U.S. patent number RE39,498 [Application Number 09/879,002] was granted by the patent office on 2007-02-27 for treatment for cardiovascular and related diseases.
This patent grant is currently assigned to Aruba International Pty. Ltd.. Invention is credited to Bill Elliot Cham, Karim Rouan Cham.
United States Patent |
RE39,498 |
Cham , et al. |
February 27, 2007 |
Treatment for cardiovascular and related diseases
Abstract
A method for the removal of cholesterol, triglycerides and other
lipids from animal plasma, serum or other suitable blood fractions,
as a discontinuous flow system, the method comprising withdrawing
blood from a subject, separating the required fraction from the
blood and mixing with a solvent mixture which extracts the lipids
from the fraction, after which the delipidated fraction is
recombined with the blood cells and returned to the subject,
wherein the solvent extraction step is carried out separately and
remote from the subject. The delipidated fraction is washed with a
second solvent before being recombined with the blood cells. To
ensure that the delipidated fraction is free from all extraction
solvent, the fraction is mixed with an absorbent specific for the
solvent that is being removed. The preferred absorbent is a
macroporous polymeric bead contained in the pores of a sintered
glass or plastic sphere, the being capable of absorbing organic
molecules from an aqueous solution. By treating the plasma, serum
or other suitable blood fraction of a patient by these methods, the
blood rheology of a patient with impaired blood circulation can be
improved. Further, a rapid regression of coronary atherosclerosis
occurs.
Inventors: |
Cham; Karim Rouan (Redland Bay,
AU), Cham; Bill Elliot (Sheldon, AU) |
Assignee: |
Aruba International Pty. Ltd.
(AU)
|
Family
ID: |
3784814 |
Appl.
No.: |
09/879,002 |
Filed: |
December 22, 1995 |
PCT
Filed: |
December 22, 1995 |
PCT No.: |
PCT/AU95/00875 |
371(c)(1),(2),(4) Date: |
June 10, 1997 |
PCT
Pub. No.: |
WO96/19250 |
PCT
Pub. Date: |
June 27, 1996 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
Reissue of: |
08849543 |
Jun 10, 1997 |
05911698 |
Jun 15, 1999 |
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Foreign Application Priority Data
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Dec 22, 1994 [AU] |
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PN 0307 |
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Current U.S.
Class: |
604/5.03;
604/4.01; 422/44; 210/651; 210/645 |
Current CPC
Class: |
A61P
9/10 (20180101); A61M 1/3486 (20140204); A61M
1/3472 (20130101); A61M 2202/0456 (20130101) |
Current International
Class: |
A61M
37/00 (20060101); A61M 1/36 (20060101); B01D
11/00 (20060101); C02F 1/44 (20060101) |
Field of
Search: |
;604/4.01,5.01-5.04,6.01,85 ;422/44
;210/645,650-1,767,749,348,634,502.1
;530/422,850,412-424,539,827,829-830 ;435/5 ;424/9.1,520,529-531
;436/71 |
References Cited
[Referenced By]
U.S. Patent Documents
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1 271 708 |
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Jul 1990 |
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CA |
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1189378 |
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Aug 1998 |
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CN |
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29 44 138 |
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Jun 1981 |
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DE |
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31 18072 |
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Nov 1982 |
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DE |
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32 13390 |
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Oct 1983 |
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DE |
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33 10 263 |
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Sep 1984 |
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DE |
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0 036 283 |
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Mar 1981 |
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EP |
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0 267 471 |
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Oct 1987 |
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EP |
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2 571 971 |
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Oct 1984 |
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FR |
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127104 |
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Oct 1980 |
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JP |
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277303 |
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Oct 1993 |
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JP |
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1116396 |
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Sep 1984 |
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SU |
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1204224 |
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Jan 1986 |
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SU |
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1752187 |
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Jul 1992 |
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SU |
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WO 88/09345 |
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Dec 1988 |
|
WO |
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WO 95/03840 |
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Feb 1995 |
|
WO |
|
WO 9503840 |
|
Feb 1995 |
|
WO |
|
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|
Primary Examiner: Bianco; Patricia
Attorney, Agent or Firm: Kilpatrick Stockton LLP
Claims
We claim:
1. A method for the removal of cholesterol, triglycerides and other
lipids from animal plasma, serum, or other suitable blood fraction
containing apolipoproteins, as a discontinuous flow system, said
method comprising connecting a subject to a device for withdrawing
blood, withdrawing blood containing blood cells from the subject,
separating said fraction from the blood cells and mixing with a
solvent mixture which extracts said lipids from the fraction but
which does not extract said apolipoproteins from the fraction,
after which the delipidated fraction is recombined with the blood
cells and returned to the subject, such that the .[.solve it.].
.Iadd.solvent .Iaddend.extraction step is carried out separately
and remote from the subject while the subject is not still
connected to the device for withdrawing blood .[.frown.].
.Iadd.from .Iaddend.the subject, wherein the extraction solvent is
removed from the delipidated fraction by mixing the delipidated
fraction with an absorbent specific for the extraction solvent and
wherein the absorbent does not remove said apolipoproteins from the
.[.delipilidated.]. .Iadd.delipidated .Iaddend.fraction being
returned to the subject.
2. A method as defined in claim 1, wherein the extraction solvent
is substantially removed from the delipidated fraction by washing
with a second solvent.
3. A method as defined in claim 2, wherein the delipidated fraction
is washed four times.
4. A method as defined in claim 2, wherein the second solvent is
diethyl ether.
5. A method as defined in claim 1, wherein the absorbent is
contained in the pores of sintered spheres.
6. A method as defined in claim 5, wherein the sintered spheres are
about 2 mm to 5 mm in diameter and the pores of the spheres are
less than 50 .[..ANG..]. .Iadd..ANG. .Iaddend.in diameter.
7. A method as defined in claim 1, wherein the absorbent is a
macroporous polymeric bead for absorbing organic molecules from an
aqueous solution.
8. A method as defined in any one of claim 1, wherein the absorbent
is held in a chamber which is adapted to allow the delipidated
fraction to pass through or over the absorbent at least twice.
9. A porous sintered sphere for use in a method as defined in claim
1, said sphere containing an absorbent in its pores.
10. A sintered sphere as defined in claim 9, wherein the absorbent
is a macroporous polymeric bead for absorbing organic molecules
from an aqueous solution.
11. A method of changing the blood rheology of an animal with
impaired blood circulation whereby the plasma, serum or other
suitable blood fraction of the animal has been treated by a method
as defined in claim 1.
12. A method for rapid regression of coronary atherosclerosis in an
animal whereby the plasma, serum or other suitable blood fraction
from the animal is treated by a method as defined in claim 1.
13. A method of removing excessive adipose tissue from an animal
whereby the plasma, serum or other suitable blood fraction from the
animal is treated by a method as defined in claim 1.
14. A method of removing fat soluble toxins from an animal whereby
the plasma, serum or other suitable blood fraction from the animal
is treated by a method as defined in claim 1.
15. A method of changing the blood rheology of an animal whereby
the plasma or serum of the animal is exchanged for non-autologous
plasma or serum wherein said non-autologous plasma or serum has
been treated by a method as defined in of claim 1.
16. A method of rapidly regressing coronary atherosclerosis in an
animal whereby the plasma or serum of the animal is exchanged for
non-autologous plasma or serum wherein said non-autologous plasma
or serum has been treated by a method as defined in claim 1.
17. A method of removing excessive adipose tissue from an animal
whereby the plasma or serum of the animal is exchanged for
non-autologous plasma or serum wherein said non-autologous plasma
or serum has been treated by a method as defined in claim 1.
18. A method of removing fat soluble toxins from an animal whereby
the plasma or serum of the animal is exchanged for non-autologous
plasma or serum wherein said non-autologous plasma or serum has
been treated by a method as defined in claim 1.
19. A method for the removal of cholesterol, triglycerides and
other lipids from animal plasma, serum, or other suitable blood
fraction containing apolipoproteins, a discontinuous flow system,
said method comprising connecting a subject to a device for
withdrawing blood, withdrawing blood containing blood cells from
the subject, separating said fraction from the blood cells and
mixing with a solvent mixture which extracts said lipids from the
fraction but which does not extract said apolipoproteins from the
fraction, after which the delipidated fraction is is recombined
with the blood cells and returned to the subject such that the
solvent extraction step is carried out separately and remote from
the subject while the subject is not still connected to the device
for withdrawing blood from the subject, wherein the solvent
extraction step comprises: (a) mixing the solvent mixture
containing the fraction with beads, said beads being of a density
substantially mid-way between the density of the fraction and the
density of the solvent mixture; and (b) isolating the thus
delipidated fraction-containing phase.
20. A method as defined in claim 19, wherein the beads contain
entrapped air to obtain the density substantially midway between
the density of the fraction and the density of the solvent
mixture.
21. A method as defined in claim 20, wherein the density of the
beads is about 0.9 g/ml.
.Iadd.22. A batch method for removing lipids from a volume of
animal or human plasma, serum, or other suitable blood fraction
containing apolipoproteins comprising: providing the volume of
animal or human plasma, serum, or other suitable blood fraction;
admixing the plasma, serum, or other suitable blood fraction with
an extraction solvent which extracts the lipids from the plasma,
serum, or other suitable blood fraction without extracting the
apolipoproteins to form a delipidated fraction; introducing an
absorbent to the delipidated fraction to substantially remove the
extraction solvent from the plasma, serum, or other suitable blood
fraction; and separating the absorbent from the delipidated
fraction to form a apolipoprotein containing delipidated
fraction..Iaddend.
.Iadd.23. A batch method as claimed in claim 22, wherein the
extraction solvent is substantially removed from the delipidated
fraction by washing at least once with a second
solvent..Iaddend.
.Iadd.24. A batch method as claimed in claim 22, wherein the
extraction solvent is substantially removed from the delipidated
fraction by washing a plurality of times with a second
solvent..Iaddend.
.Iadd.25. A batch method as claimed in claim 23, wherein the second
solvent is diethyl ether..Iaddend.
.Iadd.26. A batch method as claimed in claim 22, wherein the
absorbent is disposed in the pores of sintered
spheres..Iaddend.
.Iadd.27. A batch method as claimed in claim 26, wherein the pores
of the sintered spheres are less than 50 .ANG. in
diameter..Iaddend.
.Iadd.28. A batch method as claimed in claim 22, wherein the
absorbent is a macroporous polymeric bead for absorbing organic
molecules from an aqueous solution..Iaddend.
.Iadd.29. A batch method as claimed in claim 22, wherein the
absorbent is held in a chamber which is adapted to allow the
delipidated fraction to pass through or over the absorbent at least
twice..Iaddend.
.Iadd.30. A batch method as claimed in claim 22, further
comprising: mixing the apolipoprotein containing delipidated
fraction with blood cells derived from an animal or a human to form
a treated blood..Iaddend.
.Iadd.31. A batch method as claimed in claim 30, further
comprising: introducing the treated blood into the blood stream of
an animal or a human, wherein the apolipoprotein containing
delipidated fraction and the blood cells are respectively
autologous or non-autologous to the animal or human..Iaddend.
.Iadd.32. A batch method as claimed in claim 22, further
comprising: introducing the apolipoprotein containing delipidated
fraction into the blood stream of an animal or a human, wherein the
apolipoprotein containing delipidated fraction is autologous or
non-autologous to the animal or human..Iaddend.
.Iadd.33. A method of changing the blood rheology of an animal or a
human with impaired blood circulation whereby the plasma, serum, or
other suitable blood fraction of the animal or the human has been
treated by a method as claimed in claim 31..Iaddend.
.Iadd.34. A method for rapid regression of coronary atherosclerosis
in an animal or a human whereby the plasma, serum, or other
suitable blood fraction from the animal or human is treated by a
method as claimed in claim 31..Iaddend.
.Iadd.35. A method of removing excessive adipose tissue from an
animal or a human whereby the plasma, serum or other suitable blood
fraction from the animal or human is treated by a method as claimed
in claim 31..Iaddend.
.Iadd.36. A method of removing fat soluble toxins from an animal or
a human whereby the plasma, serum or other suitable blood fraction
from the animal or human is treated by a method as claimed in claim
31..Iaddend.
.Iadd.37. A method of changing the blood rheology of an animal or a
human whereby the plasma or serum of the animal or human is
exchanged for plasma or serum which has been treated by a method as
claimed in claim 32..Iaddend.
.Iadd.38. A method of rapidly regressing coronary atherosclerosis
in an animal or a human whereby the plasma or serum of the animal
or human is exchanged for plasma or serum which has been treated by
a method as claimed in claim 32..Iaddend.
.Iadd.39. A method of removing excessive adipose tissue from an
animal or a human whereby the plasma or serum of the animal is
exchanged for plasma or serum which has been treated by a method as
claimed in claim 32..Iaddend.
.Iadd.40. A method of removing fat soluble toxins from an animal or
a human whereby the plasma or serum of the animal or human is
exchanged for plasma or serum which has been treated by a method as
claimed in claim 32..Iaddend.
.Iadd.41. A batch method for removing lipids from a volume of
animal or human plasma, serum, or other suitable blood fraction
comprising: providing the volume of animal or human plasma, serum,
or other suitable blood fraction; admixing the plasma, serum, or
other suitable blood fraction with an extraction solvent selected
from hydrocarbons, ethers, alcohols, esters, amines, or mixtures
thereof which extracts the lipids from the plasma, serum, or other
suitable blood fraction to form a delipidated fraction; introducing
an absorbent to the delipidated fraction to substantially remove
the solvent from the plasma, serum, or other suitable blood
fraction; and separating the absorbent from the delipidated
fraction to form a delipidated fraction..Iaddend.
.Iadd.42. A method as claimed in claim 41, wherein the extraction
solvent comprises a mixture of an alcohol and an
ether..Iaddend.
.Iadd.43. A method as claimed in claim 42, wherein the extraction
solvent comprises a butanol..Iaddend.
.Iadd.44. A method as claimed in claim 41, wherein the extraction
solvent comprises butan-1-ol or butan-2-ol..Iaddend.
.Iadd.45. A method as claimed in claim 41, wherein the extraction
solvent comprises an ether..Iaddend.
.Iadd.46. A method as claimed in claim 41, wherein the extraction
solvent comprises di-isopropyl ether or propyl ether..Iaddend.
.Iadd.47. A method as claimed in claim 41, wherein the extraction
solvent comprises 1-butanol and di-isopropyl ether..Iaddend.
Description
CROSS-REFERENCE TO RELATED APPLICATION
This application is a U.S. National Phase Application filed under
35 U.S.C. .sctn. 371 based on International Application No.
PCT/AU95/00875, having an International Filing Date of Dec. 22,
1995.
TECHNICAL FIELD
THIS INVENTION relates to plasma or serum delipidation in animals
(which term shall indicate humans), to a treatment for
cardiovascular disease and to removal of excess fat from the
animals. In particular, it is directed to the removal of
cholesterol, triglycerides and other lipids, and fat soluble
toxins--for example, insecticides--from the blood plasma or serum
of such animals.
BACKGROUND ART
Cardiovascular diseases are responsible for a significant number of
deaths in most industrialised countries.
One such disease is atherosclerosis which is characterised by local
fatty thickening in the inner aspects of large vessels supplying
blood to the heart, brain and other vital organs. These lesions
obstruct the lumen of the vessel and result in ischaemia of the
tissue supplied by the vessel. Prolonged or sudden ischaemia may
result in a clinical heart attack or stroke from which the patient
may or may not recover.
The relationship between dietary lipid, serum cholesterol and
atherosclerosis has long been recognised. In many epidemiological
studies it has been shown that a single measurement of serum
cholesterol has proved to be a significant predictor of the
occurrence of coronary heart disease.
Thus diet is the basic element of all therapy for hyperlipidaemia
(excessive amount of fat in plasma). However, the use of diet as a
primary mode of therapy requires a major effort on the part of
physicians, nutritionists, dieticians and other health
professionals.
If dietary modification is unsuccessful, drug therapy is an
alternative. Several drugs, used singly or in combination, are
available. However, there is no direct evidence that any
cholesterol-lowering drug can be safely administered over an
extended period.
A combination of both drug and diet may be required to reduce the
concentration of plasma lipids. Hypolipidaemic drugs are therefore
used as a supplement to dietary control.
Many drugs are effective in reducing blood lipids, but none work in
all types of hyperlipidaemia and they all have undesirable side
effects. There is no conclusive evidence that hypolipidaemic drugs
can cause regression of atherosclerosis. Thus, despite progress in
achieving the lowering of plasma cholesterol to prevent heart
disease by diet, drug therapies, surgical revascularization
procedures and angioplasty, atherosclerosis remains the major cause
of death in Western Countries.
In view of the above, new approaches have been sought to reduce the
amount of lipid in the plasma of homozygotes and that of
heterozygotes for whom oral drugs are not effective.
Plasmapheresis (plasma exchange) therapy has been developed and
involves replacement of the patient's plasma with donor plasma or
more usually a plasma protein fraction. This treatment can result
in complications due to the possible introduction of foreign
proteins and transmission of infectious diseases. Further, plasma
exchange removes all the plasma proteins as well as very low
density lipoprotein (VLDL), low density lipoprotein (LDL), and high
density lipoprotein (HDL).
It is known that HDL is inversely correlated with the severity of
coronary arterial lesions as well as with the likelihood that these
will progress. Therefore, removal of HDL is not advantageous.
Known aphaeresis techniques also exist which can remove LDL from
plasma. These techniques include absorption of LDL in
heparinagarose beads (affinity chromatography) or the use of
immobilised LDL-antibodies. Other methods presently available for
the removal of LDL involve cascade filtration absorption to
immobilised dextran sulphate and LDL precipitation at low pH in the
presence of heparin. Each method specifically removes LDL but not
HDL.
LDL aphaeresis has, however, disadvantages. Significant amounts of
other plasma proteins are removed during aphaeresis and to obtain a
sustained reduction in LDL-cholesterol, LDL aphaeresis must be
performed frequently (up to once weekly). Furthermore, LDL removal
may be counter productive as, low blood LDL levels may result in
increased cellular cholesterol synthesis.
To satisfy the need for a method of achieving a reduction in plasma
cholesterol in homozygous familial hypercholesterolemia,
heterozygous familial hypercholesterolemia and patients with
acquired hyperlipidaemia other than by diet, drug therapy, LDL
aphaeresis, or a combination of these, an extra corporeal lipid
elimination process, termed "cholesterol aphaeresis", has been
developed. In cholesterol aphaeresis, blood is withdrawn from a
subject, plasma separated from the blood and mixed with a solvent
mixture which extracts lipid from the plasma, after which the
delipidated plasma is recombined with the blood cells and returned
to the subject.
In more detail, cholesterol aphaeresis results in the removal of
fats from plasma or serum. However, unlike LDL aphaeresis, the
proteins that transport the fat (apolipoproteins) remain soluble in
the treated plasma or serum. Thus the apolipoproteins of VLDL, LDL
and HDL are present in the treated plasma or serum. These
apolipoproteins, in particular apolipoproteins A1 from the defatted
HDL in the plasma or serum, are responsible for the mobilisation of
excessive amounts of deposited fats such as cholesterol in
arteries, plaques, or excessive amounts of triglycerides, adipose
tissue, or fat soluble toxins that are present in adipose tissue.
These excessive amount of fats or toxins are transferred to the
plasma or serum, bound to the newly assembled lipoproteins. Thus by
applying another cholesterol aphaeresis procedure, these unwanted
fats or toxins are successively removed from the plasma and thus
the body.
The main advantage of this procedure is that LDL and HDL are thus
not removed from the plasma but only cholesterol, some
phospholipids and considerable triglycerides. U.S. Pat. No.
4,895,558 describes such a system.
While cholesterol aphaeresis has overcome the shortcomings of
dietary and/or drug treatments and other aphaeretic techniques,
existing apparatus for cholesterol aphaeresis does not provide a
sufficiently rapid and safe process. For use in a clinical setting,
apparatus is required which effects delipidation more efficiently.
Furthermore, flow rates of the order of 70 ml/min are required for
cholesterol aphaeresis of a human subject.
Thus the cholesterol aphaeresis described in the aforementioned
U.S. Pat. No. 4,895,558 was improved by incorporating into the
system a spinner to disperse the incoming plasma laterally into the
extracting solvent in the form of fine droplets to improve
separation efficiency. This improved system is described in
International Patent Application No. PCT/AU94/00415.
Unfortunately, practice has established that the cholesterol
aphaeresis systems described above still suffer from a number of
disadvantages.
The first disadvantage is the explosive nature of the solvents used
to delipidate this plasma. These solvents are, by the very nature
of the continuous systems, in close proximity to the patient and
medical staff. This hazard is clearly present for the duration of
the delipidation process which usually runs for several hours.
The second disadvantage is that, in the prior continuous systems, a
reliable procedure is not available to remove totally all of the
solvents used in the delipidation before the treated plasma is
returned to the patient.
In particular, the use of the preferred solvent 1-butanol in the
delipidation is of concern as it can now be established that that
solvent can be present as 1% to 5% of the treated plasma that is
returned to the patient. This is because continuous systems can
only include a single wash to remove solvents such as 1-butanol and
a single wash is now found to be sufficient. It is not possible to
provide sequential multi-washes in a continuous system because the
patient would have to supply an unacceptable volume of blood to
maintain each stage of the system overall and the patient would
also be subjected to an increased hazard factor from the prolonged
exposure to the solvents.
The long term toxicity of 1-butanol is not known, especially when
directly present in the blood stream--it may cross the blood brain
barrier. Certainly, external contact with this solvent is known to
cause irritation of mucous membranes, contact dermatitis,
headaches, dizziness and drowsiness.
A third disadvantage is that the continuous systems described above
are not suitable for the delipidation of serum. If serum can be
delipidated, there would be the advantage of favourably altering
the blood rheology in that the viscosity will decrease following
delipidation resulting in better haemodynamics for the originally
impaired blood circulation.
Yet a fourth disadvantage is that delipidation in a continuous
system is undertaken over several hours. Apart from the prolonged
exposure to the hazardous solvents as discussed above, the
equipment and staff are committed to a single patient. As the
removal of plasma or other blood fractions and their subsequent
return to the patient as individual steps each only take a few
minutes, it would be advantageous if the relatively lengthy
delipidation step could be undertaken off site, thus freeing the
patient, medical staff and equipment for other matters.
Finally, in a continuous system, clearly it is only the patient's
own blood fraction that can be returned to that patient. However,
for example, if the patient's plasma or serum could be removed and
treated remote from the patient, then either autologous or
non-autologous plasma or serum could be returned to the patient at
a later date.
SUMMARY OF THE INVENTION
It is an object of the present invention to overcome, or at least
ameliorate, the above-mentioned disadvantages in the provision of a
method for delipidating not only plasma but also serum and other
blood fractions which substantially reduces the exposure of the
patient to the potentially hazardous solvents used, which also can
effectively remove all traces of solvent(s) used in that
delipidation, and which significantly reduces the contact time
between the patient and the actual delipidation process.
It is a further object to provide a method whereby advantageous
changes to the blood rheology of the originally impaired blood
circulation of the patient can be achieved.
It is yet another object to provide a method whereby a patient's
Plasma or serum can be treated remote from that patient, thus
allowing either autologous or non-autologous plasma or serum to be
returned to the patient at a later date.
In one aspect of the present invention, there is provided a method
for the removal of cholesterol, triglycerides and other lipids from
animal plasma, serum or other suitable blood fractions, as a
discontinuous flow system, said method comprising withdrawing blood
from a subject, separating the required fraction from the blood and
mixing with a solvent mixture which extracts the said lipids from
the fraction, after which the delipidated fraction is recombined
with the blood cells and returned to the subject, characterised in
that the solvent extraction step is carried out separately and
remote from the subject.
Preferably, as part of the solvent extraction step, beads are used
when mixing the blood fractions with the solvent. More preferably,
the beads have a density substantially mid-way between the density
of the fraction and the density of the solvent mixture. This
ensures efficient mixing with a large surface area, increasing the
efficiency of the extraction and also serving as a good separator
of the plasma from the solvent when centrifugation is used to
isolate the phases after extraction.
Preferably, to obtain a density substantially mid-way between the
density of the fraction and the density of the solvent mixture, the
beads contain entrapped air.
More preferably, as the density of plasma is approximately 1.006
g/ml and the solvents used generally have a density of
approximately 0.8 g/ml, the density of the beads will be around 0.9
g/ml.
The beads may be manufactured from any acceptable material such as
glass or plastic.
Once the resultant delipidated fraction-containing phase has been
isolated, all traces of the extraction solvent must be removed
before the fraction is recombined with the blood cells and/or
returned to the subject.
One way of removing this solvent is to wash with another solvent,
preferably diethyl ether, to remove substantially all of the
original solvent used in the extraction step.
More preferably, four (4) washes are undertaken.
However, as another aspect of the present invention, efficient
removal of the extraction solvent can be achieved by mixing the
delipidated fraction with an absorbent specific for the solvent
that is being removed.
In particular, the absorbent is contained in the pores of sintered
spheres.
More preferably, the sintered spheres are approximately 2 to 5 mm
in diameter with the pores of the spheres being less than 50 .ANG.
in diameter. Most preferably, the spheres are manufactured from
glass.
Preferably, the absorbents used in the sintered spheres are the
macroporous polymeric beads for absorbing organic molecules from
aqueous solutions marketed by Bio-Rad Laboratories under the trade
name Bio-Beads SM.
If the solvent used to delipidate the fraction is 1-butanol, then
the absorbent is preferably Bio-Beads SM-2.
Preferably, the absorbent is held in a chamber which is adapted to
allow the delipidated fraction to pass through or over the
absorbent at least twice if a single pass is insufficient to remove
all of the solvent.
Preferably, as part of isolating the delipidated
fraction-containing phase, that phase is subsequently washed with
another solvent, preferably diethyl ether, to remove a substantial
amount of the original solvent before the treatment with the
absorbent.
More preferably, that phase is washed at least three (3) times.
The plasma may be human plasma or plasma from other living animals.
The plasma can be obtained from human or animal blood by known
plasma separating techniques which include centrifugal separation,
filtration and the like.
Similarly, the serum or other lipid-containing fraction can be
derived from human or other living animals by known techniques.
Suitable solvents for the extraction comprise mixtures of
hydrocarbons, ethers and alcohols. Preferred solvents are mixtures
of lower alcohols with lower ethers. The lower alcohols suitably
include those which are not appreciably miscible with the plasma
and these can include the butanols (butan-1-ol and butan-2-ol).
C.sub.1-4 ethers are also preferred and these can include the
propyl ethers (di-isopropyl ether and propyl ether). Other solvents
which may be applicable include amines, esters, hydrocarbons and
mixtures providing that the solvent can (1) rapidly and preferably
remove cholesterol from the plasma, (2) is substantially immiscible
with the plasma, (3) can be removed from the plasma, and (4) does
not denature the desired moieties. Preferred solvent compositions
are butanol with di-isopropyl ether and these may be in the ratio
of 0%-40% of the alcohol to 100%-60% of the ether.
DETAILED DESCRIPTION OF EMBODIMENTS
Materials and Methods
Animals
The roosters used in this study were of White Leghorn Hiline strain
and were obtained as one-day old chicks. All roosters from 8 weeks
old were transferred into individual cages. Water and feed were
supplied unrestricted. At eight weeks of age, 15 control birds were
fed a commercial poultry ration for 31 days and another group of 30
birds were injected subcutaneously each day with 5 mg
diethylstilboestrol (DES) in sesame oil for a period of 31 days. In
addition they were fed on the same commercial diet which was
supplemented with 2.6% (w/w) cholesterol for a period of 31 days.
Fifteen animals of the DES treated group were then subjected to
lipid aphaeresis (LA). Fifteen animals of the DES treated group had
sham treatments. Once the LA or sham treatments commenced, all
animals were fed the standard poultry ration, except during the
actual treatment itself when animals were kept off their feed for
three hours following reinfusion of their autologous blood. Animals
were sacrificed two days following the 4th treatment, LA or
sham.
Lipid Aphaeresis Procedure
Approximately 25% of the calculated blood volume was collected from
a brachial vein of the animal with a 21 gauge needle and syringe.
The total blood volume was estimated at 8 percent of the body
weight. The blood was collected in heparinized tubes and
immediately centrifuged at 900 g for 5 minutes at room temperature.
The blood cells were suspended in an amount of saline equivalent to
the plasma volume and were reinfused into the animal. The plasma
was kept refrigerated for twelve hours and was then delipidated for
20 minutes with a mixture of butanol and di-isopropyl ether (DIPE),
25:75 (v/v), in a ratio of one volume of plasma to two volumes of
butanol-DIPE mixture (organic phase). Inert plastic beads with a
density of 0.9 g/mL (1 g) were added to the mixture. After
extraction, the mixture was centrifuged at 900 g for 2 min to
separate the plasma and organic phases. The organic phase (upper
layer) was removed, free of plasma phase, by careful aspiration
with a pasteur pipette under vacuum. Traces of butanol in the
plasma phase were washed out with four volumes of diethyl ether
(DEE) for 2 min by end-over-end rotation at 30 rpm. The mixture was
then centrifuged at 900 g for 2 min to separate plasma and ether
phases. The ether phase was subsequently removed by aspiration with
a pasteur pipette. Residual ether was removed by evacuation with a
water pump aspirator at 37.degree. C. The plasma was then passed
through a 5 mL column containing Bio-Beads SM-2.
This procedure yielded delipidated plasma. The delipidated plasma
was re-mixed with the blood cells of a subsequent 25% blood
collection which was then reinfused through a brachial vein back
into the identical donor animals. The duration of the entire
procedure, that is, removal of blood from the animal to reinfusion
of treated blood back to the animal was approximately 1 hour. After
the fourth lipid aphaeresis treatment, the animals were sacrificed
and their livers and aortae were dissected. The LA treatment
procedures were repeated 3 times after the first treatment.
Sham Treatment Procedures
This was essentially the same as the LA procedure with the
exception of the plasma delipidation with the organic solvents. The
blood was collected in heparinized tubes and immediately
centrifuged at 900 g for 5 min. The plasma was separated from the
blood cells. The blood cells were mixed with saline in the same
volume of the collected plasma and reinfused into the animal. The
plasma was kept refrigerated for twelve hours and was then remixed
with blood cells of a subsequent 25% blood collection after the
second and/or subsequent plasma separations. After the fourth lipid
aphaeresis treatment, the animals were sacrificed and their livers
and aortae were dissected. The sham treatment procedures were
repeated 3 times after the first treatment.
Tissue Lipid Preparation
The livers were weighed, minced with a scalpel blade and
homogenised in 0.9% sodium chloride solution by 10-12 strokes of a
motor driven Teflon-glass homogeniser (1900 rpm). The aorta was
weighed and three times its weight of 3 mm glass beads were added
in a homogenising bottle containing 0.9% sodium chloride. The
contents were then homogenised for one minute. The lipid from the
homogenised liver and aorta samples were extracted by the Folch
procedure and weighed.
TABLE-US-00001 TABLE I Effect of LA and sham treatments on the
total lipid concentrations in livers and aortas of hyperdiedemic
roosters TREATED UNTREATED FOUR APHAFRESIS APPLICATIONS CONTROLS
SHAM LA n = 15 n = 15 n = 15 LIVER.sup.a 3.65 .+-. 0.98 5.53 .+-.
1.50.sup.b 3.72 .+-. 1.00.sup.2 AORTA.sup.c 6.01 .+-. 0.97 0.11
.+-. 2.15.sup.c 0.32 .times. 0.95.sup.d .sup.aTotal lipid
concentrations expressed as g lipid per 100 g tissue, mean .+-. SD
.sup.b,cp values were <0.05 when sham treatments were compared
with LA treatments. There were no statistical differences between
the values of corresponding tisues in the untreated control group
and the LA treated group. All animals were sacrificed two days
after the final apherical treatment.
Humans
Patients have the plasmapheresis procedure undertaken using known
transvenous techniques and plasmapheresis systems.
Plasmapheresis is performed using vein-to-vein or arteriovenous
fistula in the forearm of patients. Heparin is given at the
beginning of the procedure as a 5,000 unit bolus, and then by
continuous infusion at the rate of 700 units per hour over the
course of the procedure. Access through the antecubital veins
should provide plasma flow rates of 25 to 40 mls per minute.
Blood taken from a patient is immediately treated with ACD-A
(anticoagulant) in a ratio of between 1:8 and 1:16 (ACD-A:blood).
The plasma is separated from this solution using a conventional
plasmapheresis machine.
Twenty five percent plasma is removed from the patient. This
represents one percent of the ideal body weight.
Only the first volume of plasma collection is replaced with plasma
replacement fluid to the patient.
The plasma is kept refrigerated up until twelve hours prior to
reinfusion of delipidated plasma in exchange for another twenty
five percent plasma collection (weekly or biweekly).
The plasma is delipidated and the delipidated plasma is tested to
ensure all solvent has been removed before the clean delipidated
plasma is exchanged for new untreated plasma.
In one embodiment of the present invention, the continuous flow
system described in U.S. Pat. No. 4,895,558 (the entire content of
which is included herein) is modified to a discontinuous system by
removing the appropriate blood volume to be treated and subjecting
that volume to delipidation at a site remote from the patient.
In another embodiment of the present invention, the continuous flow
system described in International Patent Application No.
PCT/AU94/00415 (the entire content of which is included herein) is
modified to a discontinuous system by removing the appropriate
blood volume to be a site remote from the patient before the plasma
is dispersed into small droplets into the solvent by the dispersing
means.
In either of the above embodiments, the extraction step can
include, in accordance with the present invention, either multiple
washing of the extracted phase and/or using an absorbent.
For example, the plasma is delipidated with a solvent mixture
comprising 1-butanol and di-isopropyl ether. The delipidated
fraction is then washed three (3) or four (4) times with diethyl
ether. After the final wash, the diethyl ether is removed by
centrifugation and vacuum extraction at 37.degree. C. The sintered
spheres containing Bio-Beads SM-2 are then mixed with the
delipidated plasma to remove the final traces of 1-butanol.
Conclusions
DES administration to the roosters resulted in a significant amount
of fat (lipid) accumulation in the livers and aortae.
Discontinuous LA treatments corresponding to approximately one
plasma volume treated by four applications of 25% of plasma volume
treated per time resulted in significant decreases in both hepatic
and aortic lipids in hyperlipidaemic animals. Moreover, the LA
treated hyperlipidaemic animals ended up with lipid values that
were similar to control animals. (i) These experiments show that
excessive amounts of body fats in the form of adipose tissue
(triglycerides) in the liver can be removed by LA; and (ii)
regression of atherosclerosis occurs in the aorta by LA
treatments.
Similar results can be expected for human patients.
By adapting the prior art methods to discontinuous flow systems,
the present invention can remove or at least significantly reduce
any danger to patients and medical staff from the explosive nature
of the solvents employed.
Further, by using the improved solvent extraction methods of the
present invention, all of the potentially poisonous extraction
solvents can be removed before the treated blood is returned to the
patient.
Also, the improved solvent extraction method of the present
invention is not limited to plasma delipidation but also it is
applicable to the delipidation of serum, thus providing
advantageous changes to the blood rheology of the originally
impaired blood circulation of the patient.
The present invention thus provides for a rapid regression of
coronary atherosclerosis in a patient.
Finally, as the present invention is a discontinuous system, it is
not essential to return the delipidated blood fraction immediately
to the patient. It is already known that plasma or serum can be
collected and stored under sterile conditions in a refrigerator or
freezer for extended periods and that it can be returned safely to
the patient within twelve (12) hours of breaking the sterile seal.
Therefore, if necessary, reintroduction of the delipidated fraction
can occur several weeks after it was first removed from the
patient. This option leads to particular advantages such as,
economies of scale when several patients have to be treated
simultaneously, the freeing of medical staff and equipment for
other duties, and the reduction in stress for the patient whom no
longer has to be hooked up to a delipidation apparatus for several
continuous hours. Further, it enables a bank of plasma or serum to
be maintained which is free of any infection which can be
delipidated and exchanged for a patient's plasma or serum as
required. Of course, autologous or non-autologous plasma or serum
could be returned to the patient under these conditions.
The embodiments are described by way of illustrative examples only
and various changes and modifications may be made thereto without
departing from the inventive concept as defined in the following
claims.
* * * * *