U.S. patent application number 12/042924 was filed with the patent office on 2008-09-04 for methods and apparatus for creating particle derivatives of hdl with reduced lipid content.
This patent application is currently assigned to Lipid Sciences, Inc.. Invention is credited to Hassibullah Akeefe, Marc Bellotti, H. Bryan Brewer, Adam Paul Conner, Timothy Jon Perlman.
Application Number | 20080214438 12/042924 |
Document ID | / |
Family ID | 34115319 |
Filed Date | 2008-09-04 |
United States Patent
Application |
20080214438 |
Kind Code |
A1 |
Bellotti; Marc ; et
al. |
September 4, 2008 |
Methods and Apparatus for Creating Particle Derivatives of HDL with
Reduced Lipid Content
Abstract
The present invention is directed to systems, apparatus and
methods for creating derivatives of at least one form of HDL
without substantially affecting LDL. These derivatives of HDL are
particles with reduced lipid content, particularly reduced
cholesterol content. These particles have the capacity to bind
cholesterol and are administered to a patient to enhance cellular
cholesterol efflux and reduce cholesterol levels in cells, tissues,
organs, and blood vessels. The present method is useful for
treating atherogenic vascular disease and may be combined with
other therapies such as statins, inhibitors of cholesterol
absorption, niacin, anti-inflammatories, exercise and dietary
restriction.
Inventors: |
Bellotti; Marc; (Pleasanton,
CA) ; Brewer; H. Bryan; (Potomac, MD) ;
Akeefe; Hassibullah; (Antioch, CA) ; Conner; Adam
Paul; (Livermore, CA) ; Perlman; Timothy Jon;
(Pleasanton, CA) |
Correspondence
Address: |
JOHN S. PRATT, ESQ;KILPATRICK STOCKTON, LLP
1100 PEACHTREE STREET
ATLANTA
GA
30309
US
|
Assignee: |
Lipid Sciences, Inc.
|
Family ID: |
34115319 |
Appl. No.: |
12/042924 |
Filed: |
March 5, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10796691 |
Mar 8, 2004 |
7375191 |
|
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12042924 |
|
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60484690 |
Jul 3, 2003 |
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Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
A61K 38/1709 20130101;
A61P 3/06 20180101; A61P 3/10 20180101; A61P 7/00 20180101; A61P
25/28 20180101; A61K 2300/00 20130101; G01N 33/92 20130101; A61P
3/04 20180101; A61P 9/00 20180101; A61P 25/00 20180101; A61P 9/10
20180101; A61P 9/14 20180101; A61K 9/1275 20130101; C07K 14/775
20130101; A61K 38/1709 20130101; A61P 43/00 20180101; A61P 29/00
20180101; A61P 9/12 20180101 |
Class at
Publication: |
514/2 |
International
Class: |
A61K 38/02 20060101
A61K038/02 |
Claims
1-29. (canceled)
30. A method for making a particle derivative of at least one form
of high density lipoprotein wherein the particle derivative
comprises a protein shell and a lipid bilayer comprising the steps
of: a. connecting a patient to a device for withdrawing blood; b.
withdrawing blood containing blood cells from the patient; c.
separating the blood cells from the blood to yield a blood fraction
containing high density lipoprotein and low density lipoprotein; d.
separating the low density lipoprotein from the blood fraction; e.
mixing the blood fraction with a lipid removing agent which removes
lipids associated with the lipid bilayer of the high density
lipoprotein to yield a mixture of lipid, the lipid removing agent,
and the particle derivative; f. separating the particle derivative
from the lipid and the lipid removing agent; and, g. delivering the
particle derivative to the patient.
31. The method of claim 30, wherein steps c through f occur remote
from the subject.
32. The method of claim 30, wherein the separation of the low
density lipoprotein from the blood fraction is performed using an
apheresis device.
33. The method of claim 30, wherein the lipid removing agent is at
least one of an ether, di-isopropyl ether, sevoflurane, a
combination of an alcohol and an ether, or a combination of a
sevoflurane and an alcohol.
34. The method of claim 33, wherein the lipid removing agent is a
mixture of sevoflurane and n-butanol.
35. The method of claim 34, wherein the mixture comprises 95 parts
sevoflurane and 5 parts n-butanol.
36. The method of claim 30, wherein the step of separating the
particle derivative from the lipid and the lipid removing agent is
achieved by at least one of an absorbent, separator, centrifuge, or
charcoal column.
37. The method of claim 36, wherein the at least one of an
absorbent, separator, centrifuge, or charcoal column does not
modify the protein shell of the particle derivative.
38. The method of claim 30, wherein the particle derivative is
first recombined with the blood cells before delivering the
particle derivative to the patient.
39. The method of claim 30, wherein the mixing is performed using
at least one of a static mixer, vortexer or centrifuge.
40. A method for making a particle derivative of at least one form
of high density lipoprotein wherein the particle derivative
comprises a protein shell and a lipid bilayer comprising the steps
of: a. connecting a patient to a device for withdrawing blood; b.
withdrawing blood containing blood cells from the patient; c.
separating the blood cells from the blood to yield a blood fraction
containing high density lipoprotein and low density lipoprotein; d.
mixing the blood fraction with a lipid removing agent which removes
lipids associated with a lipid bilayer of the high density
lipoprotein without substantially modifying the low density
lipoprotein to yield a mixture of lipid, the lipid removing agent,
the particle derivative, and the low density lipoprotein; e.
separating the particle derivative and the low density lipoprotein
from the lipid and the lipid removing agent; and, f. delivering the
particle derivative and the low density lipoprotein to the
patient.
41. The method of claim 40, wherein steps c through e occur remote
from the subject.
42. The method of claim 40, wherein the lipid removing agent is at
least one of an ether, di-isopropyl ether, sevoflurane, a
combination of an ether and an alcohol, or a combination of
sevoflurane and an alcohol.
43. The method of claim 42, wherein the lipid removing agent is a
mixture of sevoflurane and n-butanol.
44. The method of claim 43, wherein the mixture comprises 95 parts
sevoflurane and 5 parts n-butanol.
45. The method of claim 40, wherein the step of separating the
particle derivative and low density lipoprotein from the lipid and
lipid removing agent is achieved by at least one of an absorbent,
separator, centrifuge, or charcoal column.
46. The method of claim 45, wherein the at least one of an
absorbent, separator, centrifuge, or charcoal column does not
modify the protein shell of the particle derivative.
47. The method of claim 40, wherein the particle derivative and the
low density lipoprotein are first recombined with the blood cells
before delivering the particle derivative and the low density
lipoprotein to the patient.
48. The method of claim 40, wherein the mixing is performed using
at least one of a static mixer, vortexer or centrifuge.
49-72. (canceled)
Description
PRIOR RELATED APPLICATIONS
[0001] The present application claims the benefit of priority to
U.S. provisional patent application Ser. No. 60/484,690 filed Jul.
3, 2003.
FIELD OF INVENTION
[0002] The present invention generally relates to systems,
apparatus and methods for removing lipids from HDL particles while
leaving LDL particles substantially intact, via the extracorporeal
treatment of blood plasma using either a single solvent or multiple
solvents. The method of the present invention provides a procedure
for selective removal of lipid from HDL to create a modified HDL
particle while leaving LDL particles substantially intact. The
method of the present invention provides a procedure for removing
LDL particles from plasma before treating the HDL particles to
remove lipid. This invention creates particles comprising
derivatives of HDL that may be administered to an animal or human
for therapeutic use such as treatment of arteriosclerosis and
atherosclerotic vascular diseases within an animal or human.
BACKGROUND
Introduction--Hyperlipidemia and Arteriosclerosis
[0003] Cardiovascular, cerebrovascular, and peripheral vascular
diseases are responsible for a significant number of deaths
annually in many industrialized countries. One of the most common
pathological processes underlying these diseases is
arteriosclerosis. Arteriosclerosis is characterized by lesions,
which begin as localized fatty thickenings in the inner aspects of
blood vessels supplying blood to the heart, brain, and other organs
and tissues throughout the body. Over time, these atherosclerotic
lesions may ulcerate, exposing fatty plaque deposits that may break
away and embolize within the circulation. Atherosclerotic lesions
obstruct the lumens of the affected blood vessels and often reduce
the blood flow within the blood vessels, which may result in
ischemia of the tissue supplied by the blood vessel. Embolization
of atherosclerotic plaques may produce acute obstruction and
ischemia in distal blood vessels. Such ischemia, whether prolonged
or acute, may result in a heart attack or stroke from which the
patient may or may not recover. Similar ischemia in an artery
supplying an extremity may result in gangrene requiring amputation
of the extremity.
[0004] For some time, the medical community has recognized the
relationship between arteriosclerosis and levels of dietary lipid,
serum cholesterol, and serum triglycerides within a patient's blood
stream. Many epidemiological studies have been conducted revealing
that the amount of serum cholesterol within a patient's blood
stream is a significant predictor of coronary disease. Similarly,
the medical community has recognized the relationship between
hyperlipidemia and insulin resistance, which can lead to diabetes
mellitus. Further, hyperlipidemia and arteriosclerosis have been
identified as being related to other major health problems, such as
obesity and hypertension.
Cholesterol Transport
[0005] Cholesterol circulating in the blood is carried by plasma
lipoproteins that transport lipids throughout the blood. The plasma
lipoproteins are classified in five types according to size:
chylomicrons (which are largest in size and lowest in density),
very low-density lipoproteins (VLDL), intermediate density
lipoproteins (IDL), low-density lipoproteins (LDL), and
high-density lipoproteins (HDL) (which are the smallest and most
dense). These plasma lipoproteins exhibit differences in size,
density, diameter, protein content, phospholipid content and
triacylglycerol content, known to one of ordinary skill in the art.
Of these, the low-density lipoprotein (LDL) and high-density
lipoprotein (HDL) are primarily the major cholesterol carrier
proteins. The protein component of LDL, the apolipoprotein-B (Apo
B) and its products comprise the atherogenic elements. Elevated
plasma LDL levels and reduced HDL levels are recognized as the
primary cause of coronary disease because Apo B is in highest
concentration in LDL particles and is not present in HDL particles.
Apolipoprotein A-1 (Apo A-1) and apolipoprotein A-2 (Apo A-2) are
found in HDL. Other apolipoproteins, such as Apo C and its subtypes
(C-1, C-2 and C-3), Apo D and Apo E are also found in HDL. Apo C
and Apo E are also observed in LDL particles.
[0006] Numerous major classes of HDL particles including
HDL.sub.2b, HDL.sub.2a, HDL.sub.3a, HDL.sub.3b and HDL.sub.3c have
been reported (Segrest et al., Curr. Opin. Lipidol. 11:105-115,
2000). Various forms of HDL particles have been described on the
basis of electrophoretic mobility on agarose as two major
populations, a major fraction with .alpha.-HDL mobility and a minor
fraction with migration similar to VLDL (Barrans et al., Biochemica
Biophysica Acta 1300; 73-85, 1996). This latter fraction has been
called pre-.beta. HDL and these particles are thought to be the
most efficient HDL particle subclass for inducing cellular
cholesterol efflux (Segrest et al., Curr. Opin. Lipidol.
11:105-115, 2000). The pre-.beta. HDL particles have been further
separated into pre-.beta..sub.1 HDL, pre-.beta..sub.2 HDL and
pre-.beta..sub.3 HDL. These lipoprotein particles are comprised of
Apo A-1, phospholipids and free cholesterol. The pre-.beta. HDL
particles are considered to be the first acceptors of cellular free
cholesterol and are essential in eventually transferring free and
esterified cholesterol to .alpha.-HDL (Barrans et al., Biochemica
Biophysica Acta 1300; 73-85, 1996). Pre-.beta..sub.3 HDL particles
may transfer cholesterol to .alpha.-HDL or be converted to
.alpha.-HDL. These pre-.beta. HDL particles have been characterized
in terms of their charge, molecular mass (ranging from 40 kDa--420
kDa), size (Stoke's radius 4 nm-15 nm), shape (ellipsoidal,
discoidal or spherical) and chemical composition (protein
(including Apo A-1), free cholesterol, esterified cholesterol,
phospholipids and the ratio of free cholesterol to phospholipids
(see Barrans et al., Biochemica Biophysica Acta 1300; 73-85, 1996
for additional details)). HDL levels are inversely correlated with
atherosclerosis and coronary artery disease. Accordingly, what is
needed is a method to decrease or remove cholesterol from these
various HDL particles, especially the pre-.beta. HDL particles, so
that they are available to remove additional cholesterol from
cells.
[0007] Cholesterol is synthesized by the liver or obtained from
dietary sources. LDL is responsible for transferring cholesterol
from the liver to tissues at different sites in the body. However,
if LDL collects on the arterial walls, it undergoes oxidation
caused by oxygen free radicals liberated from the body's chemical
processes and interacts deleteriously with the blood vessels. The
modified LDL causes white blood cells in the immune system to
gather at the arterial walls, forming a fatty substance called
plaque and injuring cellular layers that line blood vessels. The
modified oxidized LDL also reduces the level of nitric oxide, which
is responsible for relaxing the blood vessels and thereby allowing
the blood to flow freely. As this process continues, the arterial
walls slowly constrict, resulting in hardening of the arteries and
thereby reducing blood flow. The gradual build-up of plaque can
result in blockage of a coronary vessel and ultimately in a heart
attack.
[0008] In contrast to LDL, high plasma HDL levels are desirable
because they play a major role in "reverse cholesterol transport",
where the excess cholesterol is transferred from tissue sites to
the liver where it is catabolized and eliminated. Optimal total
cholesterol levels are 200 mg/dl or below with a LDL cholesterol
level of 160 mg/dl or below and a HDL-cholesterol level of 45 mg/dl
for men and 50 mg/dl for women. Lower LDL levels are recommended
for individuals with a history of elevated cholesterol,
atherosclerosis or coronary artery disease.
Current Methods of Treatment
[0009] Hyperlipidemia may be treated by changing a patient's diet.
However, diet as a primary mode of therapy requires a major effort
on the part of patients, physicians, nutritionists, dietitians, and
other health care professionals and thus undesirably taxes the
resources of health professionals. Another negative aspect of this
therapy is that its success does not rest exclusively on diet.
Rather, success of dietary therapy depends upon a combination of
social, psychological, economic, and behavioral factors. Thus,
therapy based only on correcting flaws within a patient's diet is
not always successful.
[0010] In instances when dietary modification has been
unsuccessful, drug therapy has been used as an alternative. Such
therapy has included use of commercially available hypolipidemic
drugs administered alone or in combination with other therapies as
a supplement to dietary control. These drugs, called statins,
include natural statins, lovastatin, pravastatin, simvastatin,
fluvastatin, atorvastatin, and cerivastatin. Statins are
particularly effective for lowering LDL levels and are also
effective in the reduction of triglycerides, apparently in direct
proportion to their LDL-lowering effects. Statins raise HDL levels,
but to a lesser extent than other anti-cholesterol drugs. Statins
also increase nitric oxide, which, as described above, is reduced
in the presence of oxidized LDL.
[0011] Bile acid resins, another drug therapy, work by binding with
bile acid, a substance made by the liver using cholesterol as one
of the primary manufacturing components. Because the drugs bind
with bile acids in the digestive tract, they are then excreted with
the feces rather than being absorbed into the body. The liver, as a
result, must take more cholesterol from the circulation to continue
constructing bile acids, resulting in an overall decrease in LDL
levels.
[0012] Nicotinic acid, or niacin, is also known as vitamin B.sub.3.
It is extremely effective in reducing triglyceride levels and
raising HDL levels higher than any other anti-cholesterol drug.
Nicotinic acid also lowers LDL-cholesterol.
[0013] Fibric acid derivatives, or fibrates, are used to lower
triglyceride levels and increase HDL when other drugs ordinarily
used for these purposes, such as niacin, are not effective.
[0014] Probucol lowers LDL-cholesterol levels, however, it also
lowers HDL levels. It is generally used for certain genetic
disorders that cause high cholesterol levels, or in cases where
other cholesterol-lowering drugs are ineffective or cannot be
used.
[0015] Hypolipidemic drugs have had varying degrees of success in
reducing blood lipid; however, none of the hypolipidemic drugs
successfully treats all types of hyperlipidemia. While some
hypolipidemic drugs have been fairly successful, the medical
community has not found any conclusive evidence that hypolipidemic
drugs cause regression of atherosclerosis. In addition, all
hypolipidemic drugs have undesirable side effects. As a result of
the lack of success of dietary control, drug therapy and other
therapies, atherosclerosis remains a major cause of death in many
parts of the world.
[0016] New therapies have been used to reduce the amount of lipid
in patients for whom drug and diet therapies were not sufficiently
effective. For example, extracorporeal procedures like
plasmapheresis and LDL-apheresis have been employed and are shown
to be effective in lowering LDL.
[0017] Plasmapheresis therapy or plasma exchange therapy, involves
replacing a patient's plasma with donor plasma or more usually a
plasma protein fraction. Plasmapheresis is a process whereby the
blood plasma is removed from blood cells by a cell separator. The
separator works either by spinning the blood at high speed to
separate the cells from the fluid or by passing the blood through a
membrane with pores so small that only the fluid component of the
blood can pass through. The cells are returned to the person
undergoing treatment, while the plasma is discarded and replaced
with other fluids.
[0018] This treatment has resulted in complications due to the
introduction of foreign proteins and transmission of infectious
diseases. Further, plasmapheresis has the disadvantage of
non-selective removal of all serum proteins, such as VLDL, LDL, and
HDL. Moreover, plasmapheresis can result in several side effects
including allergic reactions in the form of fever, chills, and rash
and possibly even anaphylaxis.
[0019] As described above, it is not desirable to remove HDL, which
is secreted from both the liver and the intestine as nascent,
disk-shaped particles that contain cholesterol and phospholipids.
HDL is believed to play a role in reverse cholesterol transport,
which is the process by which excess cholesterol is removed from
tissues and transported to the liver for reuse or disposal in the
bile.
[0020] In contrast to plasmapheresis, the LDL-apheresis procedure
selectively removes Apo B containing cholesterol, such as LDL,
while retaining HDL. Several methods for LDL-apheresis have been
developed. These techniques include absorption of LDL in
heparin-agarose beads, the use of immobilized LDL-antibodies,
cascade filtration absorption to immobilize dextran sulphate, and
LDL precipitation at low pH in the presence of heparin. Each method
described above is effective in removing LDL. This treatment
process has disadvantages, however, including the failure to
positively affect HDL or to cause a metabolic shift that can
enhance atherosclerosis and other cardiovascular diseases. LDL
apheresis merely treats patients with severe hyperlipidemia.
[0021] Yet another method of achieving a reduction in plasma
cholesterol in homozygous familial hypercholesterolemia,
heterozygous familial hypercholesterolemia and patients with
acquired hyperlipidemia is an extracorporeal lipid elimination
process, referred to as cholesterol apheresis. In cholesterol
apheresis, blood is withdrawn from a patient, the plasma is
separated from the blood, and the plasma is mixed with a solvent
mixture. The solvent mixture extracts lipids from the plasma.
Thereafter, the delipidated plasma is recombined with the patient's
blood cells and returned to the patient.
[0022] Conventional extracorporeal delipidation processes, however,
are directed toward the concurrent delipidation of LDL and HDL.
This process can have a number of disadvantages. Because LDL is
more difficult to delipidate, extracorporeal systems are designed
to subject body fluid volumes to substantial processing, possibly
through multiple stage solvent exposure and extraction steps.
Vigorous multi-stage solvent exposure and extraction can have
several drawbacks. It may be difficult to remove a sufficient
amount of solvents from the delipidated plasma in order for the
delipidated plasma to be safely returned to a patient.
[0023] Hence, existing apheresis and extracorporeal systems for
treatment of plasma constituents suffer from a number of
disadvantages that limit their ability to be used in clinical
applications. A need exists for improved systems, apparatuses and
methods capable of removing lipids from blood components in order
to provide treatments and preventative measures for cardiovascular
diseases. What is also needed is a method to selectively remove
lipid from HDL particles and thereby create modified HDL particles
with increased capacity to accept cholesterol. What is also needed
is a method to selectively remove lipid from HDL particles and
thereby create modified HDL particles with increased capacity to
accept cholesterol, without substantially affecting LDL
particles.
SUMMARY OF THE INVENTION
[0024] The present invention is directed to systems, apparatus and
methods for creating modified HDL particles without substantially
affecting LDL. These modified HDL particles are derivatives of HDL
with reduced lipid content, particularly reduced cholesterol
content. These modified HDL particles have the capacity to bind
cholesterol and may be administered to a patient to enhance
cellular cholesterol efflux and reduce cholesterol levels in cells,
tissues, organs and blood vessels.
[0025] The present invention also provides a biological fluid
comprising a modified protein distribution wherein the biological
fluid had a first state, the first state having alpha high density
lipoproteins and pre-beta high density lipoproteins, and wherein
the biological fluid has a second state, the second state having an
increased concentration of pre-beta high density lipoprotein
relative to the first state, after being exposed to a lipid
removing agent.
[0026] The present invention also provides a biological fluid
capable of enhancing an ABCA1 pathway of a patient wherein the
biological fluid is made by modifying a fluid having a first
concentration of pre-beta high density lipoproteins relative to
total protein, wherein the modification increases the concentration
of pre-beta high density lipoprotein relative to total protein.
[0027] The present invention further provides a method of enhancing
an ABCA1 pathway of a patient with a first protein distribution,
the first protein distribution having a concentration of pre-beta
high density lipoproteins relative to total protein, comprising the
step of modifying a fluid containing the first protein distribution
by exposing the fluid to a lipid removing agent, wherein the
modification increases the concentration of pre-beta high density
lipoprotein relative to total protein, and introducing the fluid
into the patient.
[0028] The present invention further provides a method of modifying
a protein distribution in a fluid wherein the protein distribution
has a first state, said first state having alpha high density
lipoproteins and pre-beta high density lipoproteins, comprising the
steps of: exposing said fluid to a lipid removing agent wherein the
exposure modifies the protein distribution from the first state
into a second state, said second state having an increased
concentration of pre-beta high density lipoprotein relative to said
first state; and removing said lipid removing agent from the
biological fluid.
[0029] The present invention discloses a method for removing lipids
from fluids, such as blood plasma, and from HDL particles without
substantially affecting LDL by treating the fluid with solvents and
adding energy to mix the solvents and fluid. Removing lipid from
HDL particles creates a modified HDL particle with reduced lipid
content, which is capable of binding additional lipid and enhancing
cellular cholesterol efflux. More particularly, the present
invention is directed toward removal of lipids from HDL particles
in blood plasma using either a single solvent or multiple solvents,
thereby creating new particles that are derivatives of HDL with
reduced lipid content.
[0030] In one embodiment of the present invention, LDL and HDL
particles are separated prior to treatment of the plasma containing
the HDL particles. LDL is extracted and the plasma is treated to
reduce the lipid content of HDL particles. Subsequent to LDL
removal, the plasma containing HDL particles is exposed to lipid
removing agents using the present methods to reduce lipid levels
and create particle derivatives of HDL with reduced lipid content.
These particles demonstrate enhanced capacity for binding
cholesterol. These particle derivatives of HDL and the plasma with
reduced lipid content may be administered to the patient in order
to enhance cellular cholesterol efflux and treat lipid-associated
diseases and conditions.
[0031] In another embodiment of the present invention, the LDL is
retained (not separated prior to treatment) and a solvent system is
employed to selectively remove lipid from HDL and create particles
comprised of derivatives of HDL with reduced lipid content while
not substantially affecting LDL. The separated plasma is mixed with
a solvent system designed to selectively decrease lipid in HDL
particles present in the plasma. Care is taken to ensure that the
solvent employed, the mixing method employed, procedure, mixing
time, and temperature create an optimal solvent system that will
selectively remove lipid from HDL, create particles comprised of
derivatives of HDL, and leave LDL substantially intact. The at
least partially or substantially delipidated plasma, which was
separated initially, is then treated appropriately for
administration to a patient.
[0032] The present invention may be employed to treat plasma
obtained from a patient for subsequent administration to the
patient or for administration into another patient. The present
invention may also be used to treat blood and plasma stored in
blood banks in order to create plasma with reduced lipid content
and containing particles comprised of derivatives of HDL with
reduced lipid content. This treated plasma containing particles
comprised of derivatives of HDL with reduced lipid content may be
used for heterologous administration to another individual in order
to enhance cholesterol efflux in the patient. The present invention
may also be employed to create particles comprised of derivatives
of HDL that may be collected and stored.
[0033] The present method modifies various forms of different HDL
particles. Such HDL particles include but are not limited to those
HDL particles that have been described based on a variety of
methods such as methods that measure charge, density, size and
immunoaffinity, including but not limited to electrophoretic
mobility, ultracentrifugation, immunoreactivity and other methods
known to one of ordinary skill in the art. Such HDL particles
include but are not limited to the following: VLDL, .alpha. HDL,
pre-.beta. HDL (including pre-.beta..sub.1 HDL, pre-.beta..sub.2
HDL and pre-.beta..sub.3 HDL), .beta. HDL, HDL.sub.2 (including
HDL.sub.2a and HDL.sub.2b), HDL.sub.3, VHDL, LpA-I, LpA-II,
LpA-I/LpA-II (for a review see Barrans et al., Biochemica
Biophysica Acta 1300; 73-85, 1996). Accordingly, practice of the
methods of the present invention creates modified HDL particles.
These modified HDL particles may be modified in numerous ways,
including but not limited to changes in one or more of the
following metabolic and or physico-chemical properties: molecular
mass (kDa); charge; diameter; shape; density; hydration density;
flotation characteristics; content of cholesterol; content of free
cholesterol; content of esterified cholesterol; molar ratio of free
cholesterol to phospholipids; immunoaffinity; content, activity or
helicity of one or more of the following enzymes or proteins (Apo
A-1, Apo A-2, Apo D, Apo E, Apo J, Apo A-IV, cholesterol ester
transfer protein (CETP), lecithin:cholesterol acyltransferase
(LCAT); capacity and/or rate for cholesterol binding, capacity
and/or rate for cholesterol transport. The physical-chemical
properties of HDL particles are known to one of ordinary skill in
the art. For example, pre-.beta. HDL particles have been
characterized in terms of their charge, molecular mass (ranging
from 40 kDa-420 kDa), size (Stoke's radius 4 nm-15 nm), shape
(ellipsoidal, discoidal or spherical) and chemical composition
(protein (including Apo A-1), free cholesterol, esterified
cholesterol, phospholipids and the ratio of free cholesterol to
phospholipids (see Barrans et al., Biochemica Biophysica Acta 1300;
73-85, 1996 for additional details)).
[0034] The present invention creates these modified HDL particles
without substantially affecting various metabolic and or
physico-chemical properties of LDL particles.
[0035] In another aspect of the present invention, the modified HDL
derivative particles made with the disclosed method are
administered to a patient in order to enhance cholesterol efflux
from cells. These modified HDL particles may be obtained from the
same patient or a different patient who will receive the modified
HDL particles. These particles may be combined with plasma treated
with the methods of the present invention and containing
substantially reduced levels of lipid and then administered to a
patient.
[0036] The present invention also provides a modified Apo A-1
protein produced by treating plasma with the method of the present
invention, wherein the modified Apo A-1 protein has reduced lipid
content. The modified Apo A-1 protein is purified and may be
administered to a patient either alone or in conjunction with the
modified HDL particles with reduced lipid content to enhance
cholesterol efflux.
[0037] These modified HDL particles may also be combined with
heterologous plasma treated with the methods of the present
invention and containing substantially reduced levels of lipid and
then administered to a patient. These particles may be combined
with other plasma constituents, and optionally with red blood cells
before administration into the vascular system. Administration of
these particles occurs as frequently as necessary to effectuate
cholesterol efflux from cells.
[0038] The modified HDL particles of the present invention are
administered to patients in order to reduce cellular levels of
cholesterol, and are indicated for a variety of conditions,
including but not limited to atherosclerosis, arteriosclerosis,
hyperlipidemia, hypercholesterolemia, obesity, hypertension,
stroke, neuroprotection following stroke, inflammation, Alzheimer's
disease, diabetes, low endogenous HDL levels, high LDL levels,
cardiovascular disease (including atherosclerosis of the coronary
arteries, carotid arteries, subclavian, brachial, aorta, iliac,
renal, femoral, popliteal, tibial or any other artery in the
cardiovascular system), cerebrovascular disease (including
atherosclerosis of the internal carotid, middle cerebral, anterior
cerebral, posterior cerebral, basilar, cerebellar, and/or spinal
arteries, or any branch of these arteries, cerebral cortical end
arteries, or any other artery supplying the central nervous
system).
[0039] The modified HDL particles of the present invention are
administered to a patient according to any schedule that is
effective to enhance cellular cholesterol efflux. In one
non-limiting example, a liter of plasma is treated with the methods
of the present invention each week and the treated plasma
containing the modified HDL particles is returned to the patient
each week for four to six weeks. Alternatively, the modified HDL
particles may be separated from the treated plasma and administered
in an acceptable vehicle.
[0040] It is to be understood that the modified HDL particles of
the present invention may be administered in conjunction other
regimens and treatments for treatment of the diseases and
conditions mentioned above. For example, the modified HDL particles
of the present invention may be administered in conjunction with
exercise and/or dietary restriction of fat and cholesterol
intake.
[0041] The modified HDL particles of the present invention may be
used in conjunction with administration of agents for reducing
cholesterol, reducing LDL levels and enhancing HDL levels. These
agents, such as HMG-CoA reductase inhibitors, or statins, may be
administered in dosages and according to administration schedules
commonly known to one of ordinary skill in the art. Statins include
but are not limited to cerivastatin, atorvastatin, fluvastatin,
simvastatin, pravastatin and lovastatin. For example, dosages of 10
mg, 20 mg, 40 mg or 80 mg of statins, taken once per day, are
commonly employed. Administration of the modified HDL particles of
the present invention can eliminate the need for statin therapy in
patients or reduce the required dosage of statins.
[0042] In another aspect, the modified HDL particles of the present
invention are used in conjunction with administration of agents
designed to reduce absorption of fat and cholesterol. Such agents,
for example ezetimibe, and the clinically appropriate dosages are
known to one of ordinary skill in the art.
[0043] In yet another aspect of the present invention, the modified
HDL particles are used in conjunction with administration of one or
more agents such as fibric acid derivatives (gemfibrozil),
nicotinic acid (niacin), and bile acid-binding resins
(cholestyramine, cholestipol).
[0044] In yet another aspect, the modified HDL particles of the
present invention are used in conjunction with administration of
anti-inflammatory drugs known to one of ordinary skill in the art,
such as aspirin. Anti-inflammatory drugs are often prescribed to
patients with vascular disease since it is believed that
inflammation is a causative factor of atherosclerosis and other
vascular diseases.
[0045] The modified HDL particles of the present invention are used
in conjunction with administration of agents such as statins
together with agents designed to reduce absorption of fat and
cholesterol. This combination of three therapies is effective in
enhancing cholesterol efflux from cells and permits administration
of lower dosages of statins. The modified HDL particles of the
present invention are also used in conjunction with any of the
therapeutic approaches described above.
[0046] These modified HDL particles may be stored before use. They
may be made from a patient's plasma and returned to that patient.
Alternatively, the modified HDL particles may be made from plasma
obtained from a first patient and subsequently administered to a
second patient. The present invention is useful in creating plasma
samples containing modified HDL particles for storage in a plasma
bank and subsequent administration to patients.
[0047] Accordingly, it is an object of the present invention to
provide particles comprising modified HDL particles.
[0048] It is another object of the present invention to provide
particles comprising modified HDL particles without substantially
affecting LDL.
[0049] Yet another object of the present invention is to provide
particles comprising derivatives of at least one form of HDL,
wherein the particle has a reduced cholesterol content.
[0050] It is another object of the present invention to provide
particles comprising derivatives of at least one form of HDL with a
reduced ratio of free cholesterol to phospholipid.
[0051] Another object of the present invention is to provide
particles comprising derivatives of at least one form of HDL,
wherein the particles are pre-.beta. HDL particles.
[0052] Yet another object of the present invention is to provide a
biological fluid comprising a modified protein distribution wherein
the biological fluid had a first state, the first state having
alpha high density lipoproteins and pre-beta high density
lipoproteins, and wherein the biological fluid has a second state,
the second state having an increased concentration of pre-beta high
density lipoprotein relative to the first state, after being
exposed to a lipid removing agent.
[0053] Accordingly, it is an object of the present invention to
provide a novel method for creation of particles comprising
derivatives of at least one form of HDL.
[0054] It is yet another object of the present invention to provide
a novel method for creation of particles comprising derivatives of
at least one form of HDL without substantially affecting LDL.
[0055] Another object of the present invention is to provide a
method of modifying a protein distribution in a fluid wherein the
protein distribution has a first state, said first state having
alpha high density lipoproteins and pre-beta high density
lipoproteins, comprising the steps of: exposing said fluid to a
lipid removing agent wherein the exposure modifies the protein
distribution from the first state into a second state, said second
state having an increased concentration of pre-beta high density
lipoprotein relative to said first state; and removing said lipid
removing agent from the biological fluid.
[0056] It is yet another object of the present invention to provide
a biological fluid capable of enhancing an ABCA1 pathway of a
patient wherein the biological fluid is made by modifying a fluid
having a first concentration of pre-beta high density lipoproteins
relative to total protein, wherein the modification increases the
concentration of pre-beta high density lipoprotein relative to
total protein.
[0057] Another object of the present invention is to provide a
method of enhancing an ABCA1 pathway of a patient with a first
protein distribution, the first protein distribution having a
concentration of pre-beta high density lipoproteins relative to
total protein, comprising the step of modifying a fluid containing
the first protein distribution by exposing the fluid to a lipid
removing agent, wherein the modification increases the
concentration of pre-beta high density lipoprotein relative to
total protein, and introducing the fluid into the patient.
[0058] Yet another object of the present invention is to provide a
novel method for treating diseases associate with lipid
accumulation by administering to a patient a composition comprising
particles that are derivatives of at least one form of HDL.
[0059] It is another object of the present invention to provide a
novel method for treating diseases associated with lipid
accumulation by administering to a patient a composition comprising
particles that are derivatives of at least one form of HDL in
conjunction with therapeutic administration of a statin, an
inhibitor of cholesterol or lipid uptake, niacin, fibric acid
derivatives, bile acid-binding resins, or a combination
thereof.
[0060] Yet another object of the present invention is to provide a
novel method for enhancing cellular cholesterol efflux in a patient
comprising administration of a composition comprising particles
that are derivatives of at least one form of HDL.
[0061] Still another object of the present invention is to provide
a novel method for treating atherosclerosis by administering to a
patient a composition comprising particles that are derivatives of
at least one form of HDL.
[0062] Another object of the present invention is to provide a kit
useful for treating a biological fluid in order to reduce
cholesterol and lipid and to create particles comprising
derivatives of at least one form of HDL.
[0063] These and other objects, features and advantages of the
present invention will become apparent after a review of the
following detailed description of the disclosed embodiments and
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0064] FIG. 1 is a flowchart delineating the steps of the LDL
extraction, and subsequent creation of modified HDL particles .
[0065] FIG. 2 is a flowchart delineating the steps of the selective
creation of modified HDL particles.
[0066] FIG. 3 is a schematic of an FPLC profile of a sample of
plasma from a pool of normal plasma. Total cholesterol (TC) is
represented as a continuous line; phospholipid (PPL) is the dashed
line; apolipoprotein A1 (Apo A-1) is the dashed line with a symbol;
and, apolipoprotein B (Apo B) is represented by the line with a
triangle. Shown are the amounts of these compounds (mg/dl) in each
FPLC fraction.
[0067] FIG. 4 is a schematic of an FPLC profile of an aliquot from
the pool of normal plasma which was subjected to a solvent of 100%
diisopropyl ether (DIPE) to remove lipid from HDL. Total
cholesterol (TC) in the normal sample from FIG. 3 is represented
here as a dashed line with triangles. TC in the normal plasma
subjected to DIPE is shown as a solid line. Apo A-1 in the normal
sample from FIG. 3 is represented here as a solid line with square
symbols. Apo A-1 in the normal plasma subjected to DIPE is shown as
a dashed line with a dot symbol.
[0068] FIG. 5 is a schematic of an FPLC profile of an aliquot from
the pool of normal plasma which was subjected to a solvent of 100%
DIPE to remove lipid from HDL. Apo A-1 in the normal plasma sample
from FIG. 3 is represented here as a solid line with square
symbols. Apo A-1 in the normal plasma sample subjected to DIPE is
shown as a dashed line with a dot symbol. Phospholipid (PPL) in the
normal plasma sample from FIG. 3 is represented here with the
dashed line. PPL in the normal plasma subjected to DIPE is shown as
a solid line.
[0069] FIG. 6 is a schematic of an FPLC profile of an aliquot from
the pool of normal plasma which was subjected to a solvent of 100%
DIPE to remove lipid from HDL. Apo B in the normal plasma sample is
represented by the line with a dot symbol. Apo B in the normal
plasma subjected to DIPE is shown as dashed line with triangles.
Phospholipid (PPL) in the normal sample from FIG. 3 is represented
with the solid line. PPL in the normal plasma subjected to DIPE is
shown as a dashed line.
[0070] FIG. 7 is a schematic of an FPLC profile of an aliquot from
the pool of normal plasma which was subjected to a solvent ratio of
95:5 sevoflurane to n-butanol. Total cholesterol (TC) in the normal
plasma sample from FIG. 3 is represented here as a dashed line. TC
in the normal plasma subjected to sevoflurane:n-butanol is shown as
a solid line. Apo A-1 in the normal plasma sample from FIG. 3 is
represented as a solid line with square symbols. Apo A-1 in the
normal plasma subjected to sevoflurane:n-butanol is shown as a
dashed line with a dot symbol.
[0071] FIG. 8 is a schematic of an FPLC profile of an aliquot from
the pool of normal plasma which was subjected to a solvent ratio of
95:5 sevoflurane to n-butanol. Apo A-1 in the normal sample from
FIG. 3 is represented as a solid line with square symbols. Apo A-1
in the normal plasma subjected to sevoflurane:n-butanol is shown as
a dashed line with a dot symbol. Phospholipid (PPL) in the normal
sample from FIG. 3 is represented here with the dashed line. PPL in
the normal plasma subjected to sevoflurane:n-butanol is shown as a
solid line.
[0072] FIG. 9 is a schematic of an FPLC profile of an aliquot from
the pool of normal plasma which was subjected to a solvent ratio of
95:5 sevoflurane to n-butanol. Apo B in the normal sample from FIG.
3 is represented by the line with a dot symbol. Apo B in the normal
plasma subjected to sevoflurane:n-butanol is shown as dashed line
with triangles. Phospholipid (PPL) in the normal sample from FIG. 3
is represented here with the solid line. PPL in the normal plasma
subjected to sevoflurane:n-butanol is shown as a dashed line.
[0073] FIG. 10 is a schematic representation of the effect of
treatment of a plasma sample with either DIPE or
sevoflurane:butanol on alanine aminotransferase (ALT), alkaline
phosphatase (AP), bilirubin-T, sodium, potassium, phosphorus,
albumin, globulin and the albumin/globulin (A/G) ratio were
analyzed in normal untreated plasma and in plasma treated with DIPE
or with sevoflurane:n-butanol.
[0074] FIG. 11 is a schematic representation of a Superose FPLC
profile of a normal plasma sample which acts as a control for
comparison to the treatments in FIGS. 12-15. Total cholesterol (TC)
in the normal plasma sample is represented here as a solid line.
Phospholipid (PPL) is represented with the solid line with circles.
Apo B is represented by the line with open squares. Apo A-1 is
represented as a solid line with open triangle symbols. Apo A-2 is
shown as a dashed line with a star symbol.
[0075] FIG. 12 is a schematic representation of a Superose FPLC
profile of the effect of treatment of an aliquot of control plasma
sample (FIG. 11) with DIPE (100%). Total cholesterol (TC) is
represented here as a solid line. Phospholipid (PPL) is represented
with the solid line with solid circles. Apo B is represented by the
line with solid squares. Apo A-1 is represented as a solid line
with closed triangle symbols. Apo A-2 is shown as a dashed line
with a star symbol in a solid square.
[0076] FIG. 13 is a schematic representation of a Superose FPLC
profile of the effect of treatment of an aliquot of control plasma
sample (FIG. 11) with a solvent ratio of 95:5 sevoflurane to
n-butanol. Total cholesterol (TC) is represented here as a solid
line. Phospholipid (PPL) is represented with the solid line with
solid circles. Apo B is represented by the line with solid squares.
Apo A-1 is represented as a solid line with closed triangle
symbols. Apo A-2 is shown as a dashed line with a star symbol in a
solid square.
[0077] FIG. 14 is a schematic representation of a Superose FPLC
profile of the effect of treatment of an aliquot of control plasma
sample (FIG. 11) with a solvent ratio of 75:25 DIPE to n-butanol.
Total cholesterol (TC) is represented here as a solid line.
Phospholipid (PPL) is represented with the solid line with solid
circles. Apo B is represented by the line with solid squares. Apo
A-1 is represented as a solid line with closed triangle symbols.
Apo A-2 is shown as a dashed line with a star symbol in a solid
square.
[0078] FIG. 15 is a schematic representation of a Superose FPLC
profile of the effect of treatment of an aliquot of control plasma
sample (FIG. 11) with a solvent ratio of 95:5 DIPE to n-butanol.
Total cholesterol (TC) is represented here as a solid line.
Phospholipid (PPL) is represented with the solid line with solid
circles. Apo B is represented by the line with solid squares. Apo
A-1 is represented as a solid line with closed triangle symbols.
Apo A-2 is shown as a dashed line with a star symbol in a solid
square.
[0079] FIG. 16 demonstrates the effects of various solvent
treatments of plasma on the ability of treated plasma to stimulate
cholesterol efflux in metabolic pathway ABCA1 and metabolic pathway
SRB1 as represented in cell lines COS+ and FU5AH when compared to
untreated or sham treated samples.
[0080] FIG. 17 is a representation of Apo A-1-containing HDL
subspecies determined by 3-16% native PAGE, immunoblot and image
analysis of a sample of normal lipemic plasma (left panel) and an
aliquot of this plasma treated with sevoflurane:n-butanol (95:5)
(right panel). The left panel depicts a distribution of protein of
various HDL species having a distribution primarily comprising
alpha HDL and the right panel depicts a distribution of protein of
modified HDL having a distribution primarily comprising pre-.beta.
HDL.
[0081] FIG. 18 is a representation of Apo A-1-containing HDL
subspecies determined by 3-16% native PAGE, immunoblot and image
analysis of a sample of normal lipemic plasma (left panel) and a
aliquot of this plasma treated with DIPE:n-butanol (95:5) (right
panel). The left panel depicts a distribution of protein of various
HDL species having a distribution primarily comprising alpha HDL
and the right panel depicts a distribution of protein of modified
HDL having a distribution primarily comprising pre-.beta. HDL.
[0082] FIG. 19 is a schematic representation of a plurality of
components used in the present invention to achieve the novel
delipidation processes disclosed herein.
[0083] FIG. 20 is one embodiment of a configuration of a plurality
of components used in the present invention to achieve the novel
delipidation processes disclosed herein.
[0084] FIG. 21 is another embodiment of a configuration of a
plurality of components used in the present invention to achieve
the novel delipidation processes disclosed herein.
DETAILED DESCRIPTION OF THE INVENTION
[0085] This invention relates to systems, apparatus and methods
useful for removing lipid from HDL particles derived primarily from
plasma of patients thereby creating modified HDL particles with
reduced lipid content, particularly reduced cholesterol content.
The present methods create these modified HDL particles with
reduced lipid content without substantially modifying LDL
particles.
[0086] The present invention further provides a biological fluid
comprising a modified protein distribution wherein the biological
fluid had a first state, the first state having alpha high density
lipoproteins and pre-beta high density lipoproteins, and wherein
the biological fluid has a second state, the second state having an
increased concentration of pre-beta high density lipoprotein
relative to the first state, after being exposed to a lipid
removing agent. The present invention provides a biological fluid
capable of enhancing an ABCA1 pathway of a patient wherein the
biological fluid is made by modifying a fluid having a first
concentration of pre-beta high density lipoproteins relative to
total protein, wherein the modification increases the concentration
of pre-beta high density lipoprotein relative to total protein.
[0087] The present invention provides newly formed derivatives of
HDL particles that may be administered to patients to enhance
cellular cholesterol efflux and treat diseases, particularly
arteriosclerosis, atherosclerosis, cardiovascular and other
lipid-associated diseases.
Definitions
[0088] The term "fluid" is defined as fluids from animals or humans
that contain lipids or lipid containing particles, fluids from
culturing tissues and cells that contain lipids and fluids mixed
with lipid-containing cells. For purposes of this invention,
decreasing the amount of lipids in fluids includes decreasing
lipids in plasma and particles contained in plasma, including but
not limited to HDL particles. Fluids include, but are not limited
to: biological fluids; such as blood, plasma, serum, lymphatic
fluid, cerebrospinal fluid, peritoneal fluid, pleural fluid,
pericardial fluid, various fluids of the reproductive system
including, but not limited to, semen, ejaculatory fluids,
follicular fluid and amniotic fluid; cell culture reagents such as
normal sera, fetal calf serum or serum derived from any animal or
human; and immunological reagents, such as various preparations of
antibodies and cytokines from culturing tissues and cells, fluids
mixed with lipid-containing cells, and fluids containing
lipid-containing organisms, such as a saline solution containing
lipid-containing organisms. A preferred fluid treated with the
methods of the present invention is plasma.
[0089] The term "lipid" is defined as any one or more of a group of
fats or fat-like substances occurring in humans or animals. The
fats or fat-like substances are characterized by their insolubility
in water and solubility in organic solvents. The term "lipid" is
known to those of ordinary skill in the art and includes, but is
not limited to, complex lipid, simple lipid, triglycerides, fatty
acids, glycerophospholipids (phospholipids), true fats such as
esters of fatty acids, glycerol, cerebrosides, waxes, and sterols
such as cholesterol and ergosterol.
[0090] The term "extraction solvent" is defined as one or more
solvents used for extracting lipids from a fluid or from particles
within the fluid. This solvent enters the fluid and remains in the
fluid until removed by other subsystems. Suitable extraction
solvents include solvents that extract or dissolve lipid, including
but not limited to phenols, hydrocarbons, amines, ethers, esters,
alcohols, halohydrocarbons, halocarbons, and combinations thereof
Preferred extraction solvents are ethers, esters, alcohols,
halohydrocarbons, or halocarbons which include, but are not limited
to di-isopropyl ether (DIPE), which is also referred to as
isopropyl ether, diethyl ether (DEE), which is also referred to as
ethyl ether, lower order alcohols such as butanol, especially
n-butanol, ethyl acetate, dichloromethane, chloroform, isofluorane,
sevoflurane (1,1,1,3,3,3-hexafluoro-2-(fluoromethoxy) propane-d3),
perfluorocyclohexanes, trifluoroethane, cyclofluorohexanol, and
combinations thereof.
[0091] The term "patient" refers to animals and humans, which may
be either a fluid source to be treated with the methods of the
present invention or a recipient of derivatives of HDL particles
and or plasma with reduced lipid content.
[0092] The term "HDL particles" encompasses several types of
particles defined based on a variety of methods such as those that
measure charge, density, size and immunoaffinity, including but not
limited to electrophoretic mobility, ultracentrifugation,
immunoreactivity and other methods known to one of ordinary skill
in the art. Such HDL particles include but are not limited to the
following: VLDL, .alpha. HDL, pre-.beta. HDL (including
pre-.beta..sub.1 HDL, pre-.beta..sub.2 HDL and
pre-.beta..sub.3HDL), .beta. HDL, HDL.sub.2 (including HDL.sub.2a
and HDL.sub.2b) HDL.sub.3, VHDL, LpA-I, LpA-II, LpA-I/LpA-II (for a
review see Barrans et al., Biochemica Biophysica Acta 1300; 73-85,
1996). Accordingly, practice of the methods of the present
invention creates modified HDL particles. These modified
derivatives of HDL particles may be modified in numerous ways
including but not limited to changes in one or more of the
following metabolic and or physico-chemical properties (for a
review see Barrans et al., Biochemica Biophysica Acta 1300; 73-85,
1996): molecular mass (kDa); charge; diameter; shape; density;
hydration density; flotation characteristics; content of
cholesterol; content of free cholesterol; content of esterified
cholesterol; molar ratio of free cholesterol to phospholipids;
immunoaffinity; content, activity or helicity of one or more of the
following enzymes or proteins (Apo A-1, Apo A-2, Apo D, Apo E, Apo
J, Apo A-IV, cholesterol ester transfer protein (CETP),
lecithin:cholesterol acyltransferase (LCAT); capacity and/or rate
for cholesterol binding, capacity and/or rate for cholesterol
transport.
Methods
[0093] The methods of the present invention employ techniques to
create HDL particles with reduced lipid content. These HDL
particles are obtained from fluids, such as plasma. The first
method comprises removal of LDL from plasma before treating the
plasma to decrease lipids and to create HDL particles with reduced
lipid content. The second method does not remove LDL from plasma
before exposure to solvents but employs various solvent systems for
enabling selective removal of lipids from HDL particles without
substantially affecting LDL. The various steps involved in the two
methods are described generally below. Following these general
descriptions are descriptions of various embodiments of the methods
of the present invention, including variants such as solvents
employed, mixing methods, mixing times, and optionally,
temperature.
[0094] The present invention further provides a method of modifying
a protein distribution in a fluid wherein the protein distribution
has a first state, said first state having alpha high density
lipoproteins and pre-beta high density lipoproteins, comprising the
steps of: exposing said fluid to a lipid removing agent wherein the
exposure modifies the protein distribution from the first state
into a second state, said second state having an increased
concentration of pre-beta high density lipoprotein relative to said
first state; and removing said lipid removing agent from the
biological fluid.
[0095] The present invention also provides a method of enhancing an
ABCA1 pathway of a patient with a first protein distribution, the
first protein distribution having a concentration of pre-beta high
density lipoproteins relative to total protein, comprising the step
of modifying a fluid containing the first protein distribution by
exposing the fluid to a lipid removing agent, wherein the
modification increases the concentration of pre-beta high density
lipoprotein relative to total protein, and introducing the fluid
into the patient.
[0096] As discussed above, the methods and systems of the present
invention may be composed of numerous configurations. Set forth
below are numerous components that may be combined to create the
numerous embodiments that are capable of achieving the objectives
and advantages described above. These embodiments are described to
teach the invention and are not meant to limit the scope of the
invention. Rather, each embodiment is but one of many possible
configurations that can be used to accomplish the objectives
described above.
LDL Extraction and Removal of Lipids from HDL Particles
[0097] In one embodiment of the present invention, as shown in FIG.
1, the HDL and LDL particles are separated prior to treatment. FIG.
1 is a flow chart of the process for LDL extraction and removal of
lipids from HDL particles.
[0098] In step 100 of the process for LDL extraction and removal of
lipids from HDL particles, the plasma is separated from the blood.
In a preferred embodiment, this is achieved via filtration. In
another preferred embodiment, the plasma and blood components are
separated via centrifugation. The blood can optionally be combined
with an anticoagulant, such as sodium citrate, and centrifuged at
forces approximately equal to 2,000 times gravity. The red blood
cells are then aspirated from the plasma. In step 102, the cells
are returned to the patient. In this particular embodiment of the
present invention, the LDL is separated from the plasma in step
104. This is achieved via use of an affinity column,
ultracentrifugation, or any other method known to one of ordinary
skill in the art. An exemplary method is the use of
ultracentrifugation, in which the plasma is passed through the
ultracentrifugal separator, thereby parsing out the LDL and HDL
particles. The ultracentrifugal separator uses density gradient
ultracentrifugation--a sophisticated and highly accurate process
that separates lighter portions of lipoprotein from heavier
portions by centrifugal force. The LDL is discarded in step
106.
[0099] In step 108, solvents are added to the plasma which still
contains HDL in order to remove lipids. The solvent types, ratios,
and concentrations can vary. The plasma and solvent are introduced
into at least one apparatus for mixing, agitating, or otherwise
contacting the plasma with the solvent. The plasma may be
transported using a continuous or batch process. Furthermore
various sensing means may be included to monitor pressures,
temperatures, flow rates, solvent levels, and the like (discussed
in more detail below).
[0100] In step 110, energy is introduced to the system. The various
forms of energy employed involve mixing methods, time, and speed
(variants of which are discussed in further detail below).
Centrifugation is employed in step 112 to remove the residual bulk
solvent. The remaining soluble solvent is removed in step 114. This
is achieved via charcoal adsorption, evaporation, or HFC
pervaporation, as discussed below. In optional step 116, the
mixture is tested for residual solvent via gas chromatography (GC)
or any other similar means. Optionally, this step is eliminated
with statistical validation. In step 118, the plasma with reduced
lipid content is returned to the patient. This plasma with at least
partially or substantially reduced lipid levels, which was
separated initially, is then treated appropriately and subsequently
reintroduced into the body.
Selective Removal of Lipidfrom HDL and Formation of Modified HDL
Particles
[0101] FIG. 2 presents a flowchart delineating the steps of the
another preferred embodiment of the present invention. In step 200,
plasma is separated from the blood via filtration, centrifugation
or any other means known to one of ordinary skill in the art. In a
preferred embodiment, the blood is passed through a centrifugal
separator, which separates the blood into blood cells and plasma.
In step 202, the cells are returned to the patient. Solvents are
added to the separated plasma in step 204 in order to extract
lipids. The solvent system is optimally designed such that only the
HDL particles are treated to reduce their lipid levels and LDL
remains at least substantially intact. The solvent system includes
factoring in variables such as solvent employed, mixing method,
time, and temperature. Solvent type, ratios and concentrations may
vary in this step. The plasma and solvent are introduced into at
least one apparatus for mixing, agitating, or otherwise contacting
the plasma with the solvent. The plasma may be transported using a
continuous or batch process. Further, various sensing means may be
included to monitor pressures, temperatures, flow rates, solvent
levels, and the like (discussed in more detail below).
[0102] In step 206, energy is introduced into the system in the
form of varied mixing methods, time, and speed. Bulk solvents are
removed in step 208 via centrifugation. In step 210 the remaining
soluble solvent is removed via charcoal adsorption, evaporation, or
HFC pervaporation. In step 212, the mixture is optionally tested
for residual solvent via use of GC, or similar means. The test for
residual solvent may optionally be eliminated based on statistical
validation. In step 214, the treated plasma (preferably containing
modified HDL particles with reduced lipid content), which was
separated initially, is treated appropriately and subsequently
returned to the patient.
[0103] One of ordinary skill in the art would appreciate that
although the processes shown in FIGS. 1 and 2 depict only the main
steps of the processes and reference primary elements of the
systems, they may optionally contain other elements such as a blood
pump for maintaining proper blood volume, a blood pressure meter, a
blood anticoagulant agent injecting device, a drip chamber for
eliminating air bubbles in the blood, and a heater or cooler for
maintaining an appropriate temperature for the blood while it is
outside the body.
Variables to be Considered in Employing the Methods of the Present
Invention
[0104] The present invention employs one of many optimally
configured solvent systems designed to remove lipids from HDL
particles while not substantially affecting LDL. In the first
embodiment, LDL is removed from the plasma before treating the
plasma with solvent(s) to create HDL particles with reduced lipid
content while retaining the composition of the plasma proteins. In
the second embodiment, care is taken to selectively remove lipids
from HDL particles without substantially affecting LDL particles.
These variables include solvent choice, mixing methods, time, and
temperature.
Plasma Separation Procedures
[0105] Typical plasma separation procedures are well known to those
of ordinary skill in the art and preferably include, but are not
limited to, filtration, centrifugation, and aspiration.
LDL Extraction
[0106] Methods of LDL extraction are well known to those of
ordinary skill in the art. For purposes of the present invention,
two preferred methods are, but not limited to, use of an affinity
column and ultracentrifugation. The ultracentrifugal separator uses
density gradient ultra centrifugation--a sophisticated and highly
accurate process that separates lighter portions of lipoprotein
from heavier portions by centrifugal force.
Solvents Employed in the Process of Removing Lipids
[0107] Numerous organic solvents may be used in the method of this
invention for removal of lipid from fluids and HDL particles,
provided that the solvents are effective in solubilizing lipids.
Suitable solvents comprise mixtures of aromatic, aliphatic, or
alicyclic hydrocarbons, ethers, phenols, esters, alcohols,
halohydrocarbons, and halocarbons. Preferred solvents are ethers,
for example di-isopropyl ether (DIPE). Asymmetrical ethers and
halogenated ethers may be used. Particularly preferred, as at least
one component, are the C.sub.4-C.sub.8 containing-ethers, including
but not limited to, diethyl ether, and propyl ethers, including but
not limited to DIPE. Also useful in the present invention are
combinations of ethers, such as DIPE and diethyl ether. Also useful
in the present invention are combinations of ethers and alcohols,
such as DIPE and butanol. Also preferred in the present invention
are combinations of fluoroethers and alcohols, such as sevoflurane
and butanol, particularly sevoflurane and n-butanol.
[0108] Hydrocarbons in their liquid form dissolve compounds of low
polarity such as the lipids in fluids. Accordingly, hydrocarbons
comprise any substantially water immiscible hydrocarbon, which is
liquid at about 37.degree. C. Suitable hydrocarbons include, but
are not limited to the following: C.sub.5 to C.sub.20 aliphatic
hydrocarbons such as petroleum ether, hexane, heptane, and octane;
haloaliphatic hydrocarbons such as chloroform,
1,1,2-trichloro-1,2,2-trifluoroethane, 1,1,1-trichloroethane,
trichloroethylene, tetrachloroethylene dichloromethane and carbon
tetrachloride; thioaliphatic hydrocarbons; perfluorocarbons, such
as perfluorocyclohexane, perfluoromethylcyclohexane, and
perfluorodimethylcyclohexane; fluoroethers such as sevoflurane;
each of which may be linear, branched or cyclic, saturated or
unsaturated; aromatic hydrocarbons such as benzene; alkylarenes
such as toluene, haloarenes, haloalkylarenes and thioarenes. Other
suitable solvents may also include: saturated or unsaturated
heterocyclic compounds such as water insoluble derivatives of
pyridine and aliphatic, thio or halo derivatives thereof; and
perfluorooctyl bromide. Another suitable solvent is
perfluorodecalin.
[0109] Suitable esters which may be used include, but are not
limited to, ethyl acetate, propylacetate, butylacetate and
ethylpropionate. Suitable exemplary ketones which may be used
include, but are not limited to, methyl ethyl ketone.
[0110] Suitable surfactants which may be used, include but are not
limited to the following: sulfates, sulfonates, phosphates
(including phospholipids), carboxylates, and sulfosuccinates. Some
anionic amphiphilic materials useful with the present invention
include but are not limited to the following: sodium dodecyl
sulfate (SDS), sodium decyl sulfate, bis-(2-ethylhexyl) sodium
sulfosuccinate (AOT), cholesterol sulfate and sodium laurate.
[0111] The alcohols which are preferred for use in the present
invention, when used alone, include those alcohols which are not
appreciably miscible with plasma or other biological fluids. When
alcohols are used in combination with another solvent, for example,
ether, a hydrocarbon, an amine or a combination thereof,
C.sub.1-C.sub.8 containing alcohols may be used. Preferred alcohols
for use in combination with another solvent include lower alcohols
such as C.sub.4-C.sub.8 containing alcohols. Accordingly, preferred
alcohols that fall within the scope of the present invention are
preferably butanols, pentanols, hexanols, heptanols and octanols,
and iso forms thereof. Particularly preferred are the butanols
(1-butanol and 2-butanol), also referred to as n-butanol. As stated
above, the most preferred alcohol is the C.sub.4 alcohol, butanol.
The specific choice of alcohol will depend on the second solvent
employed. In a preferred embodiment, lower alcohols are combined
with lower ethers.
[0112] Ethers, used alone, or in combination with other solvents,
preferably alcohols, are another preferred solvent for use in the
method of the present invention. Particularly preferred are the
C.sub.4-C.sub.8 ethers, including but not limited to, ethyl ether,
diethyl ether, and propyl ethers, including but not limited to
di-isopropyl ether (DIPE). Also useful in the present invention are
combinations of ethers, such as di-isopropyl ether and diethyl
ether. When ethers and alcohols are used in combination as a first
solvent for removing lipid, any combination of alcohol and ether
may be used provided the combination is effective to partially or
completely remove lipid. When alcohols and ether are combined as a
solvent for removing lipid from a fluid, acceptable ratios of
alcohol to ether in this solvent are about 0.01 parts to 99.99
parts alcohol to about 99.99 parts to 0.01 parts ether, including a
ratio of about 1 part to 25 parts alcohol with about 75 parts to 99
parts ether, a ratio of about 3 parts to 10 parts alcohol with
about 90 parts to 97 parts ether, and a preferred ratio of 5 parts
alcohol with 95 parts ether. An especially preferred combination of
alcohol and ether is the combination of butanol and di-isopropyl
ether.
[0113] In sum, the particularly preferred solvents include 100
parts di-isopropyl ether, a combination of 95 parts di-isopropyl
ether per 5 parts n-butanol, and a combination of 95 parts
sevoflurane per 5 parts n-butanol. Acceptable ranges of sevoflurane
and n-butanol also include about 0.01 parts to 99.99 parts
sevoflurane per about 99.99 parts to 0.01 parts n-butanol, 0.1
parts to 99.9 parts sevoflurane per about 99.9 parts to 0.1 parts
n-butanol; 1.0 parts to 99.0 parts sevoflurane per about 99.0 parts
to 1.0 parts n-butanol, 10.0 parts to 90.0 parts sevoflurane per
about 90.0 parts to 10.0 parts n-butanol, 15.0 parts to 85.0 parts
sevoflurane per about 85.0 parts to 15.0 parts n-butanol. Preferred
combinations include about 95 parts sevoflurane per about 5.0 parts
n-butanol, about 90 parts sevoflurane per about 10 parts n-butanol,
about 85 parts sevoflurane per about 15 parts n-butanol, and, more
particularly, 97.5 parts sevoflurane per 2.5 parts n-butanol.
[0114] Acceptable ratios of solvent to plasma include any
combination of solvent and plasma. Most preferred ratios are 2
parts plasma to 1 part solvent, 1 part plasma to 1 part solvent,
and 1 part plasma to 2 parts solvent. For example, when using a
solvent comprising 95 parts sevoflurane to 5 parts n-butanol, it is
preferred to use two parts solvent per one part plasma.
[0115] Additionally, when employing a solvent containing n-butanol,
the present invention can also use a ratio of solvent to plasma
that yields at least 3% n-butanol in the final solvent/plasma
mixture. A particularly preferred final concentration of n-butanol
in the final solvent/plasma mixture is 3.33%.
Processes to Remove Lipids from Fluids and HDL Particles
[0116] The processes employed in the methods of the present
invention to reduce lipids in fluids and HDL particles relate
directly to energy input. The procedure employed must be designed
such that HDL particles are treated to reduce their lipid levels
without destruction of plasma proteins or substantially affecting
LDL particles. Note that the methods described below may be used to
achieve the steps of both of the preferred embodiments of the
present invention as described above.
Mixing Methods
[0117] The plasma and the solvent are subjected to at least one
mixing method for mixing, agitating or otherwise contacting the
biological fluid with the solvent. The mixing method employed in
the present invention may be one of, but is not limited to, an
in-line static mixer, a rotating flask, a vortexer, a centrifuge, a
sonicated flask, a high shear tube, a homogenizer, a blender,
hollow fiber contactor, a centrifugal pump, a shaker table, a
swirling process, a stirring process, an end-over-end rotation of a
sealed container, or other suitable devices, or any combination of
these devices or processes.
Mixing Duration
[0118] The amount of time required for adequate mixing of the
solvent with the fluid is related to the mixing method employed.
Fluids are mixed for a period of time sufficient to permit intimate
contact between the organic and aqueous phases, and for the solvent
to at least partially or completely solubilize the lipid. Another
consideration is temperature. The balance between the mixing time
and temperature must be designed such that it does not encourage
the contamination of or deterioration of the blood sample. The time
and temperature system is ideally balanced such that the blood
sample is still viable and does not deteriorate.
[0119] Typically, mixing will occur for a period of about 1 second
to about 24 hours, possibly about 1 second to about 2 hours,
possibly approximately 1 second to approximately 10 minutes, or
possibly about 30 seconds to about 1 hour, depending on the mixing
method employed. Non-limiting examples of mixing durations
associated with different methods include 1) gentle stirring and
end-over-end rotation for a period of about 1 second to about 24
hours, 2) vigorous stirring and vortexing for a period of about 1
second to about 30 minutes, 3) swirling for a period of about 1
second to about 2 hours, or 4) homogenization for a period of about
1 second to about 10 minutes.
Temperature
[0120] As described above, temperature is also an important
consideration. The temperature is usually set at less that
37.degree. C. so as not to denature the plasma. Optionally, cooler
temperatures may also be employed. There are various methods for
achieving temperature regulation in this system.
Solvent Extraction Methods
Removal of Residual Bulk Solvent
[0121] In a preferred embodiment of the present invention, the
residual bulk solvent is removed via centrifugation.
Removal of Remaining Soluble Solvent
[0122] Another preferred method of separating solvent is through
the use of charcoal, preferably activated charcoal. This charcoal
is optionally contained in a column. Alternatively, the charcoal
may be in slurry form. Various biocompatible forms of charcoal may
be used in these columns.
HFC Pervaporation
[0123] Hollow fiber contactors (HFCs) can successfully reduce total
concentrations of solvents, such as di-isopropyl ether and di-ethyl
ether, in water and plasma, using different HFCs, pressures, and
flow rates. HFCs may have a total surface area of permeable
membrane formed by the hollow fibers between about 4,200 square
centimeters and about 18,000 square centimeters, depending on the
type of HFC used. Further, the gas flow rate was varied in these
experiments from between about 2 liters per minute to about 10
liters per minute, and the plasma flow rate was varied from between
about 10 mL per minute to about 60 mL per minute. Operation in this
manner can reduce the initial concentrations of solvents from
between about 28,000 parts per million (ppm) and 9,000 ppm to
between about 1327 ppm and about 0.99 ppm within between about 14
minutes and 30 minutes.
[0124] In one embodiment of the solvent removal system of the
present invention, the solvent-treated plasma containing residual
soluble solvent is typically first introduced into a circulation
loop including, for instance, a recirculating vessel, a fluid
transport means such as tubing, valves, a pump, and a solvent
extracting device, such as a HFC. In this circulation loop, the HFC
functions as a recirculating, solvent-extraction device. The
plasma/solvent is circulated through the hollow fiber of the HFC,
thereby contacting the extraction solvent with a gas or a second
extraction solvent, circulating through the shell of the HFC. If a
volatile solvent is used as the first extraction solvent, any gas
capable of extracting the first extraction solvent from the
delipidated plasma may be used, including, but not limited to,
nitrogen and air.
Specific Embodiments of the Present Invention
[0125] The above-described components can be integrated into a
plurality of different embodiments to enable the practice of the
present invention. Certain specific embodiments shall be described
herein to particularly highlight defined approaches to practicing
the present invention. The embodiments listed below do not
represent every variation of the present invention and are designed
to exemplify the present invention and, in certain cases, represent
preferred approaches to practicing the present invention.
[0126] Referring to FIGS. 19 through 21, a plurality of embodiments
depicting different systems capable of practicing the present
invention are shown. It should be understood that each embodiment
has different advantages and disadvantages, from a cost and usage
perspective, and that none of the embodiments are specifically
preferred relative to other embodiments. FIG. 19 depicts a basic
component flow diagram defining elements of the HDL modification
system 1900. A fluid input is provided 1905 and connected via
tubing to a mixing device 1920. A solvent input is provided 1910
and also connected via tubing to a mixing device 1920. Preferably
valves 1915 are used to control the flow of fluid from fluid input
1905 and solvent from solvent input 1910. It should be appreciated
that the fluid input 1905 preferably contains any fluid that
includes HDL particles, including plasma having LDL particles or
devoid of LDL particles, as discussed above. It should further be
appreciated that solvent input 1910 can include a single solvent, a
mixture of solvents, or a plurality of different solvents that are
mixed at the point of solvent input 1910. While depicted as a
single solvent container, solvent input 1910 can comprise a
plurality of separate solvent containers. The types of solvents
that are used and preferred are discussed above.
[0127] The mixer 1920 mixes fluid from fluid input 1905 and solvent
from solvent input 1910 to yield a fluid-solvent mixture.
Preferably, mixer 1920 is capable of using a shaker bag mixing
method with the input fluid and input solvent in a plurality of
batches, such as 1, 2, 3 or more batches. An exemplary mixer is a
Barnstead Labline orbital shaker table. Once formed, the
fluid-solvent mixture is directed, through tubing and controlled by
at least one valve, to a separator 1925. In a preferred embodiment,
separator 1925 is capable of performing bulk solvent separation
through gravity separation in a funnel-shaped bag.
[0128] In the separator 1925, the fluid-solvent mixture separates
into a first layer and second layer. The first layer comprises a
mixture of solvent and lipid that has been removed from the HDL
particles. The second layer comprises a mixture of residual
solvent, modified HDL particles, and other elements of the input
fluid. One of ordinary skill in the art would appreciate that the
composition of the first layer and the second layer would differ
based upon the nature of the input fluid. Once the first and second
layers separate in separator 1925, the second layer is transported
through tubing to a solvent extraction device 1940. Preferably, a
pressure sensor 1930 and valve is positioned in the flow stream to
control the flow of the second layer to the solvent extraction
device 1940.
[0129] The opening and closing of valves to enable the flow of
fluid from input containers 1905, 1910 is preferably timed using
mass balance calculations derived from weight determinations of the
fluid inputs 1905, 1910 and separator 1925. For example, the valves
between separator 1925 and waste container 1935 and between
separator 1925 and solvent extraction device 1940 open after the
input masses (fluid and solvent) substantially balances with the
mass in separator 1925 and a sufficient period of time has elapsed
to permit separation between the first and second layers, as
discussed above. Depending on what solvent is used, and therefore
which layer settles to the bottom of the separator 1925, either the
valve between separator 1925 and waste container 1935 is opened or
between separators 1925 and solvent extraction device 1940 is
opened. One of ordinary skill in the art would appreciate that the
timing of the opening is dependent upon how much fluid is in the
first and second layers and would further appreciate that it is
preferred to keep the valve between separator 1925 and waste
container 1935 open just long enough to remove all of the first
layer and some of the second layer, thereby ensuring that as much
solvent as possible has been removed from the fluid being sent to
the solvent extraction device 1940.
[0130] Preferably, a glucose input 1955 and saline input 1960 is in
fluid communication with the fluid path leading from the separator
1925 to the solvent extraction device 1940. A plurality of valves
is also preferably incorporated in the flow stream from the glucose
input 1955 and saline input 1960 to the tubing providing the flow
path from the separator 1925 to the solvent extraction device 1940.
Glucose and saline are incorporated into the present invention in
order to prime the solvent extraction device 1940 prior to
operation of the system. Where such priming is not required, the
glucose and saline inputs are not required. Also, one of ordinary
skill in the art would appreciate that the glucose and saline
inputs can be replaced with other primers if the solvent extraction
device 1940 requires it.
[0131] The solvent extraction device 1940 is preferably a charcoal
column designed to remove the specific solvent used in the solvent
input 1910. An exemplary solvent extraction device 1940 is an Asahi
Hemosorber charcoal column. A pump 1950 is used to move the second
layer from the separator 1925, through the solvent extraction
device 1940, and to an output container 1945. The pump is
preferably a peristaltic pump, such as a Masterflex Model
77201-62.
[0132] The first layer is directed to a waste container 1935 that
is in fluid communication with separator 1925 through tubing and at
least one valve. Additionally, other waste, if generated, can be
directed from the fluid path connecting solvent extraction device
1940 and output container 1945 to waste container 1935.
[0133] Preferably, an embodiment of the present invention uses
gravity, wherever practical, to move fluid through each of the
plurality of components. For example, preferably gravity is used to
drain the input plasma 1905 and input solvent 1910 into the mixer
1920. Where the mixer 1920 comprises a shaker bag and separator
1925 comprises a funnel bag, fluid is moved from the shaker bag to
the funnel bag and, subsequently, to the waste container 1935, if
appropriate, using gravity.
[0134] In an additional step, not shown in FIG. 19, the output
fluid in output container 1945 would be subjected to a solvent
detection system, or lipid removing agent detection system, to
determine if any solvent, or other undesirable component, is in the
output fluid. In one embodiment, the output fluid is subjected to
sensors that are capable of determining the concentrations of
solvents introduced in the solvent input, such as n-butanol or
di-isopropyl ether. This is an important measurement because the
output fluid is returned to the bloodstream of the patient and the
solvent concentrations must be below a predetermined level to carry
out this operation safely. The sensors are preferably capable of
providing such concentration information on a real-time basis and
without having to physically transport a sample of the output
fluid, or air in the headspace, to a remote device.
[0135] In one embodiment, molecularly imprinted polymer technology
is used to enable surface acoustic wave sensors. A surface acoustic
wave sensor receives an input, through some interaction of its
surface with the surrounding environment, and yields an electrical
response, generated by the piezoelectric properties of the sensor
substrate. To enable the interaction, molecularly imprinted polymer
technology is used. Molecularly imprinted polymers are plastics
programmed to recognize target molecules, like pharmaceuticals,
toxins or environmental pollutants, in complex biological samples.
The molecular imprinting technology is enabled by the
polymerization of one or more functional monomers with an excess of
a crosslinking monomer in presence of a target template molecule
exhibiting a structure similar to the target molecule that is to be
recognized, i.e. the target solvent.
[0136] The use of molecularly imprinted polymer technology to
enable surface acoustic wave sensors is preferred relative to other
technological approaches because they can be made more specific to
the concentrations of targeted solvents and are capable of
differentiating such targeted solvents from other possible
interferents. As a result, the presence of acceptable interferents
that may have similar structures and/or properties to the targeted
solvents would not prevent the sensor from accurately reporting
existing respective solvent concentrations.
[0137] Alternatively, if the input solvent comprises certain
solvents, such as n-butanol, electrochemical oxidation could be
used to measure the solvent concentration. Electrochemical
measurements have several advantages. They are simple, sensitive,
fast, and have a wide dynamic range. The instrumentation is simple
and not affected by humidity. In one embodiment, the target
solvent, such as n-butanol, is oxidized on a platinum electrode
using cyclic voltammetry. This technique is based on varying the
applied potential at a working electrode in both the forward and
reverse directions, at a predefined scan rate, while monitoring the
current. One full cycle, a partial cycle, or a series of cycles can
be performed. While platinum is the preferred electrode material,
other electrodes, such as gold, silver, iridium, or graphite, could
be used. Although, cyclic voltammetric techniques are used, other
pulse techniques such as differential pulse voltammetry or square
wave voltammetry may increase the speed and sensitivity of
measurements. The alternative, a Taguchi sensor, is not preferred
because it does not effectively operate in humid conditions.
[0138] The present invention expressly covers any and all forms of
automatically sampling and measuring, detecting, and analyzing an
output fluid, or the headspace above the output fluid. For example,
such automated detection can be achieved by integrating a mini-gas
chromatography (GC) measuring device that automatically samples air
in the output container, transmits it to a GC device optimized for
the specific solvents used in the delipidation process, and, using
known GC techniques, analyzes the sample for the presence of the
solvents.
[0139] Referring back to FIG. 19, suitable materials for use in any
of the apparatus components as described herein include materials
that are biocompatible, approved for medical applications that
involve contact with internal body fluids, and in compliance with
U.S. PV1 or ISO 10993 standards. Further, the materials should not
substantially degrade from, for instance, exposure to the solvents
used in the present invention, during at least a single use. The
materials should typically be sterilizable either by radiation or
ethylene oxide (EtO) sterilization. Such suitable materials should
be capable of being formed into objects using conventional
processes, such as, but not limited to, extrusion, injection
molding and others. Materials meeting these requirements include,
but are not limited to, nylon, polypropylene, polycarbonate,
acrylic, polysulphone, polyvinylidene fluoride (PVDF),
fluoroelastomers such as VITON, available from DuPont Dow
Elastomers L.L.C., thermoplastic elastomers such as SANTOPRENE,
available from Monsanto, polyurethane, polyvinyl chloride (PVC),
polytetrafluoroethylene (PTFE), polyphenylene ether (PFE),
perfluoroalkoxy copolymer (PFA), which is available as TEFLON PFA
from E.I. du Pont de Nemours and Company, and combinations
thereof.
[0140] The valves used in each embodiment may be composed of, but
are not limited to, pinch, globe, ball, gate or other conventional
valves. Preferably, the valves are occlusion valves such as Acro
Associates' Model 955 valve. However, the invention is not limited
to a valve having a particular style. Further, the components of
each system described below may be physically coupled together or
coupled together using conduits that may be composed of flexible or
rigid pipe, tubing or other such devices known to those of ordinary
skill in the art.
[0141] Referring to FIG. 20, a specific configuration 2000 of the
present invention is shown. A preferred configuration 2000
comprises an enclosed housing 2005 capable of safely containing
volatile fluids, such as solvents. In a preferred embodiment, the
enclosed housing 2005 comprises a door 2015 with a clear door to
observe the delipidation process in operation, a mobile base 2025,
a control display 2020, a clear top 2010, and a filter and air
circulating system [not shown]. The interface screen of the control
display 2020 is preferably functional when the door 2015 is open to
enable system set up and priming a solvent extraction device but
does not permit the system to delipidate an input fluid until the
door 2015 is closed and, preferably, locked. It is also preferred
to have a waste or overflow tray capable of trapping any fluid
leaks, overflows, or other spills at the base of the enclosed
housing 2005 in a manner that permits the tray to be readily
removed without opening the housing 2005.
[0142] Referring to FIG. 21, a configuration of basic components of
the HDL modification system 2100 is shown. A fluid input is
provided 2105 and connected via tubing to a mixing device 2120. A
solvent input is provided 2110 and also connected via tubing to a
mixing device 2120. Preferably valves are used to control the flow
of fluid from fluid input 2105 and solvent from solvent input 2110.
It should be appreciated that the fluid input 2105 preferably
contains any fluid that includes HDL particles, including plasma
having LDL particles or devoid of LDL particles, as discussed
above. It should further be appreciated that solvent input 2110 can
include a single solvent, a mixture of solvents, or a plurality of
different solvents that are mixed at the point of solvent input
2110. While depicted as a single solvent container, solvent input
2110 can comprise a plurality of separate solvent containers. The
types of solvents that are used and preferred are discussed
above.
[0143] The mixer 2120 mixes fluid from fluid input 2105 and solvent
from solvent input 2110 to yield a fluid-solvent mixture.
Preferably, mixer 2120 is capable of using a shaker bag mixing
method with the input fluid and input solvent in a plurality of
batches, such as 1, 2, 3 or more batches. Once formed, the
fluid-solvent mixture is directed, through tubing and controlled by
at least one valve, to a separator 2125. In a preferred embodiment,
separator 2125 is capable of performing bulk solvent separation
through gravity separation in a funnel-shaped bag.
[0144] In the separator 2125, the fluid-solvent mixture separates
into a first layer and second layer. The first layer comprises a
mixture of solvent and lipid that has been removed from the HDL
particles. The second layer comprises a mixture of residual
solvent, modified HDL particles, and other elements of the input
fluid. One of ordinary skill in the art would appreciate that the
composition of the first layer and the second layer would differ
based upon the nature of the input fluid. Once the first and second
layers separate in separator 2125, the second layer is transported
through tubing to a solvent extraction device 2140. Preferably, a
pressure sensor and valve is positioned in the flow stream to
control the flow of the second layer to the solvent extraction
device 2140.
[0145] Preferably, a glucose input 2130 and saline input 2150 is in
fluid communication with the fluid path leading from the separator
2125 to the solvent extraction device 2140. A plurality of valves
is also preferably incorporated in the flow stream from the glucose
input 2130 and saline input 2150 to the tubing providing the flow
path from the separator 2125 to the solvent extraction device 2140.
Glucose and saline are incorporated into the present invention in
order to prime the solvent extraction device 2140 prior to
operation of the system. Where such priming is not required, the
glucose and saline inputs are not required. Also, one of ordinary
skill in the art would appreciate that the glucose and saline
inputs can be replaced with other primers if the solvent extraction
device 2140 requires it.
[0146] The solvent extraction device 2140 is preferably a charcoal
column designed to remove the specific solvent used in the solvent
input 2110. An exemplary solvent extraction device 2140 is an Asahi
Hemosorber charcoal column. A pump 2135 is used to move the second
layer from the separator 2125, through the solvent extraction
device 2140, and to an output container 2115. The pump is
preferably a peristaltic pump, such as a Masterflex Model
77201-62.
[0147] The first layer is directed to a waste container 2155 that
is in fluid communication with separator 2125 through tubing and at
least one valve. Additionally, other waste, if generated, can be
directed from the fluid path connecting solvent extraction device
2140 and output container 2115 to waste container 2155.
[0148] Preferably, an embodiment of the present invention uses
gravity, wherever practical, to move fluid through each of the
plurality of components. For example, preferably gravity is used to
drain the input plasma 2105 and input solvent 2110 into the mixer
2120. Where the mixer 2120 comprises a shaker bag and separator
2125 comprises a funnel bag, fluid is moved from the shaker bag to
the funnel bag and, subsequently, to the waste container 2155, if
appropriate, using gravity.
[0149] In general, the present invention preferably comprises
configurations wherein all inputs, such as input plasma and input
solvents, disposable elements, such as mixing bags, separator bags,
waste bags, solvent extraction devices, and solvent detection
devices, and output containers are in easily accessible positions
and can be readily removed and replaced by a technician.
[0150] To enable the operation of the above described embodiments
of the present invention, it is preferable to supply a user of such
embodiments with a packaged set of components, in kit form,
comprising each component required to practice the present
invention. Such a kit would preferably include an input fluid
container (i.e. a high density lipoprotein source container), a
lipid removing agent source container (i.e. a solvent container),
disposable components of a mixer, such as a bag or other container,
disposable components of a separator, such as a bag or other
container, disposable components of a solvent extraction device
(i.e. a charcoal column), an output container, disposable
components of a waste container, such as a bag or other container,
solvent detection devices, and, a plurality of tubing and a
plurality of valves for controlling the flow of input fluid (high
density lipoprotein) from the input container and lipid removing
agent (solvent) from the solvent container to the mixer, for
controlling the flow of the mixture of lipid removing agent, lipid,
and particle derivative to the separator, for controlling the flow
of lipid and lipid removing agent to a waste container, for
controlling the flow of residual lipid removing agent, residual
lipid, and particle derivative to the extraction device, and for
controlling the flow of particle derivative to the output
container.
[0151] In one embodiment, a kit comprises a plastic container
having disposable components of a mixer, such as a bag or other
container, disposable components of a separator, such as a bag or
other container, disposable components of a waste container, such
as a bag or other container, and, a plurality of tubing and a
plurality of valves for controlling the flow of input fluid (high
density lipoprotein) from the input container and lipid removing
agent (solvent) from the solvent container to the mixer, for
controlling the flow of the mixture of lipid removing agent, lipid,
and particle derivative to the separator, for controlling the flow
of lipid and lipid removing agent to a waste container, for
controlling the flow of residual lipid removing agent, residual
lipid, and particle derivative to the extraction device, and for
controlling the flow of particle derivative to the output
container. Disposable components of a solvent extraction device
(i.e. a charcoal column), the input fluid, the input solvent, and
solvent extraction devices are provided separately.
Administration Schedule
[0152] The modified HDL particles of the present invention may be
administered according to any schedule that is effective in
promoting cellular cholesterol efflux.
[0153] In one embodiment, blood is withdrawn from a patient in a
volume sufficient to produce about 1 liter of plasma. The blood is
separated into plasma and red blood cells using methods commonly
known to one of skill in the art, such as plasmapheresis, and the
red blood cells are stored in an appropriate storage solution or
returned to the patient during plasmapheresis. The red blood cells
are preferably returned to the patient during plasmapheresis.
Physiological saline is also optionally administered to the patient
to replenish volume. The 1 liter of plasma is treated with any of
the methods of the present invention to create HDL particles with
reduced lipid content while not substantially affecting LDL. The
resulting treated plasma containing the HDL particles with reduced
lipid and substantially unaffected LDL content is optionally
combined with the patient's red blood cells, if the red cells were
not already returned during plasmapheresis, and administered to the
patient. One route of administration is through the vascular
system, preferably intravenously. This treatment regimen is
repeated weekly for about 5 to 6 weeks. Enhanced cholesterol efflux
is observed in the patient after the treatment.
[0154] In another embodiment, blood is withdrawn from a patient in
a volume sufficient to produce about 1 liter of plasma. The blood
is separated into plasma and red blood cells using methods commonly
known to one of skill in the art, such as plasmapheresis, and the
red blood cells are stored in an appropriate storage solution or
returned to the patient during plasmapheresis. The 1 liter of
plasma is treated to remove the LDL component before further
treatment of the plasma. The 1 liter of plasma is treated with the
method of the present invention to create HDL particles with
reduced lipid content. The resulting treated plasma containing the
HDL particles with reduced lipid is optionally combined with the
patient's red blood cells, if the red cells were not already
returned during plasmapheresis, and administered to the patient.
One route of administration is through the vascular system,
preferably intravenously. This treatment regimen is repeated weekly
for about 5 to 6 weeks.
[0155] It is to be understood that other volumes of plasma may be
treated with the method of the present invention, and administered
to a patient on various administration schedules. For a batch
process, volumes of 100 ml to 3500 ml of plasma may be treated with
the present method. The frequency of treatment may also vary from
between several times per week to once a month or less, depending
on the volume to be treated and the severity of the condition of
the patient.
[0156] In another approach of the present invention, following
removal of a desired volume of a patient's blood, separation of the
blood into plasma and red blood cells and treatment of the plasma
to reduce lipid levels, the HDL particles with reduced lipid
content are isolated from the plasma and administered to the
patient in an acceptable vehicle.
[0157] In yet another embodiment, heterologous plasma may be
obtained, treated with the method of the present invention and the
treated plasma containing HDL particles with reduced lipid content
administered to a patient who was not the source of the plasma. In
a further embodiment, heterologous plasma may be obtained, treated
with the method of the present invention and the HDL particles with
reduced lipid content are separated from the treated plasma. These
HDL particles with reduced lipid content may be administered in an
acceptable vehicle to a patient who was not the source of the
plasma.
[0158] In still another embodiment, following removal of a desired
volume of a patient's blood, the patient is permitted to recover
for 1 to 4 days in terms of producing new blood and attaining
endogenous plasma HDL levels substantially similar to plasma HDL
levels before removing the blood. The removed blood is separated
into plasma and red blood cells and the plasma is treated to reduce
lipid levels. The HDL particles with reduced lipid content are
isolated from the plasma and administered to the patient.
Alternatively, the HDL particles with reduced lipid content are not
isolated from the plasma and the treated plasma is administered to
the patient.
[0159] In another embodiment plasma is treated with the methods of
the present invention to reduce lipid levels. Next, Apo A-1 protein
is purified from this treated plasma using techniques such as
affinity chromatography. The resulting purified, modified Apo A-1
protein is administered in an acceptable vehicle to a patient
together with the modified HDL particles with reduced lipid
content. These modified HDL particles with reduced lipid content
may be provided to the patient as isolated HDL particles in an
acceptable vehicle or included with the treated plasma.
Administration with Other Therapies
[0160] The modified HDL particles of the present invention may be
administered in conjunction with one or more additional therapeutic
approaches. The modified HDL particles of the present invention may
be administered in conjunction with exercise and dietary
restriction of fat and cholesterol intake.
[0161] The modified HDL particles of the present invention may be
administered in conjunction with administration of agents for
reducing cholesterol, reducing LDL levels and enhancing HDL levels.
These agents, such as HMG-CoA reductase inhibitors, or statins, may
be administered in dosages and according to administration
schedules commonly known to one of ordinary skill in the art.
Statins include but are not limited to cerivastatin, atorvastatin,
fluvastatin, simvastatin, pravastatin and lovastatin. For example,
dosages of 10 mg, 20 mg, 40 mg or 80 mg of statins, taken once per
day, are commonly employed. Administration of the modified HDL
particles of the present invention can eliminate the need for
statin therapy in patients or reduce the required dosage of
statins.
[0162] In another aspect, the modified HDL particles of the present
invention are used in conjunction with administration of agents
designed to reduce absorption of fat and cholesterol. Such agents,
for example ezetimibe, and the clinically appropriate dosages are
known to one of ordinary skill in the art.
[0163] In yet another aspect, the modified HDL particles of the
present invention are used in conjunction with administration of
one or more agents such as fibric acid derivatives (gemfibrozil),
nicotinic acid (niacin), and bile acid-binding resins
(cholestyramine, cholestipol), and the clinically appropriate
dosages are known to one of ordinary skill in the art.
[0164] In yet another aspect, the modified HDL particles of the
present invention are used in conjunction with administration of
anti-inflammatory drugs, such as aspirin, known to one of ordinary
skill in the art. The clinically appropriate dosages of
anti-inflammatory drugs are known to one of ordinary skill in the
art. Anti-inflammatory drugs are often prescribed to patients with
vascular disease since it is believed that inflammation is a
causative factor of atherosclerosis and other vascular
diseases.
[0165] The modified HDL particles of the present invention are used
in conjunction with administration of agents such as statins and
with agents designed to reduce absorption of fat and cholesterol.
This combination of three therapies is effective in enhancing
cholesterol efflux from cells and permits administration of lower
dosages of statins. The modified HDL particles of the present
invention are also used in conjunction with any of the therapeutic
approaches described above.
[0166] The following examples will serve to further illustrate the
present invention without, at the same time, however, constituting
any limitation thereof. On the contrary, it is to be clearly
understood that resort may be had to various embodiments,
modifications and equivalents thereof which, after reading the
description herein, may suggest themselves to those skilled in the
art without departing from the spirit of the invention.
EXAMPLE 1
Separation and Characterization of Total Cholesterol,
Apolipoprotein A1 (Apo A-1), Apolipoprotein B (Apo B) and
Phospholipids in Normal Plasma
[0167] A 25 ml pool of plasma was characterized in terms of total
cholesterol, apolipoprotein A1 (Apo A-1), apolipoprotein B (Apo B)
and phospholipids. An aliquot of 1 ml of the pooled plasma was
loaded onto a Sephacryl S-300 26/60 (FPLC) column. An elution
buffer of phosphate buffered saline containing 1 mM EDTA was
applied to the column and eluted at 2 ml/min. About 96 fractions
were collected, one every 43 seconds beginning 41 minutes after
application of the plasma sample. Each fraction was characterized
in terms of total cholesterol, Apo A-1, Apo B and
phospholipids.
[0168] Apo B containing particles comprised of very low density
lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low
density lipoprotein (LDL) particles eluted in fractions 10-40. Apo
A-1 containing particles comprised of high density lipoprotein
(HDL) particles eluted in the remaining fractions. The results are
displayed in FIG. 3. An analysis of total plasma cholesterol
indicated a clear separation of the LDL particles from the HDL
particles. The distribution of Apo A-1 and Apo B in the
corresponding fractions confirmed the separation of these
particles.
EXAMPLE 2
Selective Creation of Apo A-1-Associated HDL Particles with Reduced
Cholesterol or with Reduced Cholesterol and Phospholipids Using
DIPE
[0169] This selective plasma treatment method employs a ratio of
1:1 DIPE: plasma. The sample was vortexed for 15 seconds and then
permitted to separate by gravity. Activated charcoal was used to
remove residual DIPE after the process and several different
hematological parameters were measured.
[0170] The delipidated sample was then applied to a column and
treated as explained in Example 1. This method removed about 10% of
total cholesterol, 12% of Apo B, 17% of Apo A-1 and about 11% of
phospholipids.
[0171] A comparison of the elution of the delipidated sample with
the elution of normal, non-delipidated plasma is presented in FIGS.
4 and 5. The results show a shift to the right of the Apo
A-1-associated HDL particles, indicating a smaller particle not
associated with cholesterol (FIG. 4) and also not associated with
phospholipid (FIG. 5). Accordingly, this delipidation method
created HDL particles associated with Apo A-1 that were low or
substantially devoid of cholesterol and phospholipid and therefore
had new capacity to bind with cholesterol and phospholipid. Lipid
was only slightly removed from LDL particles (Apo B) with this
method (FIG. 6).
[0172] In summary, two types of HDL particles were observed. There
was a wide range of sizes of HDL particles containing Apo A-1 and
phospholipids but no cholesterol. A relatively narrow size range of
HDL particles containing Apo A-1 but no phospholipids or
cholesterol was observed.
EXAMPLE 3
[0173] Selective Creation of Apo A-1-Associated HDL Particles with
Reduced Cholesterol or with Reduced Cholesterol and Phospholipids
Using a Sevoflurane:n-butanol Mixture
[0174] A mixture of sevoflurane and n-butanol was employed as a
solvent in a concentration of 95% sevoflurane and 5% n-butanol. The
mixture was added to plasma in a 2:1 solvent to plasma ratio. The
sample was vortexed for 15 seconds and then centrifuged. Activated
charcoal was added to remove residual solvent. The resulting
solvent-free sample was run on an FPLC column as described in
Example 1. Data concerning the percentage reduction in cholesterol,
phospholipid and Apo A-1 were obtained from quantitative
measurements and not from the FPLC elution profiles.
[0175] This method reduced total cholesterol by 9% and
phospholipids by 9%. A decrease of about 14% was observed in Apo
A-1. FIG. 7 shows that this method resulted in Apo A-1 associated
HDL particles of lower weight that were not associated with
cholesterol when compared to plasma that was not subjected to such
treatment. However, these Apo A-1 associated HDL particles were
associated with phospholipid (FIG. 8). There was little effect on
Apo B associated LDL particles as shown in FIG. 9. In summary, this
process resulted in a modified HDL particle which contained Apo A-1
and phospholipids, but little or no cholesterol.
EXAMPLE 4
Analysis of Clinical Parameters in Normal Plasma and Plasma Treated
with DIPE or Sevoflurane:n-butanol
[0176] Alanine aminotransferase (ALT), alkaline phosphatase (AP),
bilirubin-T, sodium, potassium, phosphorus, albumin, globulin and
the albumin/globulin (A/G) ratio were analyzed in normal untreated
plasma and in plasma treated with 100% DIPE or with
sevoflurane:n-butanol. The results are presented in FIG. 10.
[0177] Treatment with DIPE did not change the parameters to any
clinically significant degree. Treatment with sevoflurane:n-butanol
did not change the parameters to any clinically significant degree.
These treatments, therefore, do not substantially affect non-HDL
plasma constituent components.
EXAMPLE 5
Summary of the Efficacy of Different Solvents on Removal of
Cholesterol from HDL and Effects on LDL
[0178] FIGS. 11-15 show a Superose FPLC profile of plasma treated
with nothing, DIPE (100%), sevoflurane:n-butanol (95:5),
sevoflurane:n-butanol (75:25) and DIPE:n-butanol (95:5),
respectively, for a variety of parameters. Shown are total
cholesterol, phospholipid, Apo B, Apo A-1, and Apo A2. The data
indicate that cholesterol is reduced following solvent treatment in
the areas associated with Apo A-1 and Apo A2 (the peak on the right
side of each figure) while the Apo B associated with LDL (middle
peak) remains substantially unchanged. However, a severe solvent
treatment with a solvent ratio of 75:25 DIPE:n-butanol (FIG. 14)
dramatically reduced total cholesterol and phospholipids when
compared to untreated plasma (FIG. 11).
EXAMPLE 6
Cholesterol Efflux Studies of Plasma Treated with Various
Solvents
[0179] All solvent conditions above were employed to test the
effects of treated plasma on cholesterol efflux in ABCA1 pathway
and SRB1 pathway as measured in COS and Fu5AH cells. The methods
employed were those described by Rothblatt and colleagues (de la
Llera Moya et al., Arteriosclerosis. & Thrombosis 14:1056-1065,
1994).
[0180] The methods employed are described generally in the next
paragraphs. The tissue culture cell system was designed to
quantitate the contribution of scavenger receptor BI (SR-BI) or
ATP-binding cassette transporter 1 (ABCA1) to the efflux of
cellular cholesterol when cells are exposed to serum or isolated
lipoproteins. The general approach is to measure the release of
radiolabeled cellular cholesterol to either isolated acceptors or
whole serum. The contributions of SR-BI or ABCA1 to this efflux
process are determined by comparing the release obtained from cells
lacking the specific receptor to that observed in parallel cell
cultures expressing the receptor. Thus, to quantitate the
contribution of ABCA1 to cellular cholesterol efflux, transformed
mouse macrophage cells are grown in monolayers and prelabeled with
.sup.3H-cholesterol. One set of monolayers is treated with cAMP
which has been shown to upregulate the ABCA1 receptor, whereas a
replicate set of monolayers the are left untreated, and serve as
control cells which lack ABCA1. The sera to be tested is diluted to
an appropriate concentration and incubated with both ABCA1 positive
and negative monolayers. The release of the radiolabeled
cholesterol is determined after an appropriate incubation time
ranging from 1 to 12 hours. The ABCA1-contribution to efflux is
determined by subtracting the efflux obtained in ABCA1 negative
cultures from that obtained from the ABCA1 positive cultures.
[0181] A general assay for determining the contribution of SRBI to
cholesterol efflux uses the same approach as described above. Cell
lines serving as cholesterol donors are treated so that they either
lack SRBI or express high levels of the receptor. In the general
protocol presently used COS-7 cells are transiently transfected
when it SR-BI. These cells are pre-labeled with 3H-cholesterol and
then exposed to the test serum for appropriate periods of time.
Following this period the medium is removed and a determination is
made of the amount of radiolabeled cellular cholesterol that has
been released. The efflux of cholesterol from control, SR-BI
negative cells is subtracted from that observed with SR-BI
expressing cells. The difference obtained by this calculation
reflects the contribution of SR-BI to cholesterol efflux. An
alternative cell system that can be used for determining
SR-BI-mediated efflux is the Fu5AH rat hepatoma cell. These cells
expresses very high levels of SR-BI and the efflux of radiolabeled
cholesterol from Fu5AH is a very reliable measure of the
contribution of SR-BI to the efflux process.
[0182] The results are shown in FIG. 16 and demonstrate that plasma
treated with the various solvents stimulated efflux of cholesterol
20 to 25 times more efficiently than untreated or sham treated
plasma, taken from the same pool of starting plasma. This effect
was observed in ABCA1 cells, which possess a metabolic pathway
believed to be representative of cholesterol egress from arterial
walls, but not in COS+ or FU5AH cells which are believed to be
representative of the SRB 1 pathway in the liver. Further testing
of sevoflurane:n-butanol of three different plasma samples obtained
from different individuals produced similar results (FIG. 16, set
of histograms). By creating a modified HDL particle, the present
invention therefore positively affects the effectiveness of the
SRB1 and ABCA1 pathways. The present invention also encompasses the
modification of SRB1 and ABCA1 pathways by modifying the relative
ratio of phospholipid to Apo A-1 in HDL particles via the above
described delipidation processes.
EXAMPLE 7
Analysis of Solvent Treatment on Levels of Apo A-1 Associated pre
B-2 HDL and pre B-1 HDL Particles
[0183] The individual effects of sevoflurane:n-butanol and
DIPE:n-butanol (95:5) on Apo A-1-containing HDL subspecies were
examined using 3-16% native PAGE gels, followed by immunoblotting
and image analysis. The techniques employed are described in
Asztalos et al., Arteriosclerosis, Thrombosis and Vascular Biology;
15:1419-1423, 1995 and Asztalos et al., Arteriosclerosis,
Thrombosis and Vascular Biology; 17:1885-1893, 1997.
[0184] The left side of FIGS. 17 and 18 each show Apo
A-1-containing HDL subspecies, namely pre.beta.-2, pre.beta.-1,
.alpha. and pre-.alpha. particles in normal lipemic plasma, while
the right panel of each figure shows normal plasma treated with
sevoflurane:n-butanol and DIPE:n-butanol (95:5)
[0185] FIG. 17 demonstrates that plasma treated with
sevoflurane:n-butanol showed an increase in the Apo A-1-containing
pre.beta.-2, pre.beta.-1 HDL subspecies and a decrease in the
.alpha. HDL subspecies. A similar pattern was observed following
treatment with DIPE:n-butanol (FIG. 18). These results demonstrate
that these solvent treatments of plasma increased Apo
A-1-containing pre.beta.-2, pre.beta.-1 HDL subspecies, thereby
enhancing their availability for accepting new cholesterol and
facilitating cellular cholesterol efflux.
[0186] A similar comparison of the untreated normal plasma shown in
the left panel of FIGS. 17 and 18 and another untreated plasma
sample with slightly elevated cholesterol levels produced a similar
pattern (data not shown) with most of the immunoreactive HDL
species appearing in the .alpha.HDL form with relatively minor
density associated with pre.beta.-2 and pre.beta.-1 HDL particles.
These results provide an internal methodological control.
EXAMPLE 8
Administration of Derivatives of HDL to a Patient with Elevated
Cholesterol and Coronary Artery Disease
[0187] A 51 year old male patient presents with acute coronary
syndrome and is determined to have atherosclerosis via angiography.
A unit of blood is removed weekly from the patient. The plasma is
recovered and processed with the method of the present invention to
produce derivatives of HDL that are particles with reduced
cholesterol content while the red blood cells are returned to the
patient. These HDL particles with reduced cholesterol content in
the treated plasma are administered to the patient intravascularly
at weekly intervals for 5-10 weeks. Another angiography test after
completion of the treatment shows lower amounts of atherogenic
plaque in the coronary vessels compared to the first angiography
test.
EXAMPLE 9
Administration of Derivatives of HDL Together with Atorvastatin and
Ezetimibe to a Patient with Elevated Cholesterol and Coronary
Artery Disease
[0188] A 58 year old female presents with acute coronary syndrome
and is determined to have atherosclerosis via angiography. A unit
of blood is removed weekly from the patient. The plasma is
recovered and processed with the method of the present invention to
produce derivatives of HDL that are particles with reduced
cholesterol content while the red blood cells are returned to the
patient. These HDL particles with reduced cholesterol content in
the treated plasma are administered to the patient intravascularly
at weekly intervals for 5-10 weeks. The patient had previously
received 80 mg of atorvastatin daily with 10 mg of ezetimibe. These
drugs are continued daily together with the weekly administration
of HDL particles with reduced cholesterol content. Another
angiography test after completion of the treatment shows lower
amounts of atherogenic plaque in the coronary vessels compared to
the first angiography test.
EXAMPLE 10
Administration of Derivatives of HDL Together with Simvastatin and
Ezetimibe to an Obese Patient with Elevated Cholesterol and
Coronary Artery Disease
[0189] A 48 year old obese female patient presents with elevated
levels of LDL and cholesterol, and an angiographic test result
indicating atherosclerosis in three coronary arteries. A unit of
blood is removed weekly from the patient. The plasma is recovered
and processed with the method of the present invention to produce
derivatives of HDL that are particles with reduced cholesterol
content while the red blood cells are returned to the patient.
These HDL particles with reduced cholesterol content are combined
with the treated plasma and administered to the patient
intravascularly at weekly intervals for 5-10 weeks. The patient had
previously received 80 mg of simvastatin daily with 10 mg of
ezetimibe. These drugs are continued daily together with the weekly
administration of HDL particles with reduced cholesterol content.
The patient is placed on a moderate exercise schedule.
[0190] New blood work indicates a reduction in circulating
cholesterol, a reduction in LDL and an increase in circulating HDL.
The patient loses 15 pounds during the five month period. A new
angiographic procedure shows lower amounts of atherogenic plaque in
the coronary vessels compared to the first angiographic
procedure.
EXAMPLE 11
Administration of Derivatives of HDL to a Diabetic Patient with
Elevated Cholesterol and Coronary Artery Disease
[0191] A 44 year old diabetic female patient presents with elevated
levels of LDL and cholesterol, and an angiographic test result
indicating atherosclerosis in two coronary arteries. A unit of
blood is removed weekly from the patient. The plasma is recovered
and processed with the method of the present invention to produce
derivatives of HDL that are particles with reduced cholesterol
content while the red blood cells are returned to the patient.
These HDL particles with reduced cholesterol content are combined
with the treated plasma and administered to the patient
intravascularly at weekly intervals for 5-10 weeks. The patient had
previously received daily insulin injections. These injections are
continued daily together with the weekly administration of HDL
particles with reduced cholesterol content.
[0192] New blood work indicates a reduction in circulating
cholesterol, a reduction in LDL and an increase in circulating HDL.
A new angiographic procedure shows lower amounts of atherogenic
plaque in the coronary vessels compared to the first angiographic
procedure.
EXAMPLE 12
Administration of Derivatives of HDL to a Patient with Elevated
Cholesterol and Peripheral Vascular Disease Causing Intermittent
Claudication
[0193] A 66 year old male patient presents with elevated levels of
LDL and cholesterol and reports pain in the right lower extremity.
An angiographic test indicating atherosclerosis in the right
popliteal and posterior tibial arteries, leading to a diagnosis of
intermittent claudication. A unit of blood is removed weekly from
the patient. The plasma is recovered and processed with the method
of the present invention to produce derivatives of HDL that are
particles with reduced cholesterol content while the red blood
cells are returned to the patient. These HDL particles with reduced
cholesterol content are combined with the treated plasma and
administered to the patient intravascularly at weekly intervals for
5-10 weeks.
[0194] New blood work indicates a reduction in circulating
cholesterol, a reduction in LDL and an increase in circulating HDL.
A new angiographic procedure shows lower amounts of atherogenic
plaque in the right popliteal and posterior tibial arteries
compared to the first angiographic procedure. The patient reports
decreased levels of pain from the right lower extremity.
[0195] All patents, publications and abstracts cited above are
incorporated herein by reference in their entirety. The terms and
expressions which have been employed herein are used as terms of
description and not of limitation, and there is no intention, in
the use of such terms and expressions, of excluding any equivalents
of the features shown and described or portions thereof. It should
be understood that the foregoing relates only to preferred
embodiments of the present invention and that numerous
modifications or alterations may be made therein without departing
from the spirit and the scope of the present invention as defined
in the following claims.
* * * * *