the
ZnTPP cochleates of FIG. 25.
[0553] Study of ZnTPP-Cochleates Usine A Lipid Imaging-Agent
[0554] Cochleates were prepared with and without ZnTPP as described
above, except that prior to introduction of the ZnTPP/solvent,
liposomes were formed with 1% diolyl phosphatidylethanolamine
(DOPE) liganded to pyrene (purchased Avanti). The DOPE was
incorporated into the soy PS liposomes by dissolving both DOPE and
lipid in solvent, drying the solvent to a film, and using an
aqueous solution to form liposomes. Confocal images were taken at 1
and 24 hours after introduction to SKOV3 cells to study the uptake
and any difference in the cellular distribution of cochleate/lipid
and the ZnTPP. The study of distribution was possible because
DOPE-pyrene fluoresces blue, and ZnTPP fluoresces red. ZnTPP in the
cochleates/liposomes appears pink.
[0555] FIG. 27 is a series of images of the SKOV3 cell culture with
the empty cochleates (including DOPE-pyrene lipid) at 1 hour and 24
hours. These images indicate uptake of the empty cochleates by the
cells.
[0556] FIG. 28 is a series of images of the SKOV3 cell culture with
the ZnTPP-cochleates (including DOPE-pyrene lipid) at 1 hour and 24
hours. These images indicate uptake of the cochleates by the cells
at both 1 and 24 hours. The images also indicate a redistribution
of lipid and ZnTPP in the cells at 24 hours versus 1 hour. It
appears that a portion of the ZnTPP has separated from the
cochleates at 24 hours.
[0557] Together, FIGS. 27 and 28 indicate high uptake of cochleates
by the cell, and subsequent release of porphyrins from the
cochleates in the cell. The Figures also indicate that, once inside
the cell, the porphyrin is more stable in the cochleate than
free.
EXAMPLE 16
Preparation of NSAID Cochleates
[0558] Acetaminophen Cochleate Preparation
[0559] Acetaminophen and DOPS were mixed in a sterile,
polypropylene tube with a rubber policeman. TES buffer was added to
the tube to disperse the mixture in a ratio of 10 mg lipid/ml. The
cochleates were formed by the slow addition (10 .mu.L) of calcium
chloride (0.1M) to the suspension of liposomes at a molar ratio of
lipid to calcium of 2:1 with an external excess of 6 mM calcium and
then stored at 4.degree. C. in the absence of light.
[0560] Acetaminophen cochleates were formulated with and without
aggregation inhibitor, casein, which was added to the buffer
solution prior to the addition of calcium chloride in a casein to
lipid ratio of 1:1 by weight.
[0561] Images were taken of the cochleates formed with (FIG. 37A,
left panel) and without (FIG. 37A, right panel) the aggregation
inhibitor. As demonstrated by the images, cochleates formed in the
presence of casein did not aggregate as did the cochleates formed
without the aggregation inhibitor.
[0562] Aspirin Cochleate Preparation
[0563] Aspirin and DOPS were solublized in chloroform in a
lipid/aspirin molar ratio of 10:1 in a sterile glass tube. The
sample was blown down with nitrogen to form a film. The sample was
then resuspended in TES buffer, pH 7.4, at a ratio of 10 mg
lipid/ml. The cochleates were formed by the slow addition (10
.mu.L) of calcium chloride (0.1M) to the suspension of liposomes at
a molar ratio of lipid to calcium of 2:1 with an external excess of
6 mM calcium and then stored at 4.degree. C. in the absence of
light.
[0564] Aspirin cochleates were formulated with and without an
aggregation inhibitor, casein, which was added to the buffer
solution prior to the addition of calcium chloride in a casein to
lipid ratio of 1:1 by weight.
[0565] Images were taken of the cochleates formed with (FIG. 37B,
left panel) and without (FIG. 37B, right panel) the aggregation
inhibitor. As demonstrated by the images, cochleates formed with
the aggregation inhibitor did not aggregate as did the cochleates
without the aggregation inhibitor, which formed needle-like
structures.
[0566] Summary
[0567] The introduction of an aggregation inhibitor to the
cochleates loaded with a variety of cargo moieties inhibited
cochleate aggregation. All cochleate formulations with casein were
significantly smaller than cochleates made without casein, and
these cochleates were stable for at least two months with no
noticeable aggregation. Cochleates formed without casein aggregated
over time.
[0568] Addition of Methylcellulose
[0569] Aspirin or acetaminophen cochleates with and without casein
were prepared as described above, except that liposomes were
filtered through a 0.45 .mu.m filter followed by a 0.22 .mu.m
filter, which resulted in unilamellar liposomes which contained the
drug. Calcium chloride was added to the liposomes as also described
above. Methylcellulose in suspension (0.5% of entire formulation)
was added and the sample was vortexed.
[0570] The addition of methylcellulose at this particular
concentration did not reverse aggregation, but rather inhibited
further aggregation of cochleates. The size of cochleates
subsequent to addition of methylcellulose was observed to remain
stable.
Example 17
Inhibition of Edema in Rat Paw
[0571] A carrageenan model was employed to study the effect of
anti-inflammatory cochleates on edema in rat paws. Various aspirin
cochleates of the invention were used to treat carrageenan-induced
rat paw edema. These results were compared to free aspirin: and
indomethacin and empty cochleates to determine the efficacy of
encochleated anti-inflammatory drugs. Additionally, rats in all
groups were examined for gastric irritation.
[0572] Samples Tested
[0573] 1. Control (no treatment)
[0574] 2. Indomethacin, 6 mg/kg
[0575] 3. Aspirin control, 150 mg/kg
[0576] 4. Large, empty cochleates
[0577] 5. Small, casein cochleates
[0578] 6. Large aspirin cochleates, 45 mg/kg
[0579] 7. Large aspirin cochleates, 150 mg/kg
[0580] 8. Small, casein aspirin cochleates, 45 mg/kg
[0581] 9. Small, casein aspirin cochleates, 140 mg/kg
[0582] 10. Large casein aspirin cochleates with 0.1% Vit E, 45
mg/kg
[0583] 11. Large casein cochleates with 0.1% Vit E
[0584] Samples 6-10 were prepared in accordance with Example 16 ,
except that the lipid:aspirin molar ratio was 5:1 and soy PS was
used instead of DOPS. Samples 4, 5, and 11 were prepared in a
similar manner in the absence of a cargo moiety. In all cases,
water was used in lieu of TES buffer. All samples were given by
oral gavage at 1 hour prior to injection of carrageenan on Day 0 in
a volume of 3 mL of 0.5% methylcellulose. Methylcellulose at this
concentration served to stabilize the cochleates but did not
significantly affect the size or distribution of the standard or
casein cochleates. Volumes of rat paw (in ml) were measured prior
to carrageenan injection using a semi-automated plethysmograph
(Buxco). At 0 time, 0.1 ml of 1% carrageenan in 0.9% pyrogen free
saline was injected into the right hind paw of the rat. The paw
volumes (in ml) were measured again 3 hours post carrageenan
injection to determine inhibition of paw edema. Four rats from each
of groups 2, 3, 6, 7, 8, 9 and 10 and one rat from each of groups
1, 4, 5 and 11 were bled (intravenously, jugular vein,
approximately 1 ml blood) at 30 minutes and 4 hours post drug
administration. Blood was collected in heparinized vacutainers. 4
hours post carrageenan injection, the stomachs of rats from all
test groups were removed after euthanasia by CO.sub.2 asphyxiation
to observe for gastric irritation (i.e. bleeding and
ulcerations).
[0585] Inhibition of rat paw edema for all samples is presented in
FIG. 38. In general, the control group and the groups given
cochleates not containing aspirin show no decrease in the level of
edema in the rat paw. Large aspirin cochleates (cochleates not made
with an aggregation inhibitor) show a decrease in edema only
slightly larger than that of free aspirin or indomethacin. Small
cochleates (with. an aggregation inhibitor), however, show a
significant decrease in edema in comparison to both free aspirin
and indomethacin and large aspirin cochleates. Additionally, large
aspirin cochleates with vitamin E show more of a decrease in edema
when compared to plain large aspirin cochleates.
[0586] FIG. 39 shows the incidence and level of severity of gastric
irritation produced by samples 2, 3, 7 and 9. In general,
indomethacin produced the greatest gastric irritation, followed by
unencochleated aspirin. Aspirin cochleate formulations produced
less incidence of irritation when compared to both aspirin and
indomethacin.
EXAMPLE 18
Activation of Macrophages by Anti-Inflammatory Cochleates
[0587] To examine the effects of cochleates on lipopolysaccharide
(LPS) plus IFN-.gamma. induced NO production, J774A.1 macrophages
were treated with LPS plus IFN-.gamma. in the presence and absence
of empty cochleates. Macrophages were also treated with and without
empty cochleates in the absence of LPS plus IFN-.gamma..
[0588] Since NO production requires the enzymatic activity of NOS,
its activity was measured by NO secretion using the method of
Griess (nitrite). Briefly, 100 .mu.L of sample was reacted with the
Griess reagent at room temperature for 10 minutes. Amount of
NO.sub.2.sup.- was then determined by measuring the absorbance at
540 nm in a microplate reader. The standard curve was obtained
using the known concentration of sodium nitrite. In all
experiments, NO.sub.2.sup.- concentration in wells containing
medium only was also measured as a blank control.
[0589] FIG. 40 indicates that empty cochleates (EC) are
immunologically inert. That is, they neither enhance nor inhibit NO
production induced by LPS plus IFN-.gamma. at all concentrations
assayed. In contrast, the addition of LPS plus IFN-.gamma. to the
macrophages with and without empty cochleates resulted in a
dramatic increase in iNOS production. In addition, all
concentrations of the empty cochleates showed no sign of cellular
toxicity as was observed under phase contrast microscopy.
[0590] In order to determine the in vitro efficacy of
anti-inflammatory cochleates of the invention, J774A.1 mouse
macrophages were incubated with LPS (1 .mu.g/ml) plus IFN-.gamma.
(10 .mu.g/ml) in the presence or absence of standard aspirin
cochleates and acetaminophen cochleates prepared as described in
Example 16, free aspirin, free acetaininophen and empty cochleates
(control) for 15 hrs.
[0591] As shown in FIG. 41, standard cochleates containing aspirin
and acetaminophen exhibited greater in vitro efficacy than free
aspirin and acetaminophen at inhibiting NO production.
EXAMPLE 19
Particle Size Analysis of Cochleates of the Invention
[0592] Cochleates stabilized with 1% casein were evaluated with the
N4 plus from Coulter. Briefly, 20 .mu.l of the suspension of empty
cochleates prepared in accordance with Example 17 (without NSAIDs)
were added to 2.5 ml of D.D. H.sub.2O. The samples were
equilibrated over 20 minutes. The samples were then analyzed for 2
minutes at a 90.degree. angle. Two different populations of
cochleates were observed, one centered at 25 nm, and the other one
at 350 nm (FIG. 34A). The population centered at about 25 nm likely
consists of casein micelles and not cochleates. Any such micelles
can be removed, e.g., by centrifugation.
[0593] The particle size of aggregated, standard cochleates was
also evaluated using the LS230 from Coulter. 100 .mu.l to 200 .mu.
of the sample was added to 250 ml of washing buffer in the vessel
until the PIDS (Polarization Intensity Differential Scattering)
reached 45%. The duration time for a run was 120 s and the number
of cycles was 3. Four different populations, one centered at
approximately 1 .mu.m, one at approximately 10 .mu.m, one at
approximately 30 .mu.m and the last one at approximately 50 .mu.m
were observed. (FIG. 34B)
EXAMPLE 20
Preparation of Cochleates with Various Aggregation Inhibitors
[0594] Rhodamine labeled phosphatidyl ethanolamine (Rho-PE)
liposomes were prepared by adding di-oleoyl-PS (DOPS) and Rho-PS to
chloroform at a ratio of 10 mg lipid/ml solvent. The DOPS was
present at 0.1% or 0.01% of the total lipid. The sample was blown
down under nitrogen to form a film. Once dry, the sample was
resuspended in a TES buffer at a ratio of 10 mg lipid/ml buffer.
The liposomes were then passed through a 0.22 .mu.L filter. The
homogeneous population of Rhodamine labeled liposomes were stored
at 4.degree. C. in the absence of light under nitrogen.
[0595] Sterile glass tubes, each containing 100 .mu.l fluorescent
Rhodamine cochleates in TES buffer were prepared. Cochleates were
formed by the addition of 10 .mu.l aliquots of 0.1M calcium
chloride until a molar ratio of lipid to calcium of 2:1 and an
external excess of 6 mM calcium was reached. 10 .mu.l Half and Half
was added to one tube and vortexed for 4 minutes. Whole milk, at a
1:1 ratio of whole milk to lipid, was added to a second tube and
vortexed for one minute. Evaporated fat free milk, at a 1:2 weight
ratio of evaporated milk to lipid was added to a third tube and
vortexed for 4 minutes. A fourth tube was used as the control, and
as such, no aggregation inhibitor was added.
[0596] FIGS. 35A-D are four fluorescent images of the
Rhodamine-labeled cochleates obtained. The Figures demonstrate the
effect of formulating cochleates in the presence of various
aggregation inhibitors: half and half (FIG. 35A), whole milk (FIG.
35B), and fat-free milk (FIG. 35C). FIG. 35D is an image of the
control composition of cochleates that do not include an
aggregation inhibitor.
[0597] FIG. 36 depicts the aggregated cochleates prior to the
addition of milk (left image) and after the addition of milk (right
image). These images indicate that milk caused aggregation to
reverse.
EXAMPLE 21
Uptake of Cochleates by Macrophages
[0598] Rhodamine labeled phosphatidyl ethanolamine (Rho-PE)
cochleates were prepared as described in Example 20, except that
casein was added to the formulation prior to the addition of
calcium in a casein to lipid ratio of 1:1. Additionally, no milk
products were added to the casein-coated Rho-PE cochleates.
[0599] Sterile cover slips were placed in the wells of 24-well
plates. J774.1 macrophages were harvested as described above,
counted using a hemacytometer and seeded at a concentration of
1.times.10.sup.5 , and allowed to incubate overnight to ensure
adherence. Rhodamine-PE cochleates were then added at a final
concentration of 50 .mu.g lipid/ml and 5 .mu.g lipid/ml.
[0600] The cover slips were removed at the desired time point,
generally about one hour, rinsed in DMEM to remove any free
cochleates, placed inverted on microscope slides and observed for
uptake using phase contrast and fluorescence microscopy. Standard
cochleate formulations were observed to remain within the
macrophage for several days and slowly transfer the fluorescent
lipid from endocytic vessels throughout the rest of the macrophage.
In contrast, the nanocochleates were only observed up to 36 hours
after administration. Due to their size and/or altered surface
characteristics, the cochleates with casein were taken up more
aggressively than standard cochleates (without casein) by cultured
cells. Nearly every cell incubated with the cochleate composition
prepared with casein showed intracellular fluorescence, indicating
that the cochleates were rapidly taken up by the macrophages (FIG.
33B). In contrast, standard cochleates were not taken up as
aggressively by the macrophages as is shown by intracellular
fluorescence (FIG. 33A).
EXAMPLE 22
Cochleates Formed with Protonized Vancomycin
[0601] Cochleates Formed with and without Calcium
[0602] Vancomycin cochleates are expected to increase the oral
bioavailability of vancomycin while limiting its side effects.
Vancomycin cochleates were formed with and without calcium.
[0603] 14.6 mg of Dioleoyl Phosphatidylserine (DOPS, Avanti, Ala.)
was used as the starting lipid material for each cochleate
formulation. The phospholipid powder and 7.6 mg protonized
Vancomycin (Vanco) powder were mixed in a molar ratio of 4.3:1.1 mL
of modified TES buffer (2 mM TES, 150 mM NaCl, 2 mM L-Histidine),
adjusted at pH 3 was added to each mixture. To one formulation,
calcium also was added. The mixture was vortexed for 2 minutes.
[0604] Optical microscopy, using phase contrast technique, revealed
the presence of cochleates in both the formulation without calcium
(FIG. 42), and the formulation with calcium (FIG. 43). The
cochleates were centrifuged at 3000 rpm at 4.degree. C. for 20 min.
The content of Vanco in the aggregates was assessed by OD
absorption at 282 nm with a spectrophotometer. Results showed that
the lipid associated with the Vanco such that the vancomycin
comprised about 40% of the precipitate by weight for the
formulation without calcium and about 70% of the precipitate for
the formulation with calcium.
[0605] Addition of EDTA chelating agent to the formulation with
calcium resulted in a rapid transformation of the cochleate into
opened structure (FIG. 44), suggesting that the cochleates included
stacked sheets of lipid bilayer and cationic drug.
[0606] Cochleates Formed with Alternative Acidification Step
[0607] Vanco crystals were added to preformed DOPS liposomes (FIG.
45A). The Vanco was solubilized as TES buffer (pH 7.4) was added to
disperse the mixture in a ratio of 10 mg lipid/ml. HCl (0.1N) was
used to bring the pH to 5.0 or 6.5, at which point an association
of the Vanco with the lipid were visible under the microscope. The
protonized Vanco was observed to associate with the negatively
charged bilayer surface. 10 .mu.L of calcium chloride (0.1M) then
was slowly added to the suspension of liposomes at a molar ratio of
lipid to calcium of 2:1 with an external excess of 6 mM calcium and
then stored at 4.degree. C. in the absence of light.
[0608] Cochleates Formed with and without an Aggregation
Inhibitor
[0609] Vancomycin cochleates were formulated with an acidification
step as described above with and without an aggregation inhibitor
(casein), which was added to the buffer solution prior to the
addition of calcium chloride in a casein to lipid ratio of 1:1 by
weight.
[0610] Images were taken of the cochleates formed with (FIG. 45B)
and without (FIG. 45C) casein. When EDTA was added to the
cochleates, they were opened to form liposomes as shown in FIG.
45D. The efficacy of the cochleates against Staph. aureus was
studied in vitro as described in Example 24, below.
EXAMPLE 23
Cochleates Formed with Tobramycin
[0611] Cochleates Formed with Acidification Step
[0612] Tobramycin crystals were added to pre-formed liposomes (FIG.
48A). Tobramycin was solubilized as TES buffer (pH 7.4) was added
to disperse the mixture in a ratio of 10 mg lipid/ml. HCl (0.1N)
was used to bring the pH to 5.5, at which point an association of
the tobramycin with the lipid were visible under the microscope.
The protonized tobramycin was observed to associate with the
negatively charged bilayer surface. 10 .mu.L of calcium chloride
(0.1M) was slowly added to the suspension at a molar ratio of lipid
to calcium of 2:1 with an external excess of 6 mM calcium and then
stored at 4.degree. C. in the absence of light.
[0613] Cochleates Formed with and without an Aggregation
Inhibitor
[0614] Tobramycin cochleates were formulated with and without an
aggregation inhibitor (casein), which was added to the buffer
solution prior to the addition of calcium chloride in a casein to
lipid ratio of 1:1 by weight.
[0615] Images were taken of the cochleates formed with (FIG. 48B)
and without (FIG. 48C) casein. EDTA was added to the cochleates of
FIG. 48C and the cochleates were observed to open as shown in FIG.
48D. The efficacy of the cochleates against Staph. aureus was
studied in vitro as described in Example 24, below.
EXAMPLE 24
Bactericidal Activity of Cochleates
[0616] J774A.1 is a well characterized murine macrophage-like cell
line that has been extensively used to study Staphylococcal
aureus-macrophage interactions. The J774A.1 cells were maintained
at -80.degree. C. prior to use and were prepared for the
phagocytosis assays as described above.
[0617] J774A.1 macrophages were counted using a hemacytometer,
seeded into 96-well plates and incubated overnight. Following
incubation, the macrophages were infected with Staphylococcal
aureus or Pseudomonas aeruginosa at a ratio of 1:200 with respect
to the macrophages.
[0618] Free Vanco and Vanco cochleates prepared with and without
casein as described in Example 22, were added to the macrophages
infected with Staph. A. at concentrations of 1, 5, 10, and 25
.mu.g/ml.
[0619] Free tobramycin and tobramycin cochleates prepared as
described in Example 23 with and without casein were added to the
macrophages infected with P. aeruginosa and P. aeruginosa alone at
concentrations of 1, 5, 10, and 25 .mu.g/ml.
[0620] Following incubation for 3 and 6 hours, the plates were
removed and observed. Medium was removed and replaced with 100
.mu.l cold sterile water. The plates were incubated 10 minutes, at
which point the 100 .mu.l cold sterile water was pipetted
vigorously to disrupt the cellular membrane. 25 .mu.l of this
suspension was placed onto Sabouraud Dextrose Agar plates, and
placed in a dry incubator overnight at 37.degree. C. Staphylococcal
aureus or Pseudomonas aeruginosa colony forming units (CFU's) were
counted the following day.
[0621] FIGS. 46 and 47 are graphs demonstrating the efficacy data
for the Vanco cochleates (with and without casein) against
Staphylococcal aureus versus free vancomycin at 3 and 6 hours after
administration, respectively.
[0622] FIGS. 49 and 50 are graphs demonstrating the efficacy of the
tobramycin cochleates of the invention (with and without casein)
against Pseudomonas aeruginosa versus free tobramycin at 3 and 6
hours after administration, respectively.
[0623] As FIGS. 46, 47, 49 and 50 indicate, the cochleates of the
invention increase the effectiveness of the cargo molecule against
bacteria in cells. Additionally, vancomycin and tobramycin
cochleates including an aggregation inhibitor show a significant
increase in efficacy in relation to both free drug and cochleates
formed without aggregation inhibitor.
EXAMPLE 25
Caspofungin Cochleates
[0624] Cochleates Formed with Calcium--Solvent Drip Method
[0625] Soy phosphatidylserine (Soy PS, Degussa) was used as the
starting lipid material for each cochleate formulation. 100 mg soy
PS was mixed with 10 mL water or saline, and the mixture was
vortexed, forming liposomes. 10 mg of protonized caspofungin (5 mg
for 20:1 cochleates or 20 mg for 5:1 cochleates) was then dissolved
in 1 mL DMSO. The DMSO solution was slowly added to the liposomal
solution. After the caspofungin/soy PS liposomal solution was
mixed, 1.5 mL of 0.1M calcium chloride solution was added at a rate
of 10 .mu.l/10 s in order to precipitate a solid. Resulting
formulations, along with observations about cochleate morphology
are presented in Table 1, below.
[0626] Cochleates Formed with Calcium--Aqueous Method
[0627] Numerous formulations using the aqueous drip method were
prepared using varying combinations of starting materials. Soy
phosphatidylserine (Soy PS, Degussa) and DOPS were both used as the
starting lipid material for cochleate formulations using the
aqueous drip method. Soy PS or DOPS (100 mg) was mixed with 5 mL
water, saline, or buffer and the mixture was vortexed until
liposomes formed. Protonized caspofungin (10 mg for 10:1
cochleates, 20 mg for 5:1 cochleates) was then dissolved in 5 mL
water, saline, or buffer. The caspofungin solution was added slowly
to the liposomal solution. After mixing, 1.5 mL of a 0.1 M calcium
chloride solution was added to the caspofungin/soy PS liposomal
solution at a rate of 10 .mu.l /10 s in order to precipitate a
solid caspofungin cochleate. Resultant formulations, along with
observations about cochleate morphology and measurements of "free"
caspofungin are presented in Table 1, below.
6TABLE 1 Caspofungin cochleate formulations PS:caspo Morphology
"free" Method (w/w) PS source Buffer/saline/water (+/- EDTA)
caspofungin DMSO drip 20:1 soy PS water or saline OK ND 10:1 soy PS
water or saline OK ND 5:1 soy PS water or saline OK ND aqueous drip
10:1 DOPS water OK 15% 10:1 DOPS saline OK 4% 10:1 soy PS water OK
2% 10:1 soy PS TES buffer OK 2% 10:1 soy PS saline OK 0.1% 5:1 soy
PS saline OK 1% * "free" caspo indicates caspofungin which did not
precipitate with the soy PS.
[0628] Briefly, the morphology of formulations made with both the
solvent drip method and the aqueous drip method were indicative of
cochleate structures. That is, they both demonstrated an opening to
liposomes upon addition of EDTA. Additionally, it appears that the
use of saline diminished the amount of free caspofungin in
comparison to the use of water when cochleates were formulated
using the aqueous drip method.
[0629] Cochleates Formed with Additional Acidification Step
[0630] 100 mg soy PS was combined with 5 mL saline, and the mixture
was vortexed until liposomes formed. 50 mg protonized caspofungin
was then combined with 5 mL saline buffer at pH 5.5. This pH
ensured that the caspofungin remained protonized and multivalent.
The caspofungin solution was slowly added to the soy PS liposomes.
Cochleates began to form immediately upon addition of caspofungin
because of the high valency of the protonized moiety.
[0631] These cochleates were also formed with additional calcium,
and with sterile water instead of saline. These three formulations
were then maintained at 4.degree. C. for five days in order to test
the stability of the resultant cochleates. HPLC analysis of the
cochleates and the supernatant was completed to measure
concentration of caspofungin in both, and is summarized in FIG. 56.
It is shown that in sterile water, the concentration of caspofungin
in the cochleate gradually decreases, while the concentration of
caspofungin not associated with a cochleate ("free caspofungin")
increases. This decrease of caspofungin in the cochleates and
increase in free caspofungin can also be observed for the saline
formulation with excess calcium. The formulation with saline only,
however, remained stable over the five day period.
[0632] Acidification of Solution Subsequent to Cochleate
Formation
[0633] Caspofungin cochleates formed with an alternative
acidification step (pH 5.5) as described above were subsequently
treated with varying amounts of sodium hydroxide and hydrochloric
acid in order to vary the pH from 1 to 9 (pH tested with litmus
paper). Phase contrast micrographs at pH 1, 4, 6, 7, 8, and 9 are
depicted in FIG. 57. It appears that the formulations at pH 4 and
pH 6 are cochleates, but open to form liposomes when the pH is
raised to 9.
[0634] Addition of Bovine Serum Albumen to Caspofungin
Cochleates
[0635] The particle size distribution of 10:1 and 5:1
PS:caspofungin cochleates was measured using diffraction-based
light-scattering from 0.5 to 500 microns with Beckman-Coulter
LS230. FIG. 53A and 53B (Vanco) are two graphs depicting the
particle size distributions of 10:1 PS:caspofungin cochleates and
5:1 PS:caspofungin cochleates, respectively. The 10:1
PS:caspofungin formulation was then treated with bovine serum
albumin (BSA), followed by C-5 homogenization in order to reduce
the mean particle size of the cochleates. Once again, the particle
size distribution of the caspofungin cochleates was measured. FIG.
54 depicts the particle size distribution of the caspofungin
cochleates, and demonstrates the decrease in particle size upon
homogenization and addition of the BSA.
EXAMPLE 26
Characterization of Caspofungin Cochleates
[0636] Caspofungin cochleates with a lipid:caspo ratio of 10:1 and
5:1 formulated as described in Example 25, using saline (0.9%
NaCl), with additional Vitamin E (1% w/w, Roche) added at the
liposomal stage, were characterized physically and chemically.
[0637] Morphology
[0638] The morphology of 10:1 and 5:1 caspofungin cochleates was
observed using phase contrast microscopy and are shown in FIGS. 51A
and 52A, respectively. EDTA was also added to the caspofungin
cochleates of the same formulations in order to observe the opening
of the cochleate structures. Phase contrast micrographs are given
in FIGS. 51B and 52B, and show the cochleates opening to form
liposomes.
[0639] Chemical Characterization
[0640] Concentrations of caspofungin in both the supernatant (to
determine "free" caspofungin) and in the pellet (encochleated
caspofungin) were measured using HPLC (FIG. 55), and the
concentration of soy PS was determined using a modified Bartlett
P.sub.i assay. The PS:caspofungin ratio in the cochleates was
determined using these values. The concentration of free
caspofungin in both the 10:1 formulations and the 5:1 formulations
ranged from approximately 0.7% to approximately 8.2% of total
caspofungin. A sterility test for bacterial growth on agar plates
was also investigated, and it was determined that all formulations
passed.
[0641] Equivalents
[0642] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *