U.S. patent application number 09/071980 was filed with the patent office on 2002-04-11 for method of forcing the reverse transport of cholesterol from a body part to the liver while avoiding harmful disruptions of hepatic cholesterol homeostasis and pharmaceutical compositions and kit related thereto.
Invention is credited to WILLIAMS, KEVIN JON.
Application Number | 20020041894 09/071980 |
Document ID | / |
Family ID | 21714125 |
Filed Date | 2002-04-11 |
United States Patent
Application |
20020041894 |
Kind Code |
A1 |
WILLIAMS, KEVIN JON |
April 11, 2002 |
METHOD OF FORCING THE REVERSE TRANSPORT OF CHOLESTEROL FROM A BODY
PART TO THE LIVER WHILE AVOIDING HARMFUL DISRUPTIONS OF HEPATIC
CHOLESTEROL HOMEOSTASIS AND PHARMACEUTICAL COMPOSITIONS AND KIT
RELATED THERETO
Abstract
The present invention provides various methods, systems and
compositions for forcing the reverse transport of cholesterol from
peripheral tissues to the liver in vivo while controlling plasma
LDL concentrations, and other significant components of living
biological systems. The method comprises the step of parenterally
administering a therapeutically effective amount of a multiplicity
of large liposomes comprised of phospholipids substantially free of
sterol for a treatment period whereby said liposomes pick-up said
cholesterol during said treatment period. The method optionally
includes the step of periodically assaying plasma LDL
concentrations with an assay during said treatment period to asess
said plasma LDL concentrations and obtain an LDL profile, and
adjusting said parenteral administration in response to said LDL
profile. Exemplary assays are selected from the group consisting of
an assay of plasma esterified cholesterol, an assay of plasma
apolipoprotein-B, a gel filtration assay of plasma, an
ultracentrifugal assay of plasma, a precipitation assay of plasma,
and a immuno turbidometric assay of plasma. Generally the
compositions described herein include large liposomes of a size and
shape larger than fenestrations of an endothelial layer lining
hepatic sinusoids in said liver, whereby said liposomes are too
large to readily penetrate said fenestrations. Therapeutically
effective amounts of said compositions include in the range of 10
mg to 1600 mg phospholipid per kg body weight per dose. The large
liposomes are selected from the group consisting of uni-lamellar
liposomes and multi-lamellar liposomes. In variants, the liposomes
have diameters larger than about 50 nm, diameters larger than about
80 nm, and diameters larger than about 100 nm. The methods
optionally include the step of enhancing tissue penetration of a
cholesterol acceptor by co-administration of an effective amount of
a compound, said compound selected from the group consisting of a
small acceptor of cholesterol and a drug that increases endogenous
small acceptors of cholesterol. The small acceptor is selected from
the group consisting of a high-density lipoprotein, a phospholipid
protein complex having a group selected from the group consisting
of apoA-I, apoA-II, apoA-IV, apoE, synthetic fragments thereof,
natural fragments thereof, an amphipathic protein, and an
amphipathic peptide, said protein substantially free of
phospholipid, small phospholipid liposomes, and a small cholesterol
acceptor. The includes an agent that raises physiologic HDL
concentrations, said agent selected from the group consisting of
nicotinic acid, ethanol, a fibric acid, a cholesterol synthesis
inhibitor, a drug that increases HDL concentrations, and
derivatives thereof. The invention further provides a method of,
and composition for regulating hepatic parenchymal cell cholesterol
content and gene expression by the steps described herein. Other
systems, and compositions for treating, and improving various
medical techniques are also described.
Inventors: |
WILLIAMS, KEVIN JON;
(WYNNEWOOD, PA) |
Correspondence
Address: |
PATREA L. PABST
HOLLAND & KNIGHT LLP
ONE ATLANTIC CENTER
SUITE 2000
ATLANTA
GA
30309-3400
US
|
Family ID: |
21714125 |
Appl. No.: |
09/071980 |
Filed: |
May 4, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60005090 |
Oct 11, 1995 |
|
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|
Current U.S.
Class: |
424/450 |
Current CPC
Class: |
A61M 1/28 20130101; A61P
9/10 20180101; A61K 31/685 20130101; C12Q 1/60 20130101; A61K 35/14
20130101; A61P 1/16 20180101; A61M 1/287 20130101; Y10S 514/824
20130101; A61K 31/685 20130101; A61P 3/00 20180101; A61M 1/16
20130101; A61M 1/3427 20140204; A61P 9/00 20180101; G01N 2800/52
20130101; A61M 1/1619 20140204; A61K 31/575 20130101; A61K 35/14
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/685
20130101; A61K 31/685 20130101; A61K 2300/00 20130101; A61K 38/1709
20130101; B82Y 5/00 20130101; A61M 1/3437 20140204; A61M 1/1654
20130101; A61K 35/14 20130101; A61K 38/1709 20130101; A61K 9/127
20130101 |
Class at
Publication: |
424/450 |
International
Class: |
A61K 009/127 |
Claims
1. A method of forcing the reverse transport of cholesterol from
peripheral tissues to the liver in vivo while controlling plasma
LDL concentrations comprising the step of: parenterally
administering a therapeutically effective amount of a multiplicity
of large liposomes comprised of phospholipids substantially free of
sterol for a treatment period whereby said liposomes pick-up said
cholesterol during said treatment period.
2. The method in accordance with claim 1 further comprising the
step of periodically assaying plasma LDL concentrations with an
assay during said treatment period to assess said plasma LDL
concentrations and obtain an LDL profile, and adjusting said
parenteral administration in response to said LDL profile.
3. The method in accordance with claim 2 in which said assay is
selected from the group consisting of an assay of plasma esterified
cholesterol, an assay of plasma apolipoprotein-B, a gel filtration
assay of plasma, an ultracentrifugal assay of plasma, a
precipitation assay of plasma, and a immuno turbidometric assay of
plasma.
4. The method in accordance with claim 1 in which the large
liposomes are of a size and shape larger than fenestrations of an
endothelial layer lining hepatic sinusoids in said liver, whereby
said liposomes are too large to readily penetrate said
fenestrations.
5. The method in accordance with claim 1 in which the
therapeutically effective amount is in the range of 10 mg to 1600
mg phospholipid per kg body weight per dose.
6. The method in accordance with claim 1 in which the liposomes are
given periodically during said treatment period.
7. The method in accordance with claim 1 in which the large
liposomes are selected from the group consisting of uni-lamellar
liposomes and multi-lamellar liposomes.
8. The method in accordance with claim 1 in which the liposomes
have diameters larger than about 50 nm.
9. The method in accordance with claim 1 in which the liposomes
have diameters larger than about 80 nm.
10. The method in accordance with claim 1 in which the liposomes
have diameters larger than about 100 nm.
11. The method in accordance with claim 1 in which parenteral
administration is selected from the group of intravenous
administration, intra-arterially administration, intramuscular
administration, subcutaneous administration, transdermal
administration, and intraperitoneal administration.
12. The method in accordance with claim 1 further comprising the
step of enhancing tissue penetration of a cholesterol acceptor by
co-administration of an effective amount of a compound, said
compound selected from the group consisting of a small acceptor of
cholesterol and a drug that increases endogenous small acceptors of
cholesterol.
13. The method in accordance with claim 12 in which said small
acceptor is selected from the group consisting of a high-density
lipoprotein, a phospholipid protein complex having a group selected
from the group consisting of apoA-I, apoA-II, apoA-IV, apoE,
synthetic fragments thereof, natural fragments thereof, an
amphipathic protein, and an amphipathic peptide, said protein
substantially free of phospholipid, small phospholipid liposomes,
and a small cholesterol acceptor; said drug including an agent that
raises physiologic HDL concentrations, said agent selected from the
group consisting of nicotinic acid, ethanol, a fibric acid, a
cholesterol synthesis inhibitor, a drug that increases HDL
concentrations, and derivatives thereof.
14. The method in accordance with claim 12 in which said
co-administration of said compound is simultaneous with said
parenteral administration of said large liposomes.
15. The method in accordance with claim 14 in which said
co-administration of said compound is separated in time from said
parenteral administration of said therapeutically effective amount
of a multiplicity of said large liposomes by an effective time
period.
16. The method in accordance with claim 13 in which said effective
time period is in the range of 1 minute to two weeks.
17. A method of regulating hepatic parenchymal cell cholesterol
content, said cell having at least one gene selected from the group
consisting of a gene for an LDL receptor, a gene for HMG-CoA
reductase, a gene for cholesterol 7-alphahydroxylase, and a gene
regulating a function involved in cholesterol homeostasis; and,
homeostasis thereof, comprising the step of: parenterally
administering a therapeutically effective amount of a multiplicity
of large liposomes comprised of phospholipids substantially free of
sterol during a treatment period.
18. The method in accordance with claim 17 further comprising the
steps of periodically assaying plasma LDL concentrations with an
assay during said treatment period to assess said plasma LDL and to
obtain an LDL profile, and adjusting said parenteral administration
in response to said LDL profile.
19. The method in accordance with claim 18 in which said assay is
selected from the group consisting of an assay of plasma esterified
cholesterol, an assay of plasma apolipoprotein-B, a gel filtration
assay of plasma, an ultracentrifugal assay of plasma, a
precipitation assay of plasma, and a immuno turbidometric assay of
plasma.
20. The method in accordance with claim 17 further comprising the
step of enhancing tissue penetration of a cholesterol acceptor with
co-administration of an effective amount of a compound, said
compound selected from the group consisting of a small acceptor of
cholesterol and a drug that increases endogenous small acceptors of
cholesterol.
21. The method in accordance with claim 20 in which said small
acceptor is selected from the group consisting of a high-density
lipoprotein, a phospholipid protein complex having a group selected
from the group consisting of apoA-I, apoA-II, apoA-IV, apoE,
synthetic fragments thereof, natural fragments thereof, an
amphipathic protein, and an amphipathic peptide, said protein
substantially free of phospholipid, small phospholipid liposomes,
and a small cholesterol acceptor; said drug including an agent that
raises physiologic HDL concentrations, said agent selected from the
group consisting of nicotinic acid, ethanol, a fibric acid, a
cholesterol synthesis inhibitor, a drug that increases HDL
concentrations, and derivatives thereof.
22. The method in accordance with claim 20 in which said
co-administration of said compound is simultaneous with said
administration of said large liposomes.
23. The method in accordance with claim 20 in which said
co-administration of said compound is separated in time from said
parenteral administration of said therapeutically effective amount
of a multiplicity of said large liposomes by an effective time
period.
24. The method in accordance with claim 26 in which said effective
time period is in the range of 1 minute to two weeks.
25. The method in accordance with claim 17 in which the
therapeutically effective amount is in the range of 10 to 1600
mg/kg/dose.
26. The method in accordance with claim 17 in which the liposomes
are given in repeated doses.
27. The method in accordance with claim 17 further comprising the
step of controlling the expression of said genes by said parenteral
administration of a multiplicity of said large liposomes.
28. The method in accordance with claim 17 in which said
parenchymal cell functions in a system having hepatic sinusiods, an
endothelial layer lining said hepatic sinusoids, and fenestrations;
and, in which the large liposomes are of a size and shape larger
than said fenestrations of said endothelial layer lining said
hepatic sinusoids, whereby said large liposomes are too large to
readily penetrate said fenestrations.
29. The method in accordance with claim 17 in which the large
liposomes are selected from the group consisting of uni-lamellar
liposomes and multi-lamellar liposomes.
30. The method in accordance with claim 17 in which the liposomes
have diameters larger than about 50 nm.
31. The method in accordance with claim 17 in which the liposomes
have diameters larger than about 80 nm.
32. The method in accordance with claim 17 in which the liposomes
have diameters larger than about 100 nm.
33. The method in accordance with claim 17 in which parenteral
administration is selected from the group of intravenous
administration, intra-arterial administration, intramuscular
administration, subcutaneous administration, transdermal
administration, and intraperitoneal administration.
34. A method of suppressing hepatic expression of a cholesterol
ester transfer protein gene in vivo comprising the step of:
parenterally administering a therapeutically effective amount of a
multiplicity of large liposomes comprised of phospholipids, said
phospholipids selected from the group of phospholipids
substantially free of sterol and phospholipids containing sterol,
for an effective period of time, whereby plasma LDL and HDL are
controlled as a result of said administration.
35. The method in accordance with claim 34 further comprising the
step of periodically assaying plasma LDL concentrations with an
assay during said treatment period to assess said plasma LDL.
36. The method in accordance with claim 35 in which said assay is
selected from the group consisting of an assay of plasma esterified
cholesterol, an assay of plasma apolipoprotein-B, a gel filtration
assay of plasma, an ultracentrifugal assay of plasma, a
precipitation assay of plasma, and a immuno turbidometric assay of
plasma.
37. The method in accordance with claim 34 in which the liposomes
are given periodically.
38. The method in accordance with claim 34 in which the
therapeutically effective amount is in the range of 10 mg/kg/dose
to 1600 mg/kg/dose.
39. The method in accordance with claim 34 further comprising the
step of enhancing tissue penetration of a cholesterol acceptor with
co-administration of an effective amount of a compound, said
compound selected from the group consisting of a small acceptor of
cholesterol and a drug that increases endogenous small acceptors of
cholesterol.
40. The method in accordance with claim 39 in which said small
acceptor is selected from the group consisting of a high-density
lipoprotein, a phospholipid protein complex having a group selected
from the group consisting of apoA-I, apoA-II, apoA-IV, apoE,
synthetic fragments thereof, natural fragments thereof, an
amphipathic protein, and an amphipathic peptide, said protein
substantially free of phospholipid, small phospholipid liposomes,
and a small cholesterol acceptor, said drug including an agent that
raises physiologic HDL concentrations, said agent selected from the
group consisting of nicotinic acid, ethanol, a fibric acid, a
cholesterol synthesis inhibitor, a drug that increases HDL
concentrations, and derivatives thereof.
41. The method in accordance with claim 39 further comprising the
step of periodically assaying plasma LDL concentrations with an
assay during said treatment period to assess said plasma LDL.
42. The method in accordance with claim 41 in which said assay is
selected from the group consisting of an assay of plasma esterified
cholesterol, an assay of plasma apolipoprotein-B, a gel filtration
assay of plasma, an ultracentrifugal assay of plasma, a
precipitation assay of plasma, and a immuno turbidometric assay of
plasma.
43. The method in accordance with claim 39 in which said
co-administration of said compound with the administration of said
large liposomes is simultaneous.
44. The method in accordance with claim 39 in which said
co-administration of said compound is separated in time from said
parenteral administration of said therapeutically effective amount
of a multiplicity of said large liposomes by an effective time
period.
45. The method in accordance with claim 44 in which said separation
in time is in the range of 1 minute to two weeks.
46. The method in accordance with claim 34 in which the large
liposomes are of a size and shape larger than fenestrations of an
endothelial layer lining hepatic sinusoids, whereby said large
liposomes are too large to readily penetrate said
fenestrations.
47. The method in accordance with claim 34 in which the large
liposomes are selected from the group consisting of uni-lamellar
liposomes and multi-lamellar liposomes.
48. The method in accordance with claim 34 in which the liposomes
have diameters larger than about 50 nm.
49. The method in accordance with claim 34 in which the liposomes
have diameters larger than about 80 nm.
50. The method in accordance with claim 34 in which the liposomes
have diameters larger than about 100 nm.
51. A method of suppressing the rise in plasma LDL concentrations
after administration of an agent having small acceptors of
cholesterol comprising the step of: co-administering an effective
amount of a multiplicity of an agent having large liposomes
comprised of phospholipids substantially free of sterol with said
administration of said agent having said small acceptors.
52. The method in accordance with claim 51 in which said agent
having small acceptors consists essentially of small acceptors and
in which said agent having large liposomes consists essentially of
large liposomes.
53. The method in accordance with claim 51 in which said
co-administration of said agent having large liposomes is
simultaneous with said administration of said agent having small
acceptors.
54. The method in accordance with claim 51 in which said
co-administration of said agent having large liposomes is separated
in time from said administration of said agent having small
acceptors by an effective time period.
55. The method in accordance with claim 54 in which said effective
time period is in the range of 1 minute to two weeks.
56. A method of diagnosing a side-effect of reverse transport of
cholesterol from peripheral tissues to the liver in vivo
accompanying parenteral administration of a multiplicity of large
liposomes and small liposomes during a treatment period, comprising
the step of: periodically assaying plasma LDL concentrations with
an assay to obtain an assayed LDL concentration, said assay
selected from the group consisting of an assay of plasma esterified
cholesterol, an assay of plasma apolipoprotein-B, a gel filtration
assay of plasma, an ultracentrifugal assay of plasma, and a
precipitation assay having a component, said component selected
from the group consisting of polyanions, divalent cations, and
antibodies, an assay of triglyceride in plasma, and an
immunoturbidometric assay or plasma, to determine the level of a
therapeutically effective amount of each of said liposomes, whereby
a side effect of administration of said liposomes is diagnosed and
effectively regulated.
57. A method of diagnosing and treating a side-effect of reverse
transport of cholesterol from peripheral tissues to the liver in
vivo accompanying parenteral administration of a multiplicity of
large liposomes and small liposomes during a treatment period,
comprising the steps of: periodically assaying plasma LDL
concentrations with an assay to obtain an assayed LDL
concentration, said assay selected from the group consisting of an
assay of plasma esterified cholesterol, an assay of plasma
apolipoprotein-B, a gel filtration assay of plasma, an
ultracentrifugal assay of plasma, and a precipitation assay having
a component, said component selected from the group consisting of
polyanions, divalent cations, and antibodies, an assay of
triglyceride in plasma, and an immunoturbidometric assay or plasma,
to determine the level of a therapeutically effective amount of
each of said liposomes; and, adjusting said therapeutically
effective amount of each of said small and large liposomes in
response to said assayed LDL concentration during said treatment
period.
58. The method in accordance with claim 57 further comprising the
steps of co-administering a pharmaceutical agent to lower said LDL
concentration with said liposome administration, and adjusting the
administration of said agent responsive to said assayed LDL
concentration.
59. An improved method of dialysis treatment of a patient in which
said improvement comprises the step of: administering a
therapeutically effective amount of an agent, said agent selected
from the group consisting of a multiplicity of large liposomes
comprised of phospholipids substantially free of sterol and small
acceptors, during treatment of said patient.
60. The method in accordance with claim 59 in which said dialysis
treatment is selected from the group consisting of hemodialysis,
peritoneal dialysis, and rectal dialysis.
61. The method in accordance with claim 59 in which said agent is
added directly to blood or blood plasma of said patient.
62. The method in accordance with claim 59 in which said
administration of said agent is selected from the group consisting
of extracorporeal administration and intracorporal
administration.
63. The method in accordance with claim 59 in which said agent is
added directly to dialysate of said patient.
64. The method in accordance with claim 59 in which plasma
component concentrations are periodically assayed, said components
selected from the group consisting of LDL, HDL, unesterified
cholesterol, phospholipid, and liposome acceptors.
65. The method in accordance with claim 59 in which said patient
has cells, and further comprising the steps of treating said
patient's cells, together or after separation into erythrocytes,
leukocytes, and platelets, extracorporeally with liposomes, and
periodically assaying a component, said component selected from the
group consisting of cellular cholesterol, phospholipid, fluidity,
fragility, and cell function.
66. The method in accordance with claim 63 in which said dialysate
is assayed for cholesterol, the determine the effectiveness of said
treatment.
67. An improved method of angioplasty in which said improvement
comprises the step of: administering a therapeutically effective
amount of an agent, said agent selected from the group consisting
of a large liposome comprised of phospholipids substantially free
of sterol and small acceptors, during the treatment of said
patient.
68. The method in accordance with claim 67 in which said
administration of said agent occurs at an effective period of
time.
69. The method in accordance with claim 67 in which said
administration of said agent occurs simultaneously with said
angioplasty.
70. The method in accordance with claim 68 in which said effective
period of time is in the range of 1 minute to two years from the
time of said angioplasty.
71. An improved method of reducing the lipid content of arterial
lesions comprising the steps of: inducing the reverse transport of
cholesterol from peripheral tissues to the liver in vivo by
administering a therapeutically effective amount of an agent to a
subject, said agent selected from the group consisting of large
liposomes comprised of phospholipids substantially free of sterol
and small acceptors; periodically monitoring plasma LDL
concentrations of said subject to obtain an LDL concentration
profile; adjusting said therapeutically effective amount of said
agent responsive to said LDL concentration profile; and,
administering a pharmaceutical agent to said subject, said agent
selected from the group consisting of compounds to lower LDL
concentrations, small acceptors, and compounds to raise HDL
concentrations, responsive to said LDL concentration profile,
whereby said reduction in lipid content of said arterial lesions is
effectively treated and monitored over a treatment period.
72. The method in accordance with claim 71 in which said arterial
lesions comprise lipid rich, rupture prone type IV and type V
arterial lesions, whereby plaque rupture, thrombosis, and tissue
infarction are greatly reduced.
73. An improved method of assessing the efficiency of a treatment
for reducing the lipid content of arterial lesions, said lesions
coming into contact with plasma and a component thereof comprising
the steps of: inducing the reverse transport of cholesterol from
peripheral tissues to the liver in vivo by administering a
therapeutically effective amount of an agent to a subject, said
agent selected from the group consisting of large liposomes
comprised of phospholipids substantially free of sterol and small
acceptors; and, periodically monitoring said plasma component with
an assay, said assay selected from the group consisting of an assay
for plasma unesterified cholesterol and phospholipid, an assay of
plasma cholesterol ester transfer protein activity, an assay of
bile acids and cholesterol in stool an assay of bile acids and
cholesterol in bile, an assay of hepatic gene expression in a liver
biopsy, an assay of gene expression in a peripheral blood
leukocytes, said gene comprising a gene involved in cholesterol
metabolism, an assay of plasma LDL concentration, and a vascular
imaging technique.
74. The method in accordance with claim 73 in which said vascular
imaging technique is selected from the group consisting of cardiac
catherization, magnetic resonance imaging, ultrasound, and
ultrafast CT.
75. A method of beneficially altering arterial function and
controlling plasma LDL concentrations and hepatic cholesterol
homeostasis in vivo comprising the step of: parenterally
administering a therapeutically effective amount of a multiplicity
of large liposomes comprised of phospholipids substantially free of
sterol for a treatment period.
76. The method in accordance with claim 75 further comprising the
step of taking a measurement of arterial function, said measurement
selected from the group consisting of a measurement of
endothelial-derived relaxing factor, a measurement of intracellular
calcium concentration in arterial cells, a measurement of arterial
cell proliferation, an assay of arterial enzymes, an assay in the
presence of calcium channel blockers, an assay of arterial uptake
of liposomes, an assay of arterial accumulation of liposomes, and
an assay of arterial retention of liposomes.
77. The method in accordance with claim 76 in which the measurement
of endothelial-derived relaxing factor is selected from the group
consisting of a functional determination of endothelial-dependant
arterial relaxation and chemical determination of production of
said endothelial relaxing factor.
78. A method of beneficially altering blood platelet function while
controlling plasma LDL concentrations, arterial function, hepatic
cholesterol homeostasis and said platelet function in vivo
comprising the step of: parenterally administering a
therapeutically effective amount of a multiplicity of large
liposomes comprised of phospholipids substantially free of sterol
for a treatment period.
79. The method in accordance with claim 78 further comprising the
step of taking a measurement of platelet function, said measurement
selected from the group consisting of a measurement of a ratio of
cholesterol to phospholipid in said platelets, a measurement of
platelet reactivity, a measurement of platelet metabolic markers, a
measurement of platelet calcium fluxes, a measurement of
intracellular calcium, a measurement of platelet aggregability, and
a measurement of platelet granule release.
80. The method in accordance with claim 78 further comprising the
step of taking a measurement of said arterial function, said
measurement selected from the group consisting of a measurement of
endothelial-derived relaxing factor, a measurement of intracellular
calcium concentration in arterial cells, a measurement of arterial
cell proliferation, and an assay of arterial enzymes.
81. The method in accordance with claim 80 in which the measurement
of endothelial relaxing factor is selected from the group
consisting of a functional determination of endothelial-dependant
arterial relaxation and chemical determination of production of
said endothelial relaxing factor.
82. A method of forcing the reverse transport of cholesterol from
peripheral tissues to the liver in vivo and delivering said
cholesterol to said liver while controlling plasma LDL
concentrations, comprising the step of: delivering the cholesterol
to the liver at a sufficiently slow rate so that hepatic
cholesterol homeostasis is free of substantial disruption by
administration of an agent, said agent selected from the group
consisting of large liposomes and small acceptors to a subject,
said large liposomes being substantially incapable of penetrating
endothelial fenestrae of said liver and substantially incapable of
interacting with hepatic parenchymal cells.
83. The method in accordance with claim 82 in which the large
liposomes are chemical compositions of liposomes of a size so that
said liposomes are cleared slowly by the liver.
84. The method in accordance with claim 82 in which the step of
delivering comprises slowly infusing said liposomes.
85. The method in accordance with claim 82 in which the step of
delivering comprises administering small doses of said liposomes,
separated in time, to avoid increasing said LDL concentration.
86. The method in accordance with claim 82 further comprising the
step of periodically assaying said plasma LDL concentrations with
an assay to obtain an assayed LDL concentration, said assay
selected from the group consisting of an assay of plasma esterified
cholesterol, an assay of plasma apolipoprotein-B, a gel filtration
assay of plasma, an ultracentrifugal assay of plasma, and a
precipitation assay having a component, said component selected
from the group consisting of polyanions, divalent cations, and
antibodies, an ultracentrifugal assay of plasma, a precipitation
assay of plasma, and a immuno turbidometric assay of plasma, to
determine the level of a therapeutically effective amount of each
of said liposomes.
87. A method of forcing the reverse transport of cholesterol from
peripheral tissues to the liver in vivo and delivering said
cholesterol to primarily to hepatic Kupffer cells rather than
hepatic parenchymal cells so that hepatic cholesterol homeostasis
is not harmfully disrupted and controlling said homeostasis and
effecting a plasma component, comprising the step of: administering
an effective amount of liposomes, said liposomes selected from the
group of liposomes being substantially incapable of penetrating
endothelial fenestrae of said liver and incapable of subsally
interacting with hepatic parenchymal cells and said liposomes being
of a size, composition or shape so that are directed away from said
hepatic parenchymal cells.
88. The method in accordance with claim 87 further comprising the
step of periodically monitoring said plasma component with an
assay, said assay selected from the group consisting of an assay
for plasma unesterified cholesterol and phospholipid, an assay of
plasma cholesterol ester transfer protein activity, an assay of
bile acids and cholesterol in stool, an assay of bile acids and
cholesterol in bile, an assay of hepatic gene expression in a liver
biopsy, an assay of hepatic gene expression in a peripheral blood
leukocytes, said gene comprising a gene involved in cholesterol
metabolism, an assay of plasma LDL concentration, and a vascular
imaging technique.
89. A method of catabolizing cholesterol with macrophages in vivo
and also affecting a plasma component or structural aspects of an
artery, comprising the step of: administering an effective amount
of liposomes to a subject substantially free of cholesterol and
being of a size and composition such that said liposomes are
capable of being taken up by said marcrophages and capable of being
catabolized by said macrophages, whereby said cholesterol is
mobilized by said liposomes resulting in said liposomes being taken
up by said macrophages and catabolized.
90. The method in accordance with claim 89 further comprising the
step of periodically monitoring said plasma component with an
assay, said assay selected from the group consisting of an assay
for plasma unesterified cholesterol and phospholipid, an assay of
plasma cholesterol ester transfer protein activity, an assay of
bile acids and cholesterol in stool, an assay of bile acids and
cholesterol in stool, an assay of hepatic gene expression in a
liver biopsy, an assay of gene expression in a peripheral blood
leukocytes, said gene comprising a gene involved in cholesterol
metabolism, an assay of plasma LDL concentration, and a vascular
imaging technique.
91. A method of forcing the reverse transport of cholesterol from a
body part, said body part selected from the group of peripheral
tissues, cells, and platelets, in vivo comprising the step of:
administering for a treatment period a therapeutically effective
amount of a multiplicity of non-liposomal particles for cholesterol
depletion of peripheral tissues while avoiding harmful disruptions
of hepatic cholesterol homeostasis, said particles being selected
from the group of a particle substantially free of cholesterol and
particles free of cholesterol.
92. The method in accordance with claim 91 in which said
non-liposomal particles are selected from the group consisting of
triglyceride-phospholipid emulsions, said emulsions selected from
the group of emulsions that are not taken up rapidly by hepatic
parenchymal cells, emulsions that are not taken up to a large
extent by parenchymal cells, and triglyceride-phospholipid-protein
emulsions.
93. A method of delivering a drug in vivo and avoiding harmful
disruptions of hepatic cholesterol homeostasis, comprising the
steps of: entrapping said drug with an agent, said agent selected
from the group consisting of a cholesterol poor liposome, a
cholesterol free liposome, an emulsion, a liposome primarily taken
up slowly by hepatic parenchymal cells, an emulsion primarily taken
up slowly by hepatic parenchymal cells, said agent selected from
the group consisting of an agent with a protein and an agent
without protein to obtain an entrapped drug; and, administering a
therapeutically effective amount of said entrapped drug for a
treatment period.
94. The method in accordance with claim 93 in which the step of
administering comprises the step of slowly infusing said entrapped
drug.
95. The method in accordance with claim 93 in which the step of
administering comprises the step of administering small doses of
said agent, appropriately separated in time, to avoid harmfull
disruptions in hepatic cholesterol homeostasis.
96. The method in accordance with claim 93 in which the step of
administering includes using low doses of said agent, whereby
disrupting hepatic cholesterol homeostasis is avoided.
97. A method of controlling plasma LDL levels in vivo and lowering
blood viscosity, comprising the step of: parenteral administering a
therapeutically effective amount of a multiplicity of large
liposomes comprised of phospholipids substantially free of sterol
for a treatment period, said effective amount administered in a
dosage, said dosage selected from a single dose and repeated
doses.
98. The method in accordance with claim 97 further comprising the
steps of taking a measurement, said measurement selected from the
group consisting of a measurement of blood flow in a carotid
artery, measurement of blood flow in a coronary artery, a
measurement of blood flow in a lower limb, an ultrasound
measurement of blood flow in other vessels, an MRI measurement of
blood flow, a radioisotope tracer measurement of blood flow, and a
measurement of blood viscosity.
99. A method of controlling plasma LDL levels in vivo and reducing
the sphingomyelin to phosphatidylcholine ratio in a cell membrane
and cell aging, comprising the step of: parenterally administering
a therapeutically effective amount of a multiplicity of large
liposomes comprised of phospholipids substantially free of sterol
for a treatment period, said effective amount administered in a
dosage, said dosage selected from a single dose and repeated
doses.
100. The method in accordance with claim 99 further comprising the
step of periodically assaying said cells with an assay, said assay
selected from the group consisting of an assay of sphingomyelin, an
assay of phosphatidylcholine, an assay of membrane function, and an
assay of cellular function.
101. A method of controlling hepatic secretion of apolipoprotein-B
while forcing the reverse transport of cholesterol from peripheral
tissues to the liver, comprising the step of: parenterally
administering a therapeutically effective amount of a multiplicity
of large liposomes comprised of phospholipids substantially free of
sterol for a treatment period, said effective amount administered
in a dosage, said dosage selected from a single dose and repeated
doses.
102. A method of controlling hepatic expression of 7-alpha
hydroxylase while forcing the reverse transport of cholesterol from
peripheral tissues to the liver, comprising the step of:
parenterally administering a therapeutically effective amount of a
multiplicity of large liposomes comprised of phospholipids
substantially free of sterol for a treatment period, said effective
amount administered in a dosage, said dosage selected from a single
dose and repeated doses.
103. A method of controlling plasma LDL levels, hepatic cholesterol
homeostasis, arterial enzymes, platelet function, and hepatic gene
expression, comprising the step of: parenterally administering a
therapeutically effective amount of a multiplicity of large
liposomes comprised of phospholipids substantially free of sterol
for a treatment period, said effective amount administered in a
dosage, said dosage selected from a single dose and repeated
doses.
104. The method in accordance with claim 103 further comprising the
step of diagnosing the efficacy of said administration by taking a
measurement of enzyme activity, said measurement selected from the
group consisting of a measurement of an arterial lipase activity, a
measurement of arterial triglyceride lipase activity, a cholesterol
esterase activity, a measurement of lysophospholipase activity, a
cholesterol synthesis inhibitor, a drug that increases HDL
concentrations, and derivatives thereof.
125. An improved dialysis apparatus for the treatment of a patient
in which said improvement comprises: means for administering a
therapeutically effective amount of an agent, said agent selected
from the group consisting of a multiplicity of large liposomes
comprised of phospholipids substantially free of sterol and small
acceptors, during treatment of said patient.
126. The apparatus of claim 125 in which said means for
administering said agent is selected from the group consisting of
means for extracorporeal administration and means for intracorporal
administration.
127. An improved angioplasty apparatus in which said improvement
comprises: means for administering a therapeutically effective
amount of an agent, said agent selected from the group consisting
of a large liposome comprised of phospholipids substantially free
of sterol and small acceptors, during the treatment of said
patient.
128. A pharmaceutical composition for reducing the size of arterial
lesions that enters the liver of a subject consisting essentially
of a multiplicity of non-liposomal particles for cholesterol
depletion of peripheral tissues while avoiding harmful disruptions
of hepatic cholesterol homeostasis, said particles being selected
from the group of particles substantially free of cholesterol and
particles free of cholesterol for a treatment period.
129. The pharmaceutical composition of claim 128 in which said
non-liposomal particles are selected from the group consisting of
triglyceride-phospholipid emulsions, said emulsions selected from
the group of emulsions that are not taken up rapidly by hepatic
parenchymal cells, emulsions that are not taken up to a large
extent by parenchymal cells, and triglyceride-phospholipid-protein
emulsions.
130. A pharmaceutical composition for reducing the size of arterial
lesions that enters the liver of a subject consisting essentially
of a drug entrapped within an agent, said agent selected from the
group consisting of a cholesterol poor liposome, a cholesterol free
liposome, an emulsion, a liposome primarily taken up slowly by
hepatic parenchymal cells, an emulsion primarily taken up slowly by
hepatic parenchymal cells, said agent selected from the group
consisting of an agent with a protein and an agent without
protein.
131. A pharmaceutical composition for increasing plasma HDL
concentrations, while controlling plasma LDL levels, hepatic
cholesterol homeostasis, and hepatic gene expression, comprising a
first agent, said first agent comprising a multiplicity of small
liposomes to raise HDL concentrations, and a second agent, said
second agent comprising large liposomes comprised of phospholipids
substantially free of sterol.
132. An improved pharmaceutical composition for reducing the size
of arterial lesions that enters the liver of a subject, said
composition consisting essentially of liposomes, in which said
improvement comprises an anti-oxidant and derivatives thereof.
133. The pharmaceutical composition of claim 132 in which said
anti-oxidant is selected from the group consisting of vitamin E,
ascorbate, probucol, carotenoids, derivates thereof, and a material
resistant to oxidation.
134. An improved pharmaceutical composition for reducing the size
of arterial lesions that enters the liver of a subject, said
improvement comprising palmitoyl-oleyl-phosphatidyl choline.
135. An improved pharmaceutical composition for reducing the size
of arterial lesions that enters the liver of a subject, said
improvement comprising a double bond in a second fatty acyl chain
of components of said composition, whereby said composition is more
fluid and more readily accepts cholesterol transfer from said
arterial lesions.
136. An improved pharmaceutical composition for reducing the size
of arterial lesions that enters the liver of a subject, said
improvement comprising PEG-liposomes.
137. A method of treating atherosclerosis in a subject comprising
the step of administering a liposome composition to said subject,
said liposome composition selected from the group consisting of
unilamellar liposomes and multilamellar liposomes, said liposomes
having an average diameter of about 50-150 nanometers, in which LDL
levels in said subject do not increase.
138. A method of controlling cholesterol metabolism in hepatic
parenchymal cells in a subject in vivo through cell-cell
communication from Kupffer cells to said parenchymal cells,
comprising the step of administering a liposome composition to said
subject, said liposome composition selected from the group
consisting of large unilamellar liposomes and large multilamellar
liposomes, said liposomes having an average diameter of about
50-150 nanometers, in which LDL levels in said subject do not
increase.
139. A method in accordance with claim 138 further comprising the
steps of diagnosing the efficacy of said control of said
cholesterol metabolism by assaying an indicator in said subject,
said indicator selected from the group consisting of plasma LDL
concentrations of said subject, hepatic gene expression of said
subject, plasma CETP levels of said subject, sterol excretion of
controlling cholesterol metabolism in hepatic parenchymal cells in
said subject, and sterol excretion in bile of said subject; and
adjusting said administration in response to said assay.
Description
CONTINUING DATA
[0001] This application is a continuation in part regular patent
application of pending U.S. provisional patent application serial
No. 60/005,090 filed by Kevin Jon Williams, a citizen of the United
States, residing at 425 Wister Road, Wynnewood, Pa. 19096 on Oct.
11, 1995 entitled "METHOD OF FORCING THE REVERSE TRANSPORT OF
CHOLESTEROL FROM PERIPHERAL TISSUES TO THE LIVER IN VIVO WHILE
CONTROLLING PLASMA LDL AND COMPOSITIONS THEREFOR." Pending U.S.
provisional patent application serial No. 60/005,090 filed Oct. 11,
1995 is attached to the instant regular patent application as
attachment A. Applicant expressly incorporates attachment A hereto
into the instant regular patent application by reference thereto as
if fully set forth.
BACKGROUND OF THE INVENTION
[0002] Several human conditions are characterized by distinctive
lipid compositions of tissues, cells, membranes, and extracellular
regions or structures. For example, in atherosclerosis, cholesterol
(unesterified, esterified, and oxidized forms) and other lipids
accumulate in cells and in extracellular areas of the arterial wall
and elsewhere. These lipids have potentially harmful biologic
effects, for example, by changing cellular functions, including
gene expression, and by narrowing the vessel lumen, obstructing the
flow of blood. Removal of these lipids would provide numerous
substantial benefits. Moreover, cells, membranes, tissues, and
extracellular structures will benefit in general from compositional
alterations that include increasing resistance to oxidation and
oxidative damage, such as by increasing the content and types of
anti-oxidants, removing oxidized material, and increasing the
content of material that is resistant to oxidation. In aging, cells
have been shown to accumulate sphingomyelin and cholesterol, which
alter cellular functions. These functions can be restored in vitro
by removal of these lipids and replacement with phospholipid from
liposomes. A major obstacle to performing similar lipid alterations
in vivo has been disposition of the lipids mobilized from tissues,
cells, extracellular areas, and membranes. Natural (e.g.,
high-density lipoproteins) and synthetic (e.g., small liposomes)
particles that could mobilize peripheral tissue lipids have a
substantial disadvantage: they deliver their lipids to the liver in
a manner that disturbs hepatic cholesterol homeostasis, resulting
in elevations in plasma concentrations of harmful lipoproteins,
such as low-density lipoprotein (LDL), a major atherogenic
lipoprotein. There exist a need for better methods to manipulate
the lipid content and composition of peripheral tissues, cells,
membranes, and extracellular regions in vivo.
[0003] The intravenous administration of cholesterol-poor
phospholipid vesicles (liposomes) or other particles that transport
cholesterol and other exchangeable material from lipoproteins and
peripheral tissues, including atherosclerotic arterial lesions, to
the liver produces substantial derangements of hepatic cholesterol
homeostasis, such as enhanced hepatic secretion of
apolipoprotein-B, and suppression of hepatic LDL receptors. The
hepatic derangements lead to increase plasma concentrations of LDL
and other atherogenic lipoproteins. Increased concentrations of LDL
or other atherogenic lipoproteins will accelerate, not retard, the
development of vascular complications. Deranged hepatic cholesterol
homeostasis can also be manifested by abnormal regulation of genes,
such as a gene for the LDL receptor, a gene for HMG-CoA reductase,
a gene for cholesterol 7-alpha hydroxylase, and a gene regulating a
function involved in cholesterol homeostasis. There exists a need
for methods or compounds that can produce a removal of cholesterol
and other exchangeable material, from peripheral cells, tissues,
organs, and extracellular regions, and that can produce a delivery
of material, such as phospholipids, to cells, tissues, or organs,
extracellular regions, but without harmfully disrupting hepatic
cholesterol homeostasis and plasma concentrations of atherogenic
lipoproteins.
[0004] The invention described herein provides methods and
compositions related to the removal of cholesterol and other
exchangeable material from peripheral tissues, and otherwise
altering peripheral tissue composition, while controlling plasma
concentrations of LDL and other atherogenic lipoproteins and
avoiding harmful disruptions of hepatic cholesterol
homeostasis.
SUMMARY OF THE INVENTION
[0005] The present invention provides a pharmaceutical composition,
kit, and method of forcing the reverse transport of cholesterol
from peripheral tissues to the liver in vivo while controlling
plasma LDL concentrations. The method includes the step of
administering a therapeutically effective amount of a multiplicity
of large liposomes comprised of phospholipids substantially free of
sterol for a treatment period. The method optionally includes the
step of periodically assaying plasma LDL concentrations with an
assay during the treatment period to assess plasma atherogenic
lipoprotein concentrations and obtain an atherogenic lipoprotein
profile, and adjusting the administration in response to said
profile. The large liposomes are dimensioned larger than
fenestrations of an endothelial layer lining hepatic sinusoids in
the liver so that the liposomes are too large to readily penetrate
the fenestrations. The therapeutically effective amounts are in the
range of about 10 mg to about 1600 mg phospholipid per kg body
weight per dose. A pharmaceutical composition and related kit for
mobilizing peripheral cholesterol and sphingomyelin that enters the
liver of a subject consisting essentially of liposomes of a size
and shape larger than fenestrations of an endothelial layer lining
hepatic sinusoids in the liver is also provided.
[0006] It is an object of the present invention to provide a better
method to manipulate the lipid content and composition of
peripheral tissues, cells, membranes, and extracellular regions in
vivo.
[0007] It is a further object of the invention to provide methods
or compounds that can produce a removal of cholesterol and other
exchangeable material, from peripheral cells, tissues, organs, and
extracellular regions, and that can produce a delivery of material,
such as phospholipids, to cells, tissues, or organs, extracellular
regions, but without harmfully disrupting hepatic cholesterol
homeostasis and plasma concentrations of atherogenic
lipoproteins.
[0008] The objects and features of the present invention, other
than those specifically set forth above, will become apparent in
the detailed description of the invention set forth below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 is a side cross-sectional view of a lipoprotein and a
liposome;
[0010] FIG. 2 illustrates a table of hepatic mRNA content
(pg/.mu.g) for CETP, HMG-CoAR, LDL receptors, and 7a-hydroxylase;
and LDL ChE;
[0011] FIGS. 3 and 4 illustrate plasma LDL cholesteryl ester
concentrations in response to injections of LUVs, SUVs or saline
over time in one variant:
[0012] FIG. 5 illustrates LDL receptor mRNA levels in liver in
response to injections of LUVs, SUVs or saline over time;
[0013] FIG. 6 illustrates HMG-CoA reductase mRNA levels in liver in
response to injection of LUVs, SUVs, or saline;
[0014] FIG. 7 Illustrates cholesteryl ester transfer protein mRNA
levels in liver in response to injection of LUVs, SUVs, or
saline;
[0015] FIG. 8 illustrates 7-alpha hydroxylase mRNA levels in liver
in response to injections of LUVs, SUVs, or saline;
[0016] FIG. 9 illustrates key points about LUVs and
atherosclerosis;
[0017] FIG. 10 illustrates plasma LDL unesterified cholesterol
concentrations in response to injections of LUVs, SUVs or saline
over time.
[0018] FIG. 11 illustrates plasma LDL esterified cholesterol
concentrations in response to injections of LUVs, SUVs or saline
over time;
[0019] FIG. 12 illustrates LDL esterified cholesterol
concentrations in response to injections of LUVs, SUVs or
saline;
[0020] FIG. 13 illustrates plasma VLDL esterified cholesterol
concentrations in response to injections of LUVs, SUVs or
saline;
[0021] FIGS. 14 and 15 illustrate HDL esterified cholesterol
concentrations in response to injections of LUVs, SUVs or
saline;
[0022] FIG. 16 illustrates the time course of cholesterol
mobilization following an LUV injection into control or apoE KO
mice;
[0023] FIG. 17 illustrates the time course of LUV clearance in
control mice and apoE mice;
[0024] FIG. 18 illustrates that the compositions and methods of the
present invention are effective in humans;
[0025] FIG. 19 illustrates a perspective view of an improved
hemodialysis system of the present invention and improved method of
hemodialysis;
[0026] FIG. 20 illustrates a perspective view of an improved
peritoneal dialysis system 2000 and method of peritoneal
dialysis;
[0027] FIG. 21 illustrates a perspective view of a variant of an
improved peritoneal dialysis system with assaying means 2100 and
method of peritoneal dialysis and analysis of spent fluid;
[0028] FIG. 22 illustrates a perspective view of an improved
cardiac catheterization and/or angioplasty system 2200 and method
of cardiac catheterization and/or angioplasty;.
[0029] FIG. 23 illustrates a perspective view of a variant of an
improved cardiac catheterization and/or angioplasty system 2300 and
method of cardiac catheterization and/or angioplasty;
[0030] FIG. 24 illustrates a graph of hepatic lipid contents in
response to injections of LUVs, SUVs, or saline;
[0031] FIG. 25 illustrates plasma free cholesterol concentrations
following repeated injections of SUVs or LUV (300 mg/kg) in NZW
rabbits;
[0032] FIG. 26 illustrates plasma cholesterol ester concentrations
following repeated injections of SUVs or LUV (300 mg/kg) in NZW
rabbits;
[0033] FIG. 27 illustrates alternations in plasma components after
repeated injections of SUVs; and,
[0034] FIG. 28 illustrates an agarose gel electrophoresis of whole
plasma following repeated injections of LUVs, SUVs, or saline.
DETAILED DESCRIPTION OF THE INVENTION
[0035] FIG. 1 illustrates a schematic illustration of the structure
of a normal lipoprotein 100 and a unilamellar liposome 200.
Lipoprotein 100 and liposome 200 are comprised of a phospholipid
molecule 300. Phospholipid molecules generally have polar head 500
and a fatty acyl chains 400. Molecule 600 represents a molecule of
unesterifed cholesterol. Lipoprotein 100 is comprised of a
hydrophobic core 102 composed mainly of triglycerides and
cholesteryl esters surrounded by a monolayer of phospholipid
molecules 300 with their fatty acyl side chains 400 facing the
hydrophobic core 102 and their polar heads 500 facing the
surrounding aqueous environment (not shown). Unesterified
cholesterol 600 is found largely within the phospholipid monolayer.
Apolipoprotein 700 is disposed within phospholipid molecules 300.
Artificial triglyceride emulsion particles have essentially
identical structures, either with or without protein.
[0036] Liposome 200 is comprised of phospholipid molecules 300
forming a phospholipid bilayer, e.g. one lamella, either with or
without protein, in which fatty acyl side chains 400 face each
other, the polar head groups 500 of the outer leaflet face outward
to the surrounding aqueous environment (not shown), and the polar
head groups 500 of the inner leaflet face inward to the aqueous
core 202 of the particle 200. Depending on the composition of
particle 200. phospholipid bilayers can have a large capacity for
unesterified cholesterol and other exchangeable material and
components thereof. As illustrated in FIG. 1 there is no sterol.
Typically, such liposomes can pick up unesterified cholesterol from
other lipid bilayers, such as cell membranes, and from
lipoproteins. Liposomes also pick up proteins and donate
phospholipids and other exchangeable material and components
thereof. Liposomes can also have multilamellar structures, in which
the bilayers are contained within the environment encapsulated by
an outer bilayer to form multiple lamellae. The multiple lamellae
can be arranged concentrically, like the layers of an onion, or in
another variant non-concentrically.
[0037] FIGS. 3 and 4 illustrate plasma LDL cholesteryl ester
concentrations in response to injections of LUVs, SUVs or saline
over time. Rabbits were intravenously injected on days 1, 3 and 5
as indicated by arrows 302, 304, and 306 respectively, with a bolus
of 300 mg of phosphatidylcholine per kg of body weight or a matched
volume of saline. The phosphatidylcholine was pharmaceutical grade
egg PC, in the form of either large unilamellar vesicles (LUVs)
having diameters of approximately 100 NM (preferably.about.120 NM)
prepared by extrusion (LUVs were measured at about 120 NM (123+35
NM and the extrusion membrane had pores of about 100 NM in
diameter) or small unilamellar vesicles with diameters of
approximately 30 NM (preferably 35 NM) prepared by sonication.
(SUVs were measured in the range of 34-30 NM.) Blood was drawn just
before each injection and on the sixth day at sacrifice. Plasma LDL
cholesteryl ester concentrations were determined by a gel
filtration assay of the plasma with an in-line enzymatic assay for
cholesteryl ester. Means+SEMs are shown in FIG. 3. Animals infused
with SUVs showed significantly higher plasma concentrations of LDL
cholesteryl ester at days 3, 5, and 6 compared to either
LUV-infused or saline infused animals. FIGS. 2-8, 10-15, 24 and 28
illustrate data from the same experiment in which injections were
made on days 1, 3, and 5 and then livers were taken. Gel filtration
was done of plasma to measure lipid contents of individual
lipoprotein classes. FIG. 2 illustrates a table of hepatic mRNA
content (pg/.mu.g) for CETP, HMG-CoA R (hydroxy methylglutaryl
coenzyme A reductase), LDL receptors, and cholesterol 7
alpha-hydroxylase; and LDL ChE (low density lipoprotein cholesteryl
ester) for the rabbits given saline (HEPES buffered saline)
(rabbits 1-4). LUVs (rabbits 5-8), and SUVs (rabbits 10-12) for the
experiment described for FIGS. 3 and 4. Rabbit 13 is the "Mix"
rabbit.
[0038] FIG. 4 shows an animal labeled as mix. "Mix" refers to a
single animal that received SUVs on day 1, 3 and 5, but also one
injection of LUVs on day 3. Before this injection of LUVs, the
plasma concentration of LDL cholesteryl ester rose, but after the
injection of LUVs, the LDL concentration fell, despite continued
injections of SUVs.
[0039] FIG. 5 illustrates LDL receptor mRNA levels in liver in
response to injections of LUVs, SUVs or saline over time. The
rabbits described above were sacrificed at day 6, and samples of
liver were snap-frozen in liquid nitrogen. mRNA was extracted, and
rabbit mRNA for the LDL receptor was quantified by an internal
standard/RNase protection assay (Rea T. J. et al. J. Lipid Research
34:1901-1910, 1993 and Pape M. E., Genet. Anal. 8:206-312, 1991).
Means+SEMs are shown in FIG. 5. Animals infused with SUVs showed
significant suppression of hepatic LDL receptor mRNA compared to
LUV-infused or saline-infused animals. Suppression of hepatic LDL
receptor mRNA reflects parenchymal cell overload with sterol, and
is a potentially harmful alteration from normal hepatic cholesterol
homeostasis. In contrast, LUV-infused animals showed the highest
levels of hepatic LDL receptor mRNA, though the increase above that
seen in the saline-infused animals did not reach statistical
significance. The liver from the "Mix" animal described above
showed a value of 5.28 pg LDL receptor mRNA/microgram which is
closer to the average value in the saline group than in the SUV
group. Thus, LDL receptor mRNA was stimulated by the single
injection of LUVs despite repeated injections of SUVs.
[0040] FIG. 6 illustrates HMG-CoA reductase mRNA levels in liver in
response to injections of LUVs, SUVs, or saline. The experimental
details are those as referenced above. Animals infused with SUVs
showed significant suppression of hepatic HMG-CoA reductase mRNA
compared to LUV-infused or saline infused animals. Suppression of
hepatic HMG-CoA reductase mRNA reflects parenchymal cell overload
with sterol, which can be a potentially harmful alteration from
normal hepatic cholesterol homeostasis. In contrast. LUV-infused
animals showed the highest levels of hepatic HMG-CoA reductase
mRNA, though the increase above that seen in the saline-infused
animals did not reach statistical significance.
[0041] The "mix" animal showed a value of 0.50 pg HMG-CoA reductase
mRNA/microgram, which is essentially identical to the average value
in the saline group (0.51) and substantially higher than the value
in the SUV group (0.27). Thus, HMG-CoA reductase mRNA was
stimulated to its normal value by the single injection of LUVs,
despite repeated injections of SUVs.
[0042] FIG. 7 illustrates cholesteryl ester transfer protein mRNA
levels in liver in response to injection of LUVs, SUVs, or saline.
The experimental details are those as referenced above. Animals
infused with LUVs showed significant suppression of hepatic CETP
mRNA compared to SUV infused or saline infused animals. Suppression
of CETP mRNA produce changes in the plasma lipoprotein profile
usually associated with reduced risk of atherosclerosis. The "mix"
animal showed a value of 3.18 pg CETP mRNA/microgram, which is
closer to the average value in the LUV group than in the SUV or
saline groups. Thus, CETP mRNA was suppressed by the single
injection of LUV's despite repeated injections of SUVs.
[0043] FIG. 8 illustrates cholesterol 7-alpha hydroxylase mRNA
levels in liver in response to injections of LUVs, SUVs, or saline.
The experimental details are those as reference above. Animals
infused with SUVs showed suppression of hepatic 7-alpha hydroxylase
mRNA compared to LUV infused or saline infused animals. Suppression
of 7-alpha hydroxylase can be a potentially harmful alteration from
normal hepatic homeostasis. In contrast, LUV-infused animals showed
the highest levels of hepatic 7-alpha hydroxylase mRNA, though the
increase above that seen in the saline infused animals did not
reach statistical significance. The "mix" animal showed a value of
0.51 pg 7-alpha hydroxylase mRNA/microgram, which is higher than
the average value in the SUV group. Thus, 7-alpha-hydroxylase mRNA
was stimulated by the single injection of LUVs, despite repeated
injections of SUVs.
[0044] FIG. 10 illustrates unesterified cholesterol concentrations
in whole plasma in response to injections of LUVs, SUVs, or saline
over time. The experimental details are those as referenced above.
As indicated by this figure, LUVs and SUVs significantly raised the
plasma concentrations of unesterfied cholesterol indicating
mobilization of tissue stores. The LUVs raised the concentration of
unesterifed cholesterol more than did the SUVs.
[0045] FIG. 11 illustrates esterified cholesterol concentrations in
whole plasma in response to injections of LUVs, SUVs or saline over
time. The experimental details are those as referenced above. SUVs
raised the plasma concentrations of cholesteryl ester on days 3.5,
and 6. FIG. 12 duplicates the information contained in FIG. 3.
[0046] FIG. 13 illustrates plasma VLDL esterified cholesterol
concentrations in response to injections of LUVs, SUVs, or saline.
SUVs increased the plasma concentration of VLDL cholesteryl ester
over that seen in the saline of LUV treated groups. The "mix"
animal showed a plasma VLDL cholesteryl ester concentration at day
6 of 2.4 mg/dl, which is lower than the average value in the SUV
group. The experimental details are those as referenced above.
[0047] FIGS. 14 and 15 illustrate HDL esterified cholesterol
concentrations in response to injections of LUVs, SUVs, or saline.
The experimental details are those as referenced above as in FIG.
2. Suitable phospholipid can be obtained from Avanti Polar Lipids,
Nippon Oil and Fat in Japan and Princeton Lipids, as well as other
suppliers. LUVs are made through an extruder that is commercially
available. SUVs caused a small but statistically significant rise
in HDL cholesteryl ester concentrations on days five and six.
[0048] FIG. 16 illustrates the time course of cholesterol
mobilization following an LUV injection into control or apoE KO
(knock-out) mice commercially available from Jackson Laboratories,
in Bar Harbor, Me. Control (C57/BL6) and apolipoprotein E knock-out
mice were injected at time zero with a single bolus of 300 mg LUV
phospholipid/kg body weight. The LUVs contained a tracer amount of
labeled cholesteryl hexadecylether, which remains on the liposomes
after injection into a mouse. Displayed data are for concentrations
of total cholesterol, i.e. esterified plus unesterifed, in whole
plasma. The rise in both sets of animals indicated that LUVs
mobilize cholesterol into the plasma, even in the presence of a
severe genetic hyperlipidemia.
[0049] FIG. 17 illustrates the time course of LUV clearance in
control mice and apoE mice. The experimental details are as
described in FIG. 16. The clearance of LUVs from the plasma is
unimpaired in the apoE knock-out mice, indicating mobilization
(FIG. 16) and disposal (FIG. 17) of cholesterol even in the
presence of a severe genetic hyperlipidemia. This indicates the
usefulness of this preparation in hyperlipidemias.
[0050] FIG. 18 illustrates exemplary applications for the
compositions and methods of the present invention in humans. The
therapeutic targets of the compositions and methods presented
herein are lipid-rich, rupture prone plaques, critical stenosis,
post-angioplasty re-stenosis, atherosclerosis in general, and any
membrane, cell, tissue, organ, and extracellular region and/or
structure, in which compositional and/or functional modifications
would be advantageous.
[0051] FIG. 19 illustrates a perspective view of an improved
hemodialysis system of the present invention and improved method of
hemodialysis. Blood is taken from a site for circulatory access
(shown here as arm 1900) and transported into a cell-plasma
separator 1910. The plasma is then transported to a dialysis
chamber 1920 and is divided into at least two compartments that are
separated by a semi-permeable membrane 1930. One side of the
membrane 1930 is the patient's plasma 1940 and on the other side is
the dialysate 1950. Selected molecules exchange across the membrane
1930 depending on the characteristics of the membrane (charge, pore
size, etc.). The device 1960 comprises a device for adding lipid
acceptors to the dialysate and for sampling the dialysate to allow
assays of cholesterol, phospholipid, and other components, such as
acceptors, specific lipoproteins, specific components, and to
monitor treatment. Extraction of plasma cholesterol or other
extractable material comprises several possibilities: 1) acceptors
are disposed in the dialysate that do not cross membrane 1930 into
plasma: 2) the acceptors do cross membrane 1930 and are either left
in the plasma and returned to the patient or are separated from
plasma before it is returned to the patient; and/or 3) immobilized
acceptors on a sheet (such as membrane 1930 itself), on beads,
and/or on the walls of the chamber 1920. Plasma thus treated is
returned to the patient, usually after having being re-mixed with
the blood cells. As noted, cholesterol acceptors can be added at
any stage, as an example, a device 1970 comprises acceptors and for
adding acceptors to plasma shortly before its return into the
patient is also illustrated in FIG. 19. It is further understood
that contaminating cellular material, such as platelets, in the
plasma will also become cholesterol depleted in endogenous lipids
and enriched in phospholipid. It is further understood that all
acceptors mentioned throughout this application may accept
molecules in addition to cholesterol and may donate material as
well.
[0052] The cellular concentrate from the cell-plasma separator 1910
can then be treated in any of several ways before being returned to
the patient: 1) returned to the patient with no further treatment
(this includes being mixed with plasma that has been treated as
above); 2) transferred to a second dialysis chamber (not shown) in
which the dialysate contains cholesterol acceptors to lipid deplete
the cells of endogenous lipids, such as cholesterol, before their
return to the patient; 3) mixed with a suspension or solution of
lipid acceptors to lipid deplete the cells of endogenous lipids,
then either returned to the patient with the acceptors or option 1)
and option 2) above can be performed with all cell types together,
or after further separation into specific cell types (for example,
purified platelets could be lipid depleted of endogenous lipids,
such as cholesterol, and enriched in liposomal lipids). Options 2)
and 3) can be performed with periodic assays of cellular
cholesterol, phospholipid, fluidity, viscosity, fragility, cell
composition and/or cell function. Devices 1960, 1970 include an
apparatus that allows for the periodic sampling of cells during
treatment. As with plasma, lipid acceptors can be added at any
stage of the treatment. All fluids, e.g. plasma and concentrated
cells, are moved by gravity, mechanically, by manual manipulation
(a syringe), or with pumps as needed. Of course, it is understood
that blood can be drawn for processing from any appropriate part of
the body.
[0053] FIG. 20 illustrates a perspective view of an improved
peritoneal dialysis system 2000 and method of peritoneal dialysis.
Patient's abdomen 2010 (FIGS. 20-21) receives peritoneal dialysate
2020 stored in container 2030 into the peritoneal cavity through
incision 2040 by way of channel 2050. Lipid acceptors and/or
cholesterol acceptors 2060 are optionally disposed in container
2070. In another variant, lipid acceptors are added to dialysate
2020; added to container 2030 in concentrated form shortly before
infusion; added as shown to the stream of fluid entering the
peritoneal cavity; or infused by a separate portal of entry into
the patient by any effective route. Throughout this application, it
is understood that all acceptors may accept molecules in addition
to cholesterol and may donate material such as phospholipids and
antioxidants.
[0054] FIG. 21 illustrates a perspective view of a variant of an
improved peritoneal dialysis system with assaying means 2100 and
method of peritoneal dialysis and analysis of spent fluid.
Container 2110 accepts spent fluid from abdomen 2010 by way of
channel 2120. The device 2110 provides access to diagnostic samples
of spent dialysate to allow for assay of cholesterol, phospholipid,
and other parameters as described herein showing the efficacy of
the treatments described. Optionally, assay syringe 2130 is
inserted by way of access portal 2140 into channel or tube 2120, or
into container 2110, and optional pumps (not shown) are used to
move the various fluids to appropriate locations for assay
thereof.
[0055] FIG. 22 illustrates a perspective view of an improved
cardiac catheterization and/or angioplasty system 2200 and method
of cardiac catheterization and/or angioplasty. Patient 2210
undergoes cardiac catherization and/or angioplasty. The patient
intravenously receives effective doses of lipid acceptors or
cholesterol acceptors 2230 co-administered with said treatment(s)
from container 2220. Intraarterial access of a catheter for
coronary angiography and/or angioplasty allows for ready
co-administration of cholesterol acceptors and administration of
diagnostic agents such as cholinergic agents, to assess vascular
function.
[0056] FIG. 23 illustrates a perspective view of a variant of an
improved cardiac catheterization and/or angioplasty system 2300 and
method of cardiac catheterization and/or angioplasty. Catherization
and/or angioplasty catheter 2310 has apertures 2320 that allow for
the egress of cholesterol acceptors therefrom. In a variant,
catheter 2310 has a permeable membrane that allow for the egress
for cholesterol acceptors therefrom. Phantom arrows 2330 indicate
egress sites for cholesterol acceptors and/or diagnostic agents.
Sites 2340 indicate entry sites for the acceptors or agents. The
balloon on the device 2300 can be replaced or supplemented with
other devices or can form an inner balloon layer disposed within an
outer balloon layer. The acceptors are disposed between the inner
and outer flexible balloon layers. Upon expansion of said inner
balloon layer a force is exerted against the fluid or gel-like
acceptors forcing the acceptors out of the sites 2320, and into
direct contact (forcefully) against arterial lesions more locally
directing the treatment. It will be appreciated that this variant
of the invention provides for maximal penetration of the acceptors
into the arterial lesions. The infusions can be accomplished by
gravity, manual manipulation of a syringe, or by mechanical
infusion pump 2350. The same method and system can be utilized with
standard vascular imaging techniques or vessels that include the
femorals, carotids, and mesenteric vessels by way of example.
[0057] Patient 2210 undergoes cardiac catherization and/or
angioplasty. The patient intravenously receives effective doses of
cholesterol or lipid acceptors 2230 co-administered with said
treatments(s) from container 2220. Intraarterial access of a
catheter for coronary angiography and/or angioplasty allows for
ready co-administration of lipid or cholesterol acceptors and
administration of diagnostic agents such as cholinergic agents, to
assess vascular function.
[0058] Container 2110 accepts spent fluid from abdomen 2010 by way
of channel 2120. The device 2110 provides access to diagnostic
samples of spent dialysate to allow for assay of cholesterol,
phospholipid, and other parameters as described herein showing the
efficacy of the treatments described. Optionally, assay syringe
2130 is inserted by way of access portal 2140 into channel or tube
2120, and optional pumps (not shown) are used to move the various
fluids to appropriate locations for assay thereof.
[0059] FIG. 24 illustrates a graph of hepatic lipid contents in
response to injections of LUVs, SUVs, or saline. The experimental
details are as outlined above. Liver samples were assayed for
contents of several lipids: cholesterol ester (CE); triglyceride
(TG); unesterified cholesterol (Chol); phosphatidylethanolamine
(PE); and phosphatidylcholine (PC), which are displayed in units of
.mu.g (micrograms) lipid/mg. Lower values of PE and PC in the
SUV-treated animals were produced; thus, the Chol:phospholipid
ratios in these animals was higher than in the other groups.
[0060] FIG. 25 illustrates cholesterol ester concentrations
following repeated injections of SUVs or LUVs (30 mg/kg) in NZW
rabbits (New Zealand White rabbits). The arrows indicate times of
phospholipid injection here on days 0, 3 and 5. For a given
phospholipid dose, LUVs promote a greater rise in plasma free
cholesterol concentrations.
[0061] FIG. 26 illustrates plasma free cholesterol concentrations
following repeated injections of SUV or LUV (300 mg/kg) in NZW
rabbits in the same experiment as in FIG. 25, arrows indicate times
of phospholipid injection. Repeated injections of LUV, unlike SUV,
do not provoke a dramatic rise in CE concentrations in plasma.
[0062] The rise in plasma CE concentrations that results from the
delivery of excess cholesterol to the liver may be the consequence
of two processes. It may involve an over production of CE-rich
particles or an impaired clearance of CE-rich lipoproteins. Over
production of CE-rich particles that occurs following SUV infusions
may result in the plasma or in the liver. In plasma, LCAT acts on
small unilamellar phospholipid vesicles or on phospholipid enriched
HDL generating CE which may be subsequently transferred by CETP
onto LDL. The results with gel filtration of plasma from animals
treated with SUVs indicate that CE is carried mostly or
substantially on LDL. Also, in plasma, removal of apoE from VLDL by
SUVs will slow the clearance of VLDL, thereby favoring a more
efficient conversion into LDL. In the liver, the increased delivery
of cholesterol to hepatocytes during cholesterol mobilization
stimulates an over secretion of apoB, CE-rich lipoproteins.
[0063] In a variant, the rise in plasma CE concentrations observed
is the result of an impaired clearance of CE rich atherogenic
lipoproteins. Intravenously administered liposomes that acquire
apoE compete with LDL for LDL-receptor mediated uptake. The
delivery of excess cholesterol to the liver down regulates LDL
receptors. The processes responsible for an increase in plasma CE
concentrations are different between the two liposome preparations.
LUVs. unlike SUVs, do not provoke a rise in plasma CE
concentrations. LUVs are superior preparations for mobilizing
tissue cholesterol without harmful side effects.
[0064] The method and composition of the present invention also
provides enrichment of HDL cholesterol esters by SUVs. One
contributing process is the stimulation of lecithin cholesterol
acyl transferase (LCAT) and other processes related thereto. The
ability of SUVs to increase HDL cholesterol ester is the result of
stimulation of LCAT and other processes related thereto. LCAT need
phospholipid and cholesterol to generate cholesteryl ester and
lysophosphatidylcholine; liposomes can supply extra phospholipid.
The present invention also provides for alterations in lipoprotein
(LDL, HDL, etc.) composition and function by LUVs and/or SUVs
and/or other acceptors.
[0065] The liposome compositions described herein and methods
utilizing same also include the liposomes picking up endogenous
apoE and hence blocking cellular uptake of LDL. The liposomes pick
up apolipoproteins, such as apoE and apoA-I, and that this alters
or enhances their functions. For example, the uptake of endogenous
apoA-I enhances the ability of liposomal derived phospholipid to
pick up cholesterol, and the uptake of endogenous apoE would allow
the liposomes to block certain pathways for arterial uptake of
lipoproteins. All of this is in the context of controlling LDL
levels and hepatic gene expression and cholesterol homeostasis.
[0066] LUVs and SUVs deliver cholesterol to different regulatory
pools within the liver. This conclusion is supported by the
differences in hepatic gene responses and CETP mRNA is suppressed:
the LDL receptor mRNA is unaffected or increased by LUVs but
suppressed by SUVs; and CETP is suppressed by LUVs, but unaffected
by SUVs. Further, it is understood that the arterial lesions
referenced herein include, by way of example, critical
stenoses.
[0067] The key points about LUVs and atherosclerosis are
illustrated in FIG. 9. The practical benefits of using LUVs as a
treatment for atherosclerosis are that they are straight forward to
manufacture, and non-toxic even at very high doses.
Mechanistically, LUVs promote reverse cholesterol transport in vivo
without provoking a rise in LDL concentration, and LUVs are an
optimal preparation.
[0068] The compositions that are used herein can direct clearance
away from hepatic parenchymal cells. And the various methods
described herein are utilized with slow infusions of the
compositions described, so that hepatic cells are not cholesterol
overloaded even if clearance by parenchymal cells occurs. Further,
HDL is also controlled by CETP gene suppression.
[0069] As described herein assays are performed by: assaying
fasting plasma triglyceride to estimate VLDL concentrations;
assaying plasma cholesterol (free and ester, or total minus
free=ester); precipitating LDL (& VLDL) with
polyanions-cations; assaying the supernatant which is HDL; and
computing LDL's (whole plasma value minus VLDL-HDL) sterol (or
sterol ester) in whole plasma. Liposomes will precipitate with
polyanions-cations; or optionally assaying the ester which
liposomes mostly lack. Other assays include electrophoresis,
chromatography, immune assays, electron microscopic assays,
functional assays, structural assays, and compositional assays.
[0070] In the dialysate of the present invention, any liposomes or
emulsions could be used as long as it's a cholesterol acceptor and
either it does not raise LDL or it is not returned to the patient's
circulation. In either case, one would need to assay plasma LDL and
the plasma concentration of the acceptors, and plasma
concentrations of other atherogenic lipoproteins.
[0071] With respect to the methods that require delivering the
cholesterol to the liver at a slow rate, or in low doses
administration might permit small acceptors, such as SUVs, to be
used without LUVs provided LDL levels as levels of other
atherogenic lipoproteins are monitored and regulated. To avoid
disrupting hepatic cholesterol homeostasis, the entrapped drug as
described herein need not be given at low doses, but rather the
encapsulating liposome or emulsion is given in low doses; the drug
could be present at high amounts within a small number of liposomes
or a small mass of liposomal lipid.
[0072] Alterations in HDL size, composition and function can be
accomplished by administering high or even truly low doses of large
and/or small liposomes that have little or no sterol. Liposomes
without sterol, when given in low doses are easily broken apart by
HDL and HDL apolipoproteins and then pieces are incorporated into
the HDL fraction of plasma enriching it in phospholipid. Such small
doses, e.g. 10-100 mg/kg/dose, even of SUVs without LUVs or drugs
to lower LDL levels, are unlikely to raise plasma LDL levels,
although periodic monitoring would be prudent.
[0073] Also, the method as disclosed herein of altering LDL
composition without increasing LDL concentration would be to enrich
the composition with phospholipids, like POPC
(palmitoyloleylphosphatidylcholine), that are resistant to
oxidation, enrich the composition with anti-oxidants, deplete
unesterified cholesterol, and reduce cellular or arterial uptake of
oxidized LDL by phospholipid enrichment.
[0074] Liposomes up to about 1000 NM or so would work in the
present invention. Larger liposomes would also work but extraction
of tissue lipoprotein may be less efficient. It is further possible
to concentrate or dry compositions of the present invention. These
preparations are then diluted or reconstituted at the time of
therapy or administration. In this variant, a two component kit
comprising the active material and a dilutent is provided.
Inclusion of phosphatidyl glycerol (PG) to make the liposomes
negatively charged, or charge other components of the composition,
to prevent aggregation during storage is also provided.
[0075] FIG. 27 illustrates alterations in plasma components after
repeated injections of SUVs. Watanabe Heritable Hyperlipidemic
(WHHL) rabbits were given intravenously 1000 mg of SUV phospholipid
per kg of body weight, or the equivalent volume of saline, on
Monday, Wednesday, & Friday of each week for three weeks (nine
doses total). Three days after the final dose, blood samples were
taken, and plasma components were fractionated by size by passage
over a Superose-6 gel-filtration column. Eluents were read by an
in-line spectrophotometer. The tracing on the right is from a
saline-injected rabbit, and shows VLDL around fractions #17-18, and
LDL around fraction #27. The tracing on the left is from an
SUV-injected rabbit, and shows VLDL with persistent liposomes
around fraction #16, and LDL-sized particles around fraction #25.
The tracings indicate an increase in the amount of LDL-sized
particles after repeated injections of SUVs, consistent with an
increase in LDL, which is a harmful effect. Because WHHL rabbits
have a genetic lack of LDL receptors, this result indicates that
SUVs disrupt hepatic cholesterol homeostasis not just by
suppressing LDL receptos (FIG. 5), but also by mechanisms
independent of LDL receptors (FIG. 27). LUVs avoid both LDL
receptor-dependent and independent disruptions.
[0076] FIG. 28 illustrates an agarose gel electrophoresis of whole
plasma following repeated injections of LUVs, SUVs, or saline.
Experimental details are referenced in FIGS. 2-8 & elsewhere
herein. Four-.mu.L plasma samples from two rabbits in each group at
day 6 were electrophoresed through 1% agarose then stained for
lipids with Sudan black. O: origin. .beta.: migration of an LDL
standard. The SUV-mediated increase in LDL concentration is
illustrated by the darker but otherwise unremarkable .beta.-band in
those lanes. SUVs in plasma exhibited a mobility ahead of LDL,
owing to their acquisition of plasma proteins, chiefly from HDL. In
contrast, plasma LUVs exhibited essentially the same mobility as
freshly prepared, protein-free vesicles, i.e., just above the
origin (O), indicating a substantial absence or reduction of
acquired proteins on the LUVs.
[0077] Based on the electrophoretic mobilities in FIG. 28,
quantification of the acquisition of protein by LUVs versus SUVs
was obtained. LUVs and SUVs were incubated with human HDL in vitro
for 4 hours at 37.degree. C., then separated from the HDL by gel
filtration chromatography and assayed for protein and phospholipid.
LUVs acquired 1.09 .mu.g of protein per mg of liposomal
phospholipid, whereas SUVs acquired 40.4 .mu.g/mg, i.e., almost 40
times as much. Thus, the two types of liposomes exhibit a striking
quantitative difference in protein adsorption. SUVs, but not LUVs,
avidly strip apoE from VLDL, thereby slowing its clearance from
plasma and favoring its conversion to LDL. In addition, adsorbed
proteins play a role in directing the SUVs into a hepatic metabolic
pool that disrupts hepatic cholesterol homeostasis, whereas LUVs
are not directed into such a pool. Liposomes, emulsions, or any
other particles or compounds that extract tissue lipids but do not
acquire large amounts of plasma proteins behave similarly to LUVs
in these regards.
[0078] Specific vascular genes affected by cholesterol loading of
cells include genes for prolyl-4-hydroxylase; hnRNP-K; osteopontin
(there may be a role for oxidized lipids in provoking arterial
calcifications); and Mac-2. The methods of regulating these genes
described herein effect restoration of normal vascular or arterial
function. Elevated expression of prolyl-4-hydroxylase (an enzyme in
the synthesis of collagen, a component of fibrotic plaques) and
hnRNP-K (identified in pre-mRNA metabolism and cell cycle
progression) messages were found in aortic smooth muscle cells
after cholesterol feeding. These would normalize after the liposome
treatments described herein. Other genes or enzymes that are
abnormal with cholesterol-loading and should normalize with
liposome treatment as described herein include osteopontin, nitric
oxide synthase (NOS), adhesion molecules, chemoatractants, tissue
factor, PAI-1 (plasmidigen activator inhibitor), tPA (tissue
plasmidigen activator) and Mac-2 (Ramaley et al. 1995). Other genes
affected by cholesterol, cholesterol loading, oxidized lipids would
also be corrected.
[0079] Many examples of small acceptors such as SUVs,
apolipoprotein-phospholipid disks, and HDL are commercially
available and can be used in the invention. Kilsdonk EP et al.
Cellular cholesterol efflux mediated by cyclodextrins, J. Biol.
Chem. 270:17250-17256, 1995. By way of further example, another
small acceptor includes the cyclodextrins. Small acceptors
(specifically HDL) shuttle cholesterol from cells to liposomes.
Cyclodextrins and also other small acceptors can shuttle
cholesterol and other exchangeable material from cultured cells to
LUVs, which substantially increases the removal and donation of
material between cells and LUVs.
[0080] Examples of anti-hyperlipidemic drugs include fibric acid
derivatives, HmG CoA reductase inhibitors, Niacin, probucol, bile
acid binders, other drugs and combinations thereof.
Anti-hyperlipidemic treatments also include LDL, apheresis, ileal
bypass, liver transplantation and gene therapy.
[0081] The data presented in this application support three
possible explanations for the difference in metabolic response to
LUVs versus SUVs. The three mechanisms act separately or in
combination. First, LUVs are taken up largely by Kupffer cells,
whereas SUVs are primarily directed towards hepatic parenchymal
cells. This is partly a mechanical consequence of hepatic
architecture: hepatic endothelial fenestrae are oval openings of
about 100.times.115 nm, through which SUVs of 30-nm diameter or so
can readily pass and gain access to parenchymal cells. Large
particles, such as large liposomes, of sufficient diameter will not
pass easily, and are cleared instead by the macrophage Kupffer
cells that line the liver sinusoids. While SUVs also have access to
Kupffer cells, their sheer number (.about.10 times as many SUVs as
LUVs per mg of phospholipid) appears to saturate the
reticuloendothelial system, and so parenchymal cells predominate in
their clearance. Other methods to direct artificial particles away
from parenchymal cells are also available, such as by changing the
particle structure or composition, including charge and specific
ligands for cell-specific binding.
[0082] Cholesterol clearance pathways mediated by parenchymal
versus Kupffer cells have distinct metabolic consequences. Direct
delivery of cholesterol to parenchymal cells by SUVs suppresses
sterol-responsive messages (FIGS. 5, 6, & 8). Delivery of
cholesterol to Kupffer cells can be followed by gradual transfer of
lipid to parenchymal cells, for example, via the extensions of
Kupffer cells that reach down through the space of Disse to make
physical contact with parenchymal cells. The rate of sterol
delivery to the parenchymal cells by transfer from Kupffer cells
can be slower than by direct uptake; the chemical form of the
sterol may be altered by the Kupffer cells before transfer; there
is other cell-cell communication; and, based on other pathways for
lipid transfer amongst liver cells, the process of transfer from
Kupffer to parenchymal cells may be regulated, whereas SUV
clearance does not appear to be.
[0083] The second contributing explanation for the difference in
metabolic response to LUVs versus SUVs is based solely on
differences in the kinetics of their delivery of cholesterol to the
liver. LUVs are cleared from plasma somewhat more slowly than are
SUVs, and thereby produce a relatively constant delivery of
cholesterol mass to the liver from the time of injection until the
bulk of injected material is cleared. SUVs are cleared more
rapidly, thereby delivering a large bolus of cholesterol mass to
the liver several hours after each injection, which is followed by
the sustained rise in plasma concentrations of cholesteryl ester
and atherogenic lipoproteins. The slow, steady delivery by LUVs
avoids disrupting hepatic cholesterol homeostasis, while the more
rapid uptake of SUV cholesterol overwhelms the ability of the liver
to maintain homeostasis, thereby provoking suppression of hepatic
LDL receptors. Other methods to deliver artificial particles or
their components to the liver at a proper rate are also available,
such as by changing the particle structure or composition,
including charge and specific ligand for cell-specific binding.
[0084] The third contributing explanation is based on the striking
quantitative difference in protein adsorption between the two types
of vesicles (FIG. 28), which, in that particular experiment, was a
result of their distinct surface curvatures. Thus, SUVs, but not
LUVs, would avidly strip apoE from VLDL, thereby showing its
clearance from plasma and favoring its conversion to LDL. SUVs that
acquire apoE will compete with VLDL, LDL, and other particles for
receptor mediated uptake by the liver. Also, adsorbed apoproteins
can play a role in directing phospholipid vesicles to different
hepatic metabolic pools. Other methods to reduce protein uptake by
artificial particles are also available, such as by changing the
particle structure or composition, including charge and specific
ligands for cell-specific binding.
[0085] Overall, given the observation that cholesteryl ester and
LDL concentrations do not increase after delivery of large amounts
of cholesterol and other exchangeable material to the liver by
LUVs, it was apparent that delivery was to a specific metabolic
pool or pools with unique properties that do not increase plasma
concentrations of atherogenic lipoproteins or harmfully disturb
hepatic cholesterol homeostasis, including the regulation of genes
and other functions. Thus, these inventions can be regarded in part
as a unique delivery system that brings original particle
components, such as phospholipid, plus material acquired by the
particles, such as cholesterol, to a specific delivery site for
harmless disposal and other additional benefits. The delivery
system with these characteristics will be useful in any situation
whatsoever in which control of hepatic cholesterol homeostasis,
hepatic phospholipid homeostasis, and hepatic metabolism in general
is advantageous.
[0086] For example, in a situation in which it is desirable to
modify erthyrocyte lipids, a straightforward approach would be to
administer artificial particles that can donate and remove the
appropriate lipids. If SUVs are used for this purpose, however,
they will transport cholesterol and other material to the liver in
a harmful manner, to the wrong pool and/or at the wrong rate, and
this will cause increases in plasma concentrations of atherogenic
lipoproteins, which is an undesirable side-effect that would
preclude this approach. In contrast, the use of large liposomes or
other particles with similar properties will result in the proper
delivery of original and acquired material, to the proper pool(s)
at a proper rate, so that the desired effect (modification of
erythrocyte lipids) can be achieved without harmful increases in
plasma concentrations of atherogenic lipoproteins.
[0087] As another example, it can be desirable to modify infectious
agents, such as bacteria, fungi, and viruses, using the
compositions and method described herein. Administration of large
liposomes or other particles with similar properties will remove
and donate exchangeable materials to and from these infectious
agents, and then the administered particles will be delivered to
the proper pool(s), so that the desired effect can be achieved
without harmful increases in plasma concentrations of atherogenic
lipoproteins.
[0088] As another example, a valuable therapy may provoke an
increase in plasma concentrations of atherogenic lipoproteins as an
unwanted side-effect. Administration of large liposomes or other
particles with similar properties will alter this response through
the delivery of lipids and other material to the proper hepatic
metabolic pool. The data with the "Mix" animal provides a specific
example of this effect (FIG. 4).
[0089] There are several mechanisms for affecting arterial uptake,
accumulation, and retention of lipoproteins. Liposomes can pick up
apoE from atherogenic lipoproteins, thereby reducing lipoprotein
binding to arterial cells and also competing for binding to
arterial cells. Finally, alterations in LDL size and/or composition
affect its binding to extracellular matrix and affect subsequent,
harmful alterations within the arterial wall, for example,
susceptibility to oxidation or enzymatic modifications.
[0090] The action or mode of operation of large acceptors, such as
large liposomes, can be aided by small acceptors, and vice-versa,
and this applies to both endogenous (e.g., HDL) and exogenous
(e.g., apoprotein-phospholipid complexes) small acceptors. Large
acceptors penetrate poorly into the interstitial space and appear
to inefficiently approach the cell surface under certain
circumstances. These effects impede their uptake and donation of
exchangeable material from membranes, cells, tissues, organs, and
extracellular regions and structures. Small acceptors do penetrate
well into the interstitial space and are able to approach the cell
surface, thereby allowing efficient uptake of exchangeable
material. Small acceptors have major disadvantages, however. They
have a very limited capacity to acquire or donate material (even
though the initial rate of acquisition or donation is rapid, until
their capacity becomes saturated) and, once they have acquired
material, they deliver it to the liver in a way that disrupts
hepatic cholesterol homeostasis.
[0091] Large acceptors and small acceptors together, however,
synergistically overcome each other's drawbacks through at least
three mechanisms. First, the large acceptors act as a sink (or
supply) for exchangeable material, while the small acceptors act as
a shuttle that siphons material from peripheral stores to the large
acceptors and in the other direction. Thus, for example, the small
acceptors penetrate tissue, acquire (and/or donate) material from
the tissue, and their capacity becomes at least partly saturated.
They leave the tissue and encounter the large acceptors in the
plasma, at which point the small acceptors are stripped of tissue
lipids. The capacity of the small acceptors is thereby restored, so
that when they return to the tissue, they can acquire (and/or
donate) more material. This cycle can continue many times. Second,
the large acceptors can re-model some small acceptors. For example,
large acceptors can donate phospholipid to HDL, which increases the
capacity of HDL acquire tissue cholesterol and other material.
Third, as noted elsewhere, the presence of large acceptors can
block or reduce the harmful disruptions in hepatic cholesterol
homeostasis caused by the small acceptors.
[0092] Large liposomes avoid raising plasma concentrations of
atherogenic lipoproteins in general, not just LDL. This list
includes all lipoproteins that contain apolipoprotein B (apoB),
such as LDL, IDL, VLDL, Lp(a), .beta.-VLDL, and remnant
lipoproteins.
[0093] Immune cells are also the targets for depletion using the
methods and modes of operation disclosed herein. It is understood
that administration of an HMG-CoA reductase inhibitor, pravastatin,
to cardiac transplant recipients reduced their natural-killer-cell
cytotoxicity in vitro, reduced episodes of rejection accompanied by
hemodynamic compromise, reduced coronary vasculopathy, reduced
plasma LDL levels (and increased HDL levels), and significantly
enhanced one-year survival. The effect on survival was dramatic: in
the control group, 22% died in the first year, whereas only 6% died
in the pravastatin-treated group.
[0094] Immunologic effects of HMG-CoA reductase inhibitors have
been reported in vitro. These reported immunologic effects include
the regulation of DNA in cycling cells, the inhibition of
chemotaxis by monocytes, the regulation of natural-killer-cell
cytotoxicity, and the inhibition of antibody-dependent cellular
cytotoxicity. Regulation of such inhibitors results from changes in
circulating lipids or other effects and by utilization of the
methods and modes of operation disclosed herein.
[0095] HMG-CoA reductase catalyzes an early step in cholesterol
biosynthesis and is crucial in the synthesis of molecules besides
cholesterol. Adding cholesterol to immune cells treated with
HMG-CoA reductase inhibitors does not restore function, although
the addition of mevalonate does. Although this suggests that
cholesterol depletion is not directly responsible for the immune
effects, the use of liposomes or other acceptors to remove
cholesterol from cells increases endogenous consumption of
mevalonate, as the cells try to make more cholesterol. To impede
the ability of the immune or other cells to make up their
cholesterol loss by picking up more LDL or other lipoproteins, the
methods and treatment described herein are also be done in
conjunction with therapies to lower plasma cholesterol
concentrations (including HMG-CoA reductase inhibitors, fibric
acids, niacin, bile acid binders, LDL-pheresis, etc.).
[0096] These processes include enhancement of cholesterol removal
and reduction of cholesterol influx. Levels of HDL, the apparent
natural mediator of cholesterol removal from peripheral cells,
increased in a treated group of patients, and LDL levels were
deceased. The administration of HMG-CoA reductase inhibitors in
vivo usually causes very tiny changes in reductase enzyme activity:
cells simply make more enzyme to overcome the presence of the
inhibitor. They also make more LDL receptors (especially in the
liver) and so LDL levels fall.
[0097] The invention further provides for additives to PD
(peritoneal dialysis solutions) that reduce the accelerated
atherosclerosis that occurs in renal failure.
[0098] Chemotaxis of monocytes is an important early event in
atherosclerotic lesion development: monocytes become attracted to
abnormal arterial lipid deposits, and to cellular products made in
response to the presence of these deposits, enter the vessel wall,
transform into macrophages, internalize the lipid by phagocytosis
and/or endocytosis, and become a major component of the so-called
lipid-rich foam cells of human atherosclerotic lesions. Thus,
inhibition of monocyte chemotaxis is important for atherosclerosis
as well and can be accomplished using the methods disclosed herein.
Both cellular and humoral immunity seem to be affected by reductase
inhibition: cardiac rejection accompanied by hemodynamic compromise
has often been associated with humoral rejection (i.e., that
occurring without producing marked lymphocytic infiltration in
endomyocardial-biopsy specimens).
[0099] Pravastatin may interact with cyclosporine [an important
immunosuppressive drug], which blocks the synthesis of
interleukin-2 in stimulated T-lymphocytes. The addition of
interleukin-2 restored the natural-killer-cell cytotoxicity and
partly restored the antibody-dependent cytotoxicity that were
inhibited in lovastatin-treated in vitro cell cultures. A synergy
between cyclosporine and pravastatin explains increased
immunosuppression in recipients of cardiac transplants, whereas
patients without transplants who receive HMG-CoA reductase
inhibitors for hypercholesterolemia do not have clinical
immunosuppression.
[0100] Thus, the use of safe cholesterol acceptors with other
immunosuppressives, such as cyclosporine &/or glucocorticoids
(which can also suppress IL-2) is also contemplated by this
invention.
[0101] It is also appreciated that the invention utilizes
derivatives of various compounds described herein.
[0102] Pathological specimens from patients with cardiac
transplants who have severe coronary vasculopathy have been
reported to have a high cholesterol content. Therefore, early
cholesterol lowering with pravastatin may play a part in decreasing
the incorporation of cholesterol into the coronary arteries of the
donor heart. Large liposomes or other cholesterol acceptors are
used to accomplish the same effect, quickly and directly, alone or
in combination, therewith.
[0103] Immune modulations is important in many conditions, not just
cardiac transplantation. Areas in which the above approaches could
be used also include transplantations of other organs, autoimmune
diseases (in which the body's immune system mistakenly attacks the
body's own tissues), some infections (in which the immune reaction
becomes harmful), and any other situation in which immune
modulation would be helpful.
[0104] With respect to infections, modification of the lipid
content and composition of foreign objects in the body (such as
infectious agents) while maintaining normal hepatic cholesterol
homeostasis should also be mentioned.
[0105] Oxidized lipids alter tissue function and cause damage,
including decreased EDRF, and increased adhesion molecules, cell
damage, and macrophage chemotaxis.
[0106] There are interactions between LUVs and small acceptors,
such as HDL, apoprotein phospholipid complexes, and cyclodextrins.
Liposomes remodel HDL into a better acceptor by donating extra
phospholipid, and the small acceptors act as a shuttle, carrying
cholesterol efficiently from cells to liposomes. LUVs do not
elevate LDL concentrations and do not suppress hepatic LDL receptor
gene expression. The medical utility for LUVs includes restoring
EDRF secretion by endothelial cells. High cholesterol levels
inhibit endothelial release of EDRF not through cholesterol, but
through an oxidized derivative of cholesterol. Because HDL itself
restores EDRF release, perhaps through the removal of cholesterol
or of oxidized lipids, then liposomes would be able to do the same
(the HDL ferries cellular oxidized lipids to liposomes, for
example).
[0107] The invention provides a method and mode of operation for
modifying cellular lipids, including oxidized lipids, without
provoking a rise in LDL concentrations or harmfully disturbing
hepatic homeostasis. Thus, the LUVs, presumably acting in concert
with endogenous (or exogenous) small acceptors of cholesterol (such
as HDL), pull oxidized lipids out of peripheral tissues and deliver
them to the liver for disposal. Oxidized lipids have a wide range
of harmful biological effects, including suppression of EDRF
release, induction of cell adhesion molecules, cellular damage,
chemotaxis of macrophages, and so forth.
[0108] Oxidized lipids and their harmful effects include decrease
endothelial C-type ANF; increased endothelial PAI-1 and decreased
tPA and decreased endothelial thrombomodulin. Liposomes enhance or
participate in this effect. These changes impair the body's ability
to dissolve clots. The methods disclosed herein assist in
ameliorating these harmful effects of oxidized lipids. HDL acts in
part by transporting enzymes that inactivate biologically active
oxidized lipids.
[0109] It is understood that oxidized LDL inhibits endothelial
secretion of C-type natrizuretic peptide (CNP). It is the lipid
component of oxidized LDL that mediates this effect. Most
importantly, HDL blocks the action of oxidized LDL, presumably by
picking up oxidized lipids (e.g., oxidized cholesterol).
Coincubation with high-density lipoprotein (HDL), which alone had
no effect on CNP release, significantly prevented Ox-LDL-induced
inhibition of CNP secretion by endothetial cells (ECs). Analysis by
thin-layer chromatography demonstrated that oxysterols. including
7-ketocholesterol, in Ox-LDL were transferred from Ox-LDL to HDL
during coincubation of these two lipoproteins. These results
indicate that Ox-LDL suppresses CNP secretion from ECs by
7-ketocholesterol or other transferable hydrophilic lipids in
Ox-LDL, and the suppressive effect of Ox-LDL is reversed by
HDL.
[0110] Whatever molecule HDL picks up, the presence of liposomes or
other acceptors around as described herein will allow it to do a
better job, because of remodeling of HDL by liposomes &
shuttling of oxidized lipids by HDL from tissues to liposomes
(i.e., the liposomes continuously strip the HDL). Liposomes with an
exogenous small acceptor will also work.
[0111] It is further understood that transferable lipids in
oxidized low-density lipoprotein stimulate plasminogen activator
inhibitor-1 and inhibit tissue-type plasminogen activator release
from endothelial cells. As above, it is the lipids in oxidized LDL,
such as oxidized forms of cholesterol, that produce the effect. It
is understood that oxidized low density lipoprotein reduced
thrombomodulin transcription in cultured human endothelial cells.
It is appreciated that oxidized lipids play a role in
atherosclerosis, and enzymes on HDL that inactivate oxidized lipids
may contribute to a protective effect. It is contemplated that the
methods and compositions disclosed herein will help this proposed
mechanism as well, for example, by removing end-products of these
enzymes, by otherwise altering HDL, and by providing an additional
platform for enzyme transport and action.
[0112] As such the use of large liposomes to remove harmful lipids
in general (here, oxidized lipids) from peripheral tissues, either
directly or via HDL, which would extract the lipids first, possibly
inactivate them, then deliver them or their break-down products to
liposomes in the circulation is described. Direct methods to assess
oxidation and oxidative damage in vivo include for lipids, assays
for 8-epiPGF.sub.2alpha; for DNA, assess 8-oxo-2'deoxyguanosine;
generally assess anti-oxidant enzymes in tissues; and assess
anti-oxidants levels, such as vitamin E, vitamin C, urate, and
reduced/oxidized glutathione.
[0113] Methods relating to and modes for effecting the reverse
lipid transport, from cells, organs, & tissues, including
transport of extracellular material, and any exchangeable material
in general are described herein. This covers not just cholesterol,
but also sphingomyelin, oxidized lipids, lysophophatidylcholine,
proteins, and also phospholipid donation. Some effects of oxidized
material include increased calcification in arterial cells as
described above and below.
[0114] Three potential differences between large versus small
liposome to explain their different effects on LDL and apoB levels
include: fenestral penetration (LUV<<SUV); rate of clearance
(LUV<SUV, so that LUVs produce a slow, sustained cholesterol
delivery to the liver that may be less disruptive); and protein
adsorption (LUV<<SUV).
[0115] Unesterfied cholesterol increases tissue factor expression
by macrophages. This is extremely important, because it is
macrophage-derived tissue factor that makes the material released
by unstable, rupturing plaques such a powerful stimulus for a clot
to form that then blocks the vessel leading to a heart attack. The
methods and modes of operation and compositions of the invention
act upon the expression of tissue factor.
[0116] Poor absorption of proteins by large liposomes affects LDL
levels and/or atherosclerosis by the following mechanisms: 1)
acquisition of apoE from VLDL by small liposomes impairs the
removal of VLDL from the circulation, thereby allowing it to be
more efficiently converted into atherogenic LDL; ii) absorbed
proteins on small liposomes direct these particles into the wrong
metabolic pool within the liver. Polyacrylamide gel electrophoresis
shows that liposomes (actually small liposomes) increase the size
of LDL. Liposomes are used to alter LDL size, composition and
structure to decrease its atherogenicity.
[0117] Other properties of LDL could be changed by administration
of liposomes. For example, liposomes reduce surface unesterified
cholesterol; reduce surface sphingomyelin; replace surface
phospholipids with POPC which is poorly oxidized; supplement the
LDL with antioxidants that were added to the liposomes before
administration. These changes would substantially alter arterial
entry, retention, modification and atherogenicity of LDL.
[0118] The side-effects controlled are focused on hepatic
cholesterol metabolism, hepatic expression of genes involved in
cholesterol metabolism, and plasma concentrations of
cholesterolrich atherogenic lipoproteins that contain
apolipoprotein B (chiefly, LDL). Reverse transport of
sphingomyelin, for example, changes hepatic cholesterol metabolism
(cellular sphingomyelin affects the intracellular distribution of
cholesterol, and hence its regulatory effects; also sphingomyelin
is a precursor to ceramide, which mediates intracellular
signaling), though large liposomes appear to avoid any problems in
the area. The same holds true for reverse transport of oxidized
forms of cholesterol (they are even more potent that unoxidized
cholesterol in suppressing LDL receptor gene expression).
Cyclodextrins do not pick up phospholipids.
[0119] Liposomes pick up any exchangeable lipid (actually, any
exchangeable amphipathic or hydrophobic material, which includes
lipid or protein or anything else with these characteristics). This
includes sphingomyelin, oxidized or modified lipids, such as
oxidized sterols and phospholipids. Typically, such liposomes can
pick up unesterified cholesterol and other exchangeable material
from other lipid bilayers, such as cell membranes, and from
lipoproteins. Liposomes also pick up proteins and donate
phospholipids. During and after these modifications, the liposomes
are removed from the plasma, chiefly by the liver. Throughout this
application, we will refer to this general process as "reverse
lipid transport", although it is understood that any exchangeable
material in tissues, blood, or liposomes could participate.
Specific examples of exchangeable material include unesterified
cholesterol, oxidized forms of cholesterol, sphingomyelin, and
other hydrophobic or amphipathic material.
[0120] These molecules accumulate in atherosclerosis and mediate
harmful effects (e.g., cholesterol, oxidized cholesterol, and other
material, such as lysophospholipids) or in aging (e.g.,
sphingomyelin). For example, oxidized lipids, particularly sterols,
alter many peripheral tissue functions, including stimulating
calcification by arterial cells in atherosclerosis &
stimulating endothelial plasminogen activator inhibitor-1 release
by endothelial cells; other oxidized lipid products include
lysophospholipids that stimulate endothelial expression of adhesion
molecules that attract macrophages into lesions, and sphingomyelin
accumulates in some cell-culture models of aging and, with
cholesterol, may account for some of the cellular changes. Other
changed, such as oxidation, may also mediate or accelerate aging.
Many of these molecules have been shown to be picked up by
liposomes in vitro (e.g., cholesterol, sphingomyelin, &
probably oxidized cholesterol) and many by HDL (cholesterol,
oxidized cholesterol by liposomes) but it is likely that they pick
up these other molecules as well. In terms of total mass, however,
the bulk of the acquired material is unesterified cholesterol, with
proteins in second place. Alternatively, by acquiring unesterified
cholesterol, the liposomes may reduce the amount of oxidized
cholesterol that develops, because there will be less starting
material.
[0121] The effective periods of time described herein should not be
interpreted to exclude very long courses of treatment, lasting
years, for example. Nor should it exclude repeated courses of
treatment separated by weeks, months, or years.
[0122] Side effects include overload of the liver with cholesterol
or other materials acquired by the liposomes; with subsequent
alterations in hepatic function, such as suppression of LDL
receptors, stimulation of intrahepatic cholesterol esterification,
stimulation of intrahepatic cholesterol esterification, stimulation
of hepatic secretion of atherogenic lipoproteins that contain
apolipoprotein-B, and impaired uptake of atherogenic lipoproteins
by the liver from plasma.
[0123] As used herein the word, "endogenous" indicates that the HDL
arises from within the body, and is not itself administered. HDL
and related acceptors can, however, be administered.
[0124] The data indicates another difference between large and
small liposomes in vivo. Before injection, the liposomes that are
used in our experiments were essentially electrically neutral,
indicated by a failure to migrate rapidly through a gel of agarose
when an electric field is applied. (This does not imply that
charged liposomes or other particles could not be used. The small
liposomes pick up proteins and other material, and become
electrically charged: they now rapidly migrate through agarose gels
when an electric field is applied. Agarose gels of plasma samples
we had stored from the three groups of rabbits were run. The small
liposomes became more mobile LDL in these gels. The large liposomes
were substantially less mobile, indicating a lower charge density,
reflecting a lower protein content.
[0125] Two explanations for the difference between large and small
liposomes exist: 1) small ones penetrate through hepatic
endothelial fenestrae while large ones do not (thus, large ones go
to Kupffer cells and small ones go to hepatic parenchymal cells and
cause problems); 2) large liposomes are known to be cleared by the
liver somewhat more slowly than are small liposomes (the reason is
not known), and so may not overwhelm the liver as easily. The data
on charge density provides an explanation in part: less protein,
therefore slower or altered hepatic uptake.
[0126] The delivery of cholesterol to the liver by LUVs is actually
more efficient than by SUVs, per mg of phospholipid. One difference
is that the delivery by LUVs is steady over a long period after the
injection, whereas the delivery by SUVs peaks then falls.
[0127] Some of the composition described herein include egg
phosphatidylcholine; synthetic phosphatidylcholines that are not
crystalline at body temperature (e.g., they contain at least one
double bond) yet are resistant to oxidation (e.g., they do not have
many double bonds, such as 1-palmitoyl, 2-oleyl
phosphatidylcholine, abbreviated POPC); other natural or synthetic
phospholipids alone or in mixtures; any of the preceding
supplemented or replaced with hydrophobic or amphipathic material
that still allows a liposomal or micellar structure. An extruder is
certainly not the only conceivable method for making large
liposomes or even particularly LUVs. Other methods known to
practioners in the field are available or can be adapted to make
large liposomes in general and LUVs in particular.
[0128] As used herein, a dose includes from 10 to 1600 mg of
phospholipid, in the form of large liposomes, per kg of body
weight. Other acceptable rates described herein can be determined
empirically by the response of plasma LDL concentrations.
[0129] Where there is a change in membrane composition, as well as
function, one can use an assay of membrane composition or an assay
of tissue composition. Compositional assays should include lipids,
proteins, and other components.
[0130] HDL can pick up oxidized material, and HDL-associated
enzymes may inactivate oxidized material.
[0131] The separations in time will depend on the actual dose of
material, its effects on hepatic cholesterol homeostasis, and
whether cholesterol-lowering agents are being concurrently
administered. Thus, for doses of about 300 mg of small liposomes
per kg of body weight, slight disruptions will occur after even a
single dose, and single administrations of higher doses may cause
even more disruptions. Exemplary separations in time include one
day to one month, but the precise schedules would have to be
determined by monitoring hepatic cholesterol metabolism and plasma
levels od LDL and other atherogenic lipoproteins.
[0132] The major macrophages that would be involved in liposomal
clearance would be Kupffer cells in the liver and macrophages in
the bone marrow or spleen. The catabolism here would be the
so-called alternative pathway for initiating the conversion of
cholesterol into bile acids (macrophages are known to have at least
one cholesterol-catabolizing enzyme), or would be transfer of
sterol (enzymatically altered or not) to other cells, such as
hepatic parenchymal cells that would then dispose of the
molecules.
[0133] The methods described herein also control effects of
cellular aging.
[0134] The invention includes means for assessing the efficacy of
liposomal therapy by performing assays of oxidation in vitro and in
vivo, assays of oxidative susceptibility of plasma components, and
assays of the ability of altered HDL to inhibit oxidation (by
binding oxidative products and/or through its paroxinase or other
anti-oxidant components), and the ability of HDL or plasma or serum
or blood to mobilize cholesterol and other exchangeable
material.
[0135] Large liposomes may cause the mobilization of some material
that is trapped between cells as well (this is the extracellular
space). This extracellular material causes problems a) when it
contacts cells or platelets, altering their function and b) by
simply taking up space.
[0136] Estimate rates of cholesterol mobilization can be
empirically determined. It is appreciated that the kinetics of
liposomal clearance is different in different species (the
t.sub.1/2 of LUVs in mice is about 8 h, but in rabbits it is about
24th, and in humans it is longer). Thus, rates calculated may vary
from species to species. Based on my data on injection of 300 mg of
SUVs into rabbits, the peak rate of liposomal cholesterol removal
from plasma was between 3 h and 6 h after the injection. At that
point, the liposomes had raised plasma unesterified cholesterol by
just over 2 mmol/L; assuming a total plasma volume of 90 mL in a
3-kg rabbit, the total liposomal cholesterol at that point was 180
.mu.moles; the t.sub.1/2 for SUVs in these rabbits was about h, so
roughly 10% is removed in 3 h; thus, the peak rate of liposomal
cholesterol removal was about 2 .mu.moles/h/kg, and this caused a
subsequent rise in plasma cholesteryl ester concentrations. Notice
that at other time periods after the injection, the rate of
liposomal cholesterol removal from plasma was less. Note also that
the liver is the predominant organ for clearance, but not the sole
organ for clearance.
[0137] It has been calculated that a single injection of 300 mg
LUVs/kg into 20-22-g mice mobilized about 2400 nmoles of
cholesterol in the first 24 h after injection. In contrast to the
data with SUVs in rabbits, the mobilization of cholesterol during
the first 24 h in the mice injected with LUVs was quite steady.
This calculates to about 4.7 .mu.moles/h/kg over this first 24-h
period, which is actually more than the above figure of 2
.mu.moles/h/kg, which was a peak rate. It is not fair comparison,
because the clearance of LUVs in mice is three times as fast as in
rabbits. If we take 4.7 divided by 3, we get 1.6 .mu.moles/h/kg,
which is less than 2, but these are imperfect estimates. Human
rates can be empirically determined. It is clear, however, that
LUVs deliver their cholesterol at a steady rate, whereas SUVs make
a brief, rapid push of lipid into the liver.
[0138] At body temperature, the most desirable liposomes are fluid
within the confines of the bilayer, which is called the liquid
crystalline state. Less desirable are liposomes in the gel state,
which is less fluid.
[0139] It is understood that unesterified cholesterol stimulates
macrophages to express more tissue factor, a substance known to
provoke blood clots. This explains the presence of abundant tissue
factor in rupture-prone plaques, which, when they rupture, expose
tissue factor to plasma and provoke a clot that can occlude the
vessel, causing a heart attack. This would be another example of an
abnormal cellular function that may be reversed by removal of
cholesterol by liposomes.
[0140] Several human conditions are characterized by distinctive
lipid compositions of tissues, cells, membranes and/or
extracellular regions. For example, in atherosclerosis, cholesterol
(unesterified, esterified, and oxidized forms) and other lipids
accumulated in cells and in extracellular areas of the arterial
wall and elsewhere. These lipids have potentially harmful biologic
effects, for example, by changing cellular functions and by
narrowing the vessel lumen, obstructing the flow of blood. Removal
of the lipids would provide numerous, substantial benefits.
Moreover cells, membranes, tissues and extracellular structures
would benefit from composition and alteration that include
increasing resistance to oxidation and oxidative damages, such as
by increasing the content and types of anti-oxidants, removing
oxidized material, and increasing the content of material that is
resistant to oxidation. In aging, cells have been shown to
accumulate sphingomyelin and cholesterol, which alter cellular
functions. These functions can be restored in vitro by removal of
these lipids and replacement with phospholipid from liposomes. A
major obstacle to performing similar lipid alterations in vivo has
been disposition of the lipids mobilized from tissues, cells,
extracellular areas, and membranes. Natural (e.g., high-density
lipoproteins) and synthetic (e.g., small liposomes) particles that
could mobilize peripheral tissue lipids have a substantial
disadvantage: they delivery their lipids to the liver in a manner
that disturbs hepatic cholesterol homeostasis, resulting in
elevations in plasma concentrations of harmful lipoproteins, such
as low-density lipoprotein (LDL), a major atherogenic
lipoprotein.
[0141] The invention described herein provides methods and
compositions related to the "reverse" transport of cholesterol and
other materials and compounds from peripheral tissues to the liver
in vivo while controlling plasma LDL concentration.
[0142] Agarose gel electrophoreses of plasma samples from the last
a set of rabbits injected with LUVs, SUVs, or saline (these agarose
gels separate particles by their charge, which is not the same from
one type of particle to another) were performed. Freshly made SUVs
migrate very slowly through agarose, which indicates that freshly
made liposomes have very little charge. After injection into
animals or after co-incubation with plasma or lipoproteins, SUVs
pick up proteins from lipoproteins. These proteins give more charge
to the SUVs and substantially enhance their migration through
agarose gels. SUVs after exposure to plasma migrate faster through
these gels than does LDL.
[0143] The gels showed a substantial difference between LUVs and
SUVs. As expected, the SUVs migrated ahead of LDL in these gels.
The LUVs, however, migrated almost exactly where freshly made,
protein-free liposomes migrate. This result indicates that LUVs,
unlike SUVs, do not readily pick up proteins from circulating
lipoproteins.
[0144] There is a direct verification of this difference between
the liposomes. Human HDL (which has most of the proteins that
liposomes pick up) was incubated with either LUVs or SUVs, then the
liposomes were reisolated, and assayed their
protein-to-phospholipid ratios. Per amount of liposomal
phospholipid, the SUVs picked up about 40 times as much protein as
did the LUVs. This difference appears to arise because of the
difference in surface curvature: SUVs are smaller, so their surface
is more tightly curved, thus under greater strain, proteins can
more easily insert.
[0145] There are three most likely metabolic effects of the
difference in protein uptake between the two types of liposomes are
as follows:
[0146] 1. VLDL has two metabolic fates: it can be removed from
plasma before it is fully converted to LDL by lipolytic enzymes, or
it can be fully converted into circulating LDL. SUVs strip apoE off
VLDL, thereby slowing its clearance from plasma and favoring its
conversion to LDL. In contrast, LUVs leave apoE on VLDL, and so LDL
concentrations in plasma would not rise.
[0147] 2. Absorbed apoproteins might play a role in directing
liposomes to different hepatic metabolic pools.
[0148] Here are some ways to assay effect on oxidation in vivo:
Catella F, Reilly M P, Delanty N, Lawson J A, Moran N, Meagher E,
FitzGerald G A. Physiological formation of 8-epi-PGF2 alpha in vivo
is not affected by cyclooxygenase inhibition. Adv Prostaglandin
Thromboxane Leukot Res. 23:233-236, 1995. These authors describes
8-epi-PGF.sub.2alpha, which is an end-product of lipid oxidation.
This molecule can be used, they suggest, as a measure of lipid
oxidative flux in an animal. It is superior to other commonly used
measure of oxidation in vivo, such as antioxidant levels (which are
affected by diet), thiobarituric acid reactive substances (some
sugars interfere with this assay), and short-lived oxidative
intermediates (these do not indicate total flux of material being
oxidized). Administration of LUVs, by removing oxidized lipids from
the periphery, would lower total oxidative flux in vivo, and
8-epi-PGF.sub.2alpha would be a suitable way to measure this; Cadet
J, Ravanat J L, Buchko G W, Yeo H C, Ames B N. Singlet oxygen DNA
damage: chromatographic and mass spectrometric analysis of damage
products. Methods Enzymol. 234:79-88, 1994. they describe
8-oxo-2'-deoxyguanosine, which is an end-product of DNA oxidation.
As above, this molecule can be used as a measure of DNA oxidative
flux in an animal. Administration of LUVs would lower DNA oxidative
flux in vivo, and this is a suitable way to measure this; and, Xia
E, Rao G, Van Remmen H, Heydari A R, Richardson A. Activities of
antioxidant enzymes in various tissues of male Fischer 344 rats are
altered by food restriction. J Nutr. 125(2):195-201, 1995.
Antioxidant enzymes in tissues were measured, to indicate
de-oxidant capacity. LUVs help this. Anti-oxidant levels (vitamin
E, ascorbate, urate); oxidized and reduced glutathione; and many
other measures can be used to assess peripheral oxidation and
oxidative damage. Again, these and other measures would be coupled
with LUV administration, to assess efficacy of the therapy.
[0149] Other particles that mimic there properties of large
liposomes will act similarly, to mobilize peripheral lipids and
other exchangeable materials, and deliver exchangeable materials,
while avoiding harmful disruptions in hepatic cholesterol
homeostasis. For example, these would include emulsion particles
that are two large to penetrate hepatic endothelial fenestrae, of a
composition and structure that is taken up by the liver slowly,
and/or a composition and structure that does not readily acquire
specific endogenous proteins. Such emulsions could be made with or
without proteins, and could be made from phospholipid and a neutral
lipid, such as triglycerides or another neutral lipid.
[0150] The invention also provides a pharmaceutical composition
comprised or consisting essentially of liposomes dimensioned and of
a composition so that the liposomes are taken up slowly by the
liver.
[0151] The invention also includes a method of forcing the reverse
transport of cholesterol from peripheral tissues to the liver in
vivo while controlling plasma LDL concentrations comprising the
step of parenterally administering a therapeutically effective
amount of a multiplicity of large liposomes comprised of
phospholipids substantially free of sterol for a treatment period,
whereby the liposomes pick-up the cholesterol during the treatment
period. The method includes the optional step of enhancing tissue
penetration of a cholesterol acceptor and increasing extraction of
tissue cholesterol and other exchangeable material by
co-administration of an effective amount of a compound. The
compound is selected from the group consisting of a small acceptor
of cholesterol and a drug that increases endogenous small acceptors
of cholesterol. In a variant, co-administration of the compound is
simultaneous with the parenteral administration of the large
liposomes. In another variant, co-administration of the compound is
separated in time from the parenteral administration of the
therapeutically effective amount of a multiplicity of the large
liposomes by an effective time period. The effective time period is
in the range of about 1 minute to about two weeks.
[0152] In another aspect the invention includes an improved method
of reducing the lipid content of arterial lesions comprising the
steps of inducing the reverse transport of cholesterol from
peripheral tissues to the liver in vivo by administering a
therapeutically effective amount of an agent to a subject. The
agent is selected from the group consisting of large liposomes
comprised of phospholipids substantially free of sterol and small
acceptors; periodically monitoring plasma LDL concentrations of the
subject to obtain an LDL concentration profile; adjusting the
therapeutically effective amount of the agent responsive to the LDL
concentration profile; and, administering a pharmaceutical agent to
the subject. The agent is selected from the group consisting of
compounds to lower LDL concentrations, small acceptors, and
compounds to raise HDL concentrations, responsive to the LDL
concentration profile, whereby the reduction in lipid content of
the arterial lesions is effectively treated and monitored over a
treatment period. The arterial lesions comprise lipid rich, rupture
prone, type IV and type V arterial lesions. Plaque rupture,
thrombosis, and tissue infarction are greatly reduced.
[0153] In yet another aspect the invention provides for an improved
method of assessing the efficiency of a treatment for reducing the
lipid content of arterial lesions, the lesions coming into contact
with plasma and a component thereof comprising the steps of
inducing the reverse transport of cholesterol from peripheral
tissues to the liver in vivo by administering a therapeutically
effective amount of an agent to a subject. The agent is selected
from the group consisting of large liposomes comprised of
phospholipids substantially free of sterol and small acceptors;
and, periodically monitoring the plasma component with an assay.
The assay is selected from the group consisting of an assay for
plasma unesterified cholesterol and phospholipid, an assay of bile
acids and cholesterol in stool, an assay of bile acids and
cholesterol in bile, an assay of hepatic gene expression in a liver
biopsy, an assay of gene expression in peripheral blood leukocytes,
the gene comprising a gene involved in cholesterol metabolism, an
assay of plasma LDL concentration, and a vascular imaging
technique. The vascular imaging technique is selected from the
group consisting of cardiac catherization, magnetic resonance
imaging, ultrasound, ultrafast CT and a radionuclide assay which
optionally includes a stress-thalium scan.
[0154] The invention also includes a method of beneficially
altering arterial function, blood platelet function, and
controlling plasma LDL concentrations and hepatic cholesterol
homeostasis in vivo comprising the step of parenterally
administering a therapeutically effective amount of a multiplicity
of large liposomes comprised of phospholipids substantially free of
sterol for a treatment period with or without administration of
other agents. The other agents optionally include small acceptors
and LDL lowering agents. Optionally the method includes the step of
taking a measurement of arterial function. The measurement is
selected from the group consisting of a measurement of
endothelial-derived relaxing factor, a measurement of intracellular
calcium concentration in arterial cells, a measurement of arterial
cell proliferation, an assay of arterial enzymes, an assay in the
presence of calcium channel blockers, an assay of arterial uptake,
accumulation and retention of lipoproteins, an assay of arterial
accumulation of liposomes, an assay of arterial retention of
liposomes, an assay of gene products, and an assay of arterial cell
functions. The measurement of endothelial-derived relaxing factor
is selected from the group consisting of a functional determination
of endothelial-dependent arterial relaxation, chemical
determination of production of said endothelial relaxing factor,
and an assay of nitric oxide synthase.
[0155] A method of beneficially altering blood platelet function
while controlling plasma LDL concentrations, arterial function,
hepatic cholesterol homeostasis and said platelet function in vivo
is also included. The method comprises the step of parenterally
administering a therapeutically effective amount of a multiplicity
of large liposomes comprised of phospholipids substantially free of
sterol for a treatment period, said liposomes administered with or
without other agents. The method optionally includes the step of
taking a measurement of arterial function. The measurement selected
from the group consisting of a measurement of endothelial-derived
relaxing factor, a measurement of intracellular calcium
concentration in arterial cells, a measurement of arterial cell
proliferation, an assay of arterial enzymes, and an assay of gene
products. The measurement of endothelial relaxing factor is
selected from the group consisting of a functional determination of
endothelial-dependant arterial relaxation and chemical
determination of production of said endothelial relaxing
factor.
[0156] Also included is a method of catabolizing cholesterol with
macrophages in vivo and also affecting a plasma component or
structural aspects of an artery, comprising the step of
administering an effective amount of liposomes to a subject
substantially free of cholesterol and being of a size and
composition such that the liposomes are capable of being taken up
by the macrophages and capable of being catabolized by the
macrophages. The cholesterol is mobilized by the liposomes
resulting in the liposomes being taken up by the macrophages and
catabolized. The method also can include the step of periodically
monitoring the plasma component with an assay. The assay is
selected from the group consisting of an assay for plasma
unesterified cholesterol and phospholipid, an assay of plasma
cholesterol ester transfer protein activity, an assay of bile acids
and cholesterol in stool, an assay of hepatic gene expression in a
liver biopsy, an assay of gene expression in a peripheral blood
leukocytes, the gene comprising a gene involved in cholesterol
metabolism, an assay of plasma LDL concentration, and a vascular
imaging technique.
[0157] In yet another aspect the invention includes a method of
delivering a drug in vivo and avoiding harmful disruptions of
hepatic cholesterol homeostasis, comprising the steps of entrapping
the drug with an agent. The agent is selected from the group
consisting of a cholesterol poor liposome, a cholesterol free
liposome, an emulsion, a liposome primarily taken up slowly by
hepatic parenchymal cells, an emulsion primarily taken up slowly by
hepatic parenchymal cells. The agent is selected from the group
consisting of an agent with a protein and an agent without protein
to obtain an entrapped drug. The method also includes the step of
administering a therapeutically effective amount of the entrapped
drug for a treatment period. The step of administering comprises
the step of slowly infusing said entrapped drug. In variants, the
step of administering comprises the step of administering small
doses of the agent, appropriately separated in time, to avoid
harmful disruptions in hepatic cholesterol homeostasis, and
includes using low doses of said agent, whereby disrupting hepatic
cholesterol homeostasis is avoided.
[0158] A method of controlling plasma LDL levels, hepatic
cholesterol homeostasis, arterial enzymes, arterial function, and
platelet function, and altering platelet hormone production is also
provided. The method includes the step of parenterally
administering a therapeutically effective amount of a multiplicity
of large liposomes comprised of phospholipids substantially free of
sterol for a treatment period. The effective amount is administered
in a dosage and the dosage is selected from a single dose and
repeated doses. The method optionally includes the step of
diagnosing the efficacy of the administration by taking a
measurement of the hormone production and regulating the effective
amount in response to the measurement. The measurement of hormone
production is an assay selected from the group consisting of an
assay for thromboxanes, an assay for prostacyclines, an assay of
prostaglandins, an assay for leukotrienes, and an assay for
derivatives thereof.
[0159] In yet a further aspect the invention provides a method of
increasing plasma HDL concentrations, while controlling plasma LDL
levels, hepatic cholesterol homeostasis, and hepatic gene
expression. The method comprises the step of parenterally
administering a therapeutically effective amount of a first agent.
The first agent comprising a multiplicity of small liposomes to
raise HDL concentrations for a treatment period. The method then
includes the step of coadministering a second agent. The second
agent includes large liposomes comprised of phospholipids
substantially free of sterol for a treatment period. The effective
amount is administered in a dosage selected from a single dose and
repeated doses. The co-administration acts to prevent the small
liposomes from stimulating harmful changes in hepatic cholesterol
homeostasis and an increase in plasma LDL. In a variant, the first
agent consists essentially of small liposomes and the second agent
consists essentially of large liposomes. The method also includes
the step of diagnosing the efficacy of the administration by taking
a measurement of plasma HDL and LDL levels before, during and after
the treatment period.
[0160] A method of controlling plasma LDL levels, and hepatic
cholesterol homeostasis in vivo while altering cell membrane
composition and function is also described herein. The method
includes the step of parenterally administering a therapeutically
effective amount of a multiplicity of large liposomes comprised of
phospholipids substantially free of sterol for a treatment period.
The effective amount administered in a dosage selected from a
single dose and repeated doses. The method includes the step of
co-administering a small acceptor selected from the group
consisting of a small acceptor of cholesterol, an acceptor of
sphingomyelin, an acceptor of lysophosphatidylcholine and an
acceptor of a lipid. The method can optionally include the step of
diagnosing the efficacy of the administration by performing a
measurement selected from the group consisting of a measurement of
membrane fluidity, a measurement of transmembrane ion flux said
ions selected from the group consisting of calcium ions, sodium
ions, and potassium ions an assay of membrane fragility, and an
assay of membrane function.
[0161] In a further embodiment, the invention includes a
pharmaceutical composition for mobilizing peripheral cholesterol
and sphingomyelin that enters the liver of a subject consisting
essentially of liposomes selected from the group of uni-lamellar
liposomes, multi-lamellar liposomes, combinations thereof, and
derivatives thereof, and a pharmaceutical composition for reducing
the size of arterial lesions that enters the liver of a subject
consisting essentially of a multiplicity of non-liposomal particles
for cholesterol depletion of peripheral tissues while avoiding
harmful disruptions of hepatic cholesterol homeostasis. The
particles are selected from the group of particles substantially
free of cholesterol and particles free of cholesterol.
[0162] Non-liposomal particles are selected from the group
consisting of triglyceride-phospholipid emulsions. The emulsions
include emulsions that are not taken up rapidly by hepatic
parenchymal cells, emulsions that are not taken up to a large
extent by parenchymal cells, and triglyceride-phospholipid-protein
emulsions.
[0163] Also included in the invention is a pharmaceutical
composition for reducing the size of arterial lesions that enters
the liver of a subject consisting essentially of a drug entrapped
within an agent. The agent is selected from the group consisting of
a cholesterol poor liposome, a cholesterol free liposome, an
emulsion, a liposome primarily taken up slowly by hepatic
parenchymal cells, and an emulsion primarily taken up slowly by
hepatic parenchymal cells. The agent is selected from the group
consisting of an agent with a protein and an agent without
protein.
[0164] The invention also provides for a pharmaceutical composition
for increasing plasma HDL concentrations, while controlling plasma
LDL levels, hepatic cholesterol homeostasis, and hepatic gene
expression, comprising a first agent which comprises a multiplicity
of small liposomes to raise HDL concentrations, and a second agent
which comprises large liposomes comprised of phospholipids
substantially free of sterol.
[0165] In yet another aspect a method of controlling cholesterol
metabolism in hepatic parenchymal sells in a subject in vivo
through cell-cell communication from Kupffer cells to the
parenchymal cells is included. The method includes the steps of
administering a liposome composition to the subject. The liposome
composition is selected from the group consisting of large
unilamellar liposomes and large multilamellar liposomes. The
liposomes have an average diameter of about 50-150 nanometers. The
LDL levels in the subject do not increase. The method also includes
the step of diagnosing the efficacy of the control of cholesterol
metabolism by assaying an indicator in the subject. The indicator
is selected from the group consisting of plasma LDL concentrations
of the subject, hepatic gene expression of said subject, sterol
excretion controlling cholesterol metabolism in hepatic parenchymal
cells in the subject, and sterol excretion in bile of the subject;
and adjusting the administration in response to the assay.
[0166] The present invention further provides a mode of operation
of artherogenic lipoproteins. cellular structures, and
extracellular structures that is altered by the compositions
described herein through which beneficial physiological effects are
obtained.
[0167] While only a few, preferred embodiments of the invention
have been described hereinabove, those of ordinary skill in the art
will recognize that the embodiment may be modified and altered
without departing from the central spirit and scope of the
invention. Thus, the preferred embodiment described hereinabove is
to be considered in all respects as illustrative and not
restrictive, the scope of the invention being indicated by the
appended claims, rather than by the foregoing description, and all
changes which come within the meaning and range of equivalency of
the claims are intended to be embraced herein.
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