Note: Descriptions are shown in the official language in which they were submitted.
W 0 91/OS~36 P ~ /US90/05679
j; ! 2 o 6 7 3 6 4
NOVEL OXIDIZED LIPOPROTEINS
AND METHODS FOR THEIR PREPARATION
BACKGROUND OF THE INVENTION
Cross-Reference to Related AD~lication
This application is a continuation-in-part of U.S.
Patent Application Serial Number 07/418,382 filed October 6,
1989.
Sta~ement Re~arding FederallY S~onsored Research
., ,- . .
Punding for work descrlbed herein was provided by the
Federal Government under a grant from the Department Of
Healtn and Human Services. The Government may have certain
rights in this invention.
Prior Art
The treatment of disease using oxidized lipoproteins is
described in detail in the parent application cited above
which is herein ~ncorporated by reference. Other teachings
WO9l/05536 PCT/US90/05679
20~73fi4 2
by Eric T. Fossel which are incorporated herein by reference
are: "Process for the Screening of Cancer Using Nuclear
Magnetic Resonance", U.S. Patent No. 4,912,050, March 27,
1990 and ~Process for the Detection of Cancer Using Nuclear
Magnetic Resonance", U.S. Patent No. 4,918,021, April 17,
1990.
Field of Invention
This invention relates to novel oxidized lipoprotein
compositions and especially methods and an apparatus for
preparin~ them.
Most organic compounds, including most tissue
constituents, are thermodynamically unstable in the presence
of oxygen. Due to the presence of both energy barriers and
electron-spin barriers to the direct reaction of ordinary
triplet-state oxygen with methylene groups, the reaction is
generally very slow. However, certain substances present in
the tissues react relatively readily with oxygen. Among
these are the unsaturated fatty acids.
One of the known properties of unsaturated fatty acids,
particularly the polyenoic acids, is their susceptibility to
oxidation, in particular peroxidation and autoxidation.
Autoxidation is a radical chain reaction involving molecular
oxygen as a reactant in one of the steps. The term
"peroxidation" refers ts production o~ peroxides- and their
degradation products. In general terms, oxidation involves
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WO91~05536 PCT/US90/05679
3 2~736~
one molecule of oxygen whereas peroxidation involves two
molecules of oxygen. In accordance with the present
invention, another form of oxidation, photoperoxidation is
provided. This form of oxidation is mediated by a
photosensitizer and ultraviolet light.
A study by Edelson et al., N. Enq. J. of Med., 1987,
Vol. 316, pp. 297-303 describes treatment effective for some
cutaneous lymphomas and certain leukemias whereby a
photosensitizer is added to blood and the blood irradiated
with ultraviolet light. In this study, an aromatic
compound, 8-methoxypsoralen, was ingested by a patient.
Several hours later blood was withdrawn and irradiated with
ultraviolet light (type A), and then re-infused into the
patient. The Edelson study, however, does not explain the
mechanism of this method of treatment, it simply indicates
that it is effective against certain forms of cancer.
The present invention provides several methods for
oxidizing lipoproteins and photoperoxidation is one such
method. It is believed that by adding a photosensitizer such
as 8-methoxypsoralen to blood and then irradiating the blood
with ultraviolet light produces oxidized lipoproteins. It
is further believed, that the oxidized lipoproteins are
useful as a therapeutic agent in fighting disease.
: .. , : '
WO91~05536 PCT/US90/05679
20673~4 4
Formulas for the three types of oxidation appear below.
autoxidation: o -----> O -----> product
2 2 + ~C
eroxidation: ~-O-O-H -----> H-O-O- + H ------>product
+ HC
Dhotoperoxidation: photosensitizer + hv ----->
[photosensitizer]' + O -----> o ~ + H O ----->
HOO -----> HC -----~ product 2 2
Empirically, it was ~ound that the rate of autoxidation
is directly proportional to substrate concentration and to
the partial pressure of oxygen above lO0 mm. The rate also
increases with the extent of oxidation, indicating a chain
reaction and the autocatalytic nature of the process. The
principal initial products are hydroperoxides. FIG. l
expresses these relationships, where Ko and ~ are empirical
constants, RX is unoxidized substrate, p2 the partial
pressure of oxygen and the rate of oxidation is expressed as
the derivative with respect to time: d/dt.
To date, most studies dealing with fatty acid
oxidation have concentrated on the autoxidation of
linoleate. Linoleate, a fatty acid naturally occurring in
animals, provides a good model for the mechanism of
oxidation.
~ he initiation reactions, as shown in FIG. 2, are of
particular importance since it has been shown that highly
purified polyunsaturated fatty acids are stable for long
periods of time in the presence of oxygen (Privett and
Blank, 1962). Acting as catalysts, traces of peroxides or
,
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: . . : . : :
WO 91/05536 PCl`tl IS90/05679
transition metals and ultraviolet or ionizing radiatlon, in
addition to several other factors. are known to bring about
initiation. Once initiated, the reaction continues by a
chain mechanism involving resonance-stabilized free radicals
that react readily with oxygen to form peroxy radicals,
which can then initiate new chains by the slower abstraction
of a hydrogen atom from another molecule of substrate as the
peroxy radicals are converted to hydroperoxides. -
Hydroperoxides will decompose under certain conditions
by homolysis, giving radicals that can initiate new chains.
In addition, the hydroperoxides are reasonably strong
oxidizing agents and are reduced by thiol-containing
proteins, glutathione, cysteine and particularly, by a
glutathione-dependent factor in the cytosol which apparently
has this function as part of the antioxidant dPfense of the -
cell. If not reduced to the alcohol, the hydroperoxide can
undergo homolysis, usually catalyzed by transition metal
ions, eo form a hydroperoxy or alkoxy radical, depending
upon the oxidation state of the metal as shown in FIGS. 3
and 4. The alkoxy radical formed, as shown in FIG. 3,
reacts w1th any susceptible molecule in its vicinity,
usually by abstraction of a hydrogen atom and creation of
another radical likely to initiate further reactions.
Indeed, it is this type of reaction that is responsible
~or the unpleasant odor and flavor of oxidatively rancid
fat, the odor being that of unsaturated aldehydes produced
...
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WOgl/05536 PCT/US90/05679
206736~
in ehis manner from unsaturated fats. For example,
hydroperoxides formed by autoxidation of oleic acid would be
expected to decompose in this way to give aldehydes as shown
in FIG. 5.
Additional reactions that alkoxy radicals may undergo
are shown in FIGS. 6A-6D. FIG. 6A shows abstraction of a
hydrogen atom, initiating new chains. FIG. 6B shows
disproportionation with another radical. FIG. 6C shows a
coupling: FIG. 6D depicts addition to double bonds, which
can lead to polymeric products of high order of the types
commonly found among lipid peroxidation products.
It would seem from the nature of the autoxidation
reaction that this reaction would occur most readily in
systems such as cell membranes, in which the lipids are in
an ordered arrangement that would facilitate the propagation
reactions. However, the complexity of such membranes has
made the investigation of their peroxidation difficult and
has led to the use of several model systems in order to gain
insight into the reaction involved.
one such system consists of monomolecular films of
unsaturated fatty acids adsorbed on silica gel.
Autoxidation of linoleic acid in this way, induced by small
amounts of peroxides, leads to disappearance of linoleic
acid by apparent first order kinetics. The equation shown
in FIG. 1 indicates that autoxidation of polyunsaturated
. .
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., ., . . ,: :
~ . . . ' ' . . .
W09t/05~36 PCT/US90/05679
7 ' l` 2~87~
fatty acids exhibits much more complex kinetics. The
monomolecular film thus appears to exhibit a different
mechanism of peroxidation and this is confirmed by the
finding that major products are the fatty acid epoxides.
This reaction is of interest since some of the most potent
chemical mutagens and carcinogens are epoxides.
In another model system, the unilamellar liposome,
formed by sonication of an unsaturated phosphatidylcholine
in aqueous buffered solution, the products of autoxidation
are the expected 9- and 13-hydroperoxyoctadecadienoic acids.
However, with inclusion of saturated phosphatidytlcholine in
the liposome, the product mixture becomes much more complex,
containing also epoxides, hydroxyepoxides, and di- and
trihydroxy acyl moieties (see also Fridovich and Porter,
1981). A simple membrane system from Achole~lasma
laidlawii, with a fatty acid composition of the membrane
phospholipids consisting of a 50:50 mixture of 18:2 and
16:0, gave very similar fatty acid products, indicating that
results with the model systems are indeed representative of
those occurring in biomembranes. Lipids, Mead, Plenum
Press, New York, p.83 (1986).
- Although the prior art is replete with the chemistry
for oxidizing fatty acids, including lipoproteins, it is
believed that the present invention contains novel methods
of oxidation of lipoproteins as well as novel oxidized
lipoprotein compositions.
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-: -
.
.
WO91/05536 PCT/US90/05679
` 20~73~4 8
SUMMARY OF THE INVENTION
In accordance with the present invention, it was
discovered that peroxide, such as hydrogen peroxide in
conjunction with an enzyme, such as horseradish peroxidase,
is capable of generating free radicals which in turn can
peroxidize lipoproteins, preferably low density
lipoproteins. Thus, novel oxidized lipoprotein compositions
are provided as well as methods and apparatus for preparing
them. Also, the use for such oxidized lipoproteins is
indicated.
It is thus an obiect of this invention to provide a
novel oxidized lipoprotein composition which in the most
preferred embodiment consists of low density lipoproteins,
hydrogen peroxide, horseradish peroxidase and saline
solution. Other novel compositions are provided which
include flavin, riboflavin, oxidase, lipoxidase, organic
peroxide or ditertiarybutyl peroxide as oxidants. Modified
lipoproteins can be added to the composition to enhance the
effect of the organic peroxides. Also, compositions are
provided which include chemotherapeutic agents such as
adriamycin and mitomycin-D or photosensitizers such as
a-methoxypsoralen and hematoporphyrins. Additionally, novel
lipoproteins compositions are provided which include
elemental oxygen or perfluorocarbon fluosal.
Another object of this invention is to provide methods
. . . . . . . . ............................ . .
- . - . , ~... ~ ~ ,
W091~05536 PCT/US90/OS679
9 ` 20~736~
for preparing oxidized lipoproteins. The preferred method
is to add hydrogen peroxide and horseradish peroxidase to a
solution of low density lipoproteins and then subject the
sample to carbon-13 MMR spectroscopy to determine the degree
of peroxidation. The lipoproteins can also be oxidized
using other oxidants. Additional methods include oxidizing
the lipoproteins by irradiating the sample, to which a
photosensitizer has been added, with ultraviolet light or
adding certain chemotherapeutic agents to the lipoprotein
solution.
It is further object of this invention to provide
apparatus for preparing the oxidized lipoproteins. One such
apparatus is a peroxidizing module containing an immobilized
enzyme into which a pump introduces hydrogen peroxide.
Another apparatus is a container, which holds an immobilized
enzyme, into which hydrogen peroxide is added. Yet another
apparatus is an atrioventricular shunt or arterial bypass
which is attached to a patient with a peroxidizing module
attached to the shunt.
In a separate patent application, filed on even date
with the present application, Eric ~. Fossel teaches that
oxidized lipoproteins may be helpful in fighting disease
states, such as cancer, malaria and viral infections such as
acquired immunodeficiency syndrome, which are characterized
by diseased cells with an increased number of lipoprotein
receptors or an enhanced ability to take up lipoproteins.
. .
.
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.
WO91/05~36 PCT/US90/05679
- 206736~ lO ;-`
The diseased cells have been found to be more susceptible
than healthy cells to the cytotoxic effect of oxidized
lipoproteins. Thus, therapy with oxidized lipoproteins is
believed to have the effect of killing the diseased cells.
Other objects and advantages of the invention will
become apparent from the descriPtion of the invention which
follows, made with reference to the drawings described
below.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an equation that expresses the kinetics of
oxidizing linoleic acid;
FIG. 2 shows the initial reactions in the autoxidation
of linoleic acid;
FIG. 3 shows a likely decomposition mechanism in which
metal ions readily convert hydroperoxides to alkoxy radicals -
and hydroxyl ions;
FIG. 4 shows the hydroperoxide undergoing homolysis
catalyzed by transition metal ions to form a hydroperoxy or
alkoxy radical depending on the oxidation state of the
metal;
FIG. 5 shows how hydroperoxides formed by autoxidation
.
... . .. ~ . . . ~ ~.
WO9l/05S36 PCT/US90/05679
11 ` ; 20S7364
of oleic acid are expected to decompose to give aldehydes;
FIG. 6A shows abstraction of a hydrogen atom,
initiating new chains; .
FIG. 6B shows disproportionation with another radical;
FIG. 6C shows a coupling;
FIG. 6D depicts addition to double bonds, which can
lead to polymeric products of high order of the types
commonly found among lipid peroxidation products;
FIG. 7 shows the olefinic region of a C-13 spectrum of
a normal human plasma sample;
FIG. 8A shows the olefinic region of a 125.8 MXz proton
decoupled spectrum from normal human plasma; ;~
FIG. 8B shows the olefinic region of a 125.8 MHz proton
decoupled spectrum of the same plasma as in FIG. 8A
following the addition of peroxidase (2 mg/ml), and after 3
aliquots of 3% hydrogen peroxide (100 ~ l/ml) were added at
hourly intervals;
FIG. 9 shows the apparatus of the present invention;
FIG. lOA is a schematic diagram of the mechanism for
producing peroxidized low density lipoproteins as carried
~, . ,,, . .................... , . , , ,:
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WO91/05536 PCTJUS90/05679
2n~7364 12
out by the human body in response to malignancy;
FIG. 10B is a schematic diagram of the general method
for treating cancer in accordance with the claimed
invention;
FIG. 11 shows another embodiment o~ the apparatus of
the present invention for oxidizing the lipoproteins in a
blood supply and
FIG. 12 shows a further embodiment of the apparatus of
the present invention for oxidizing the lipoproteins in a
blood supply.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
At the outset. the invention is described in its
broadest overall aspects, with a more detailed des~ription
following. In its broadest overall aspects, the invention
relates to a novel oxidized lipoprotein composition as well
as methods and apparatus for preparing the oxidized
lipoproteins.
Lipoproteins take various forms in the blood including
chylomicrons, chylomicron remnants, very low density
lipoproteins, lntermediate density lipoproteins, low density
lipoproteins, and high density lipoproteins. Certain lipids
associate with specific proteins to form lipid:protein
WO9l/05536 PCT/US90/05679
13 20S73~A
systems in which the specific physical properties of these
two classes of biomolecules are blended. There are two
major types; transport lipoproteins and membrane systems.
In these systems, the lipids and proteins are not covalently
~oined but are held together largely by hydrophobic
interactions between the nonpolar portions of the lipid and
the protein components.
The plasma lipoproteins are complexes in which the
lipids and proteins occur in a relatively fixed ratio. They
carry water-insoluble lipids between various organs via the
blood, in a form with a relatively small and constant
particle diameter and weight. Human plasma lipoproteins
occùr in four ma~or classes that differ in density as well
as particle size as shown in the table below.
Maior Classes of Human Plasma Li~o~roteins
Vo~ylo~ dondt~dondlyH4h dondty
lipoprotdn~Iipop~hln~lipopro~ln~
Chylomicnm~ r~lDL) PDL) ~HDL)
D~ty, ~ 08~ O~ OOB-~ 0~31 0~3-~ 2~
Fiobtton nb. S~ ~ 20-~oo 0-20(S~ilm~nt)
Putici~ s-t ooo 30-50 20-Z2 7 ~-lo
Pn~t~, % of dr~ 2 - 10 25 ~5-5
~docyl~lye~ol~, % of dr~r ~Ight ~0-9~ SS~ 0 3
Pho phoUpft~ of d y wd~h~ 3-B 15-2D 22 30
Clwi~t~rol, f~, % of dr~ wd~bt 1-3 10 8 3
Cbtli~ol._~d.t~ 1~ 5 37 ~5
~:t
There are pathways within the body for interconversion
among the four major classes.
.
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WO91/05536 PCT/US90/05679
20~736~ 14
As shown in the above table, the plasma lipoproteins
contain varying proportions of protein and different types
of lipid. The very low-density lipoproteins contain four
different types of polypeptide chains having distinctive
amino acid sequences. The high-density lipoproteins have
two different types of polypeptide chains, of molecular
weight 17,500 and 28,000. The polypeptide chains of the
plasma lipoproteins are believed to be arranged on the
surface of the molecules, thus conferring hydrophilic
properties. However, in the very low-density lipoproteins
and chylomicrons, there is insufficient protein to cover the
surface; presumably the polar heads of the phospholipid
components also contribute hydrophilic groups on the
surface, with the nonpolar triacylglycerols in the interior.
Biochemistrv, Lehninger, Worth Publishers, Inc., New York,
1975, pp.301.
According to the most preferred embodiment of the
present invention, an oxidized lipoprotein composition
includes a low density lipoprotein solution, hydrogen
peroxide, horseradish peroxidase and saline. In all
embodiments described herein, the term "lipoprotein
solution" is to be understood as lipoproteins in either
saline or buffer. In addition, the lipoprotein solution may
consist of LDL, HDL or VLDL whereas a lipoprotein solution
containing LDL is the preferred embodiment of this
invention. :: '
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- . .. , . . . ~ :
WO91/05536 PCT/US90/05679
1S - 20~73~
Another embodiment is a composition of a solution of
low density lipoproteins which includes one of the following
oxidants: flavin, riboflavin, oxidase, peroxidase,
horseradish peroxidase, lipoxidase, peroxide, organic
peroxide or ditertiarybutyl peroxide.
The preferred embodiment of the method for preparing
oxidized lipoproteins includes adding hydrogen peroxide and
horseradish peroxidase to a solution of low density
lipoprote1ns. The lipoproteins oxidized in this manner are
then introduced into the patient. To determine and monitor
the degree of peroxidation, the sample is subiected to
carbon-13 NMR spectroscopy.
Another embodlment of the method of the present
invention is oxidizing naturally occurring lipoproteins in a
patient. Hydrogen peroxide itself or in con;unction with an
enzyme such as peroxidase or lipoxidase is introduced into
the patient by means of an atrioventricular shunt (or
arterial bypass) or by means of an injection.
Administering non-oxidized but modified lipoproteins
should enhance the effect of the organic peroxides in
preparing the oxidized lipoproteins. The modified
lipoproteins should be administered to the patient or added
to the solution prior to oxidation of the lipoproteins.
Modified lipoproteins are prepared by enriching the content
of natural lipoproteins with triglycerides, such as
. ., , : ... . .
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.:
~VO9l/05'36 PCT/US90/05679
~Q`~7364 16
trilinoleal triglyceride, phospholipids such as dilinoleal
phosphatidylcholine or cholesterol esters such as
cholesterol ester of linoleic acid. These substances are
believed to be more easily oxidized or result in more
cytotoxic peroxidation products. The modified lipoproteins
may be included in each embodiment of the present invention.
The lipid peroxidation process of the body may be
further augmented by increasing the oxygen level in the
blood via inhalation of increased levels of elemental oxygen
during breathing. Perfluorocarbon fluosal may also be
introduced into a person by means of an intravenous
in;ection to increase the oxygen level in the blood. The
oxygen level in the blood should be increased before
oxidized lipoproteins are administered or oxidized in the
body. Both of these methods of increasing the level of
oxygen in the blood may be included in each em~odiment of
this invention.
In all the embodiments described herein, the blood may
be monitored for oxidized lipoprotein level by carbon-13 -;
nuclear magnetic resonance (NMR) spectroscopy. This
procedure is taught by Eric T. Fossel in a copending U.S.
Patent Application ~o. 557,529 filed July 24, 1990, where a
method for the detection of cancer by measuring lipid
peroxldation using NMR is disclosed. It has been discovered
that in cancer patients, lipid peroxidation occurs and the
rat~o of polyunsaturated fatty acids to monounsaturated
WO91/0~536 PCT/US90/05679
17 `'; ~,2as~.73~
fatty acids is different than in healthy subjects. MMR
parameters for lipid methyl and methylene groups are
determined and compared against a corresponding value for a
healthy person.
In all embodiments described herein the lipoproteins
may peroxidized in a sample of whole blood, plasma or serum.
- The apparatus of the invention is illustrated in FIG. 9
where the lipoproteins are peroxidized directly by the
method of the invention. A peroxidizing module or chamber
contains an immobilized enzyme 52, such as peroxidase or
lipoxidase, and an inlet 54 from a pump 55 which can very
slowly and precisely introduce a flow of hydrogen peroxide
into the module. Blood form a patient's artery is introduced
through inlet 53 into the peroxidizing module. Blood
containing peroxidized lipoproteins is returned to the
patient's vein by outlet Sl.
FIG. ll depicts another embodiment of this invention
wherein a blood 56 taken from patient 57 is stored in a
container 58. A peroxide 59 is added to a separate container
60 and stored there. The peroxide 59 is then added to
container 58 thereby causing oxidation of the lipoproteins
in the blood. The oxidized lipoprotein-containing blood 6l
is then reintroduced to the patient 50.
.
FIG. 12 illustrates yet another embodiment of the
.
. .
- .
.
WO9l/05536 PCT/US90/05679
, ~
i2 0 6~ 36 4 18
present invention. Heparinized blood 66 is added to the
bottom of a container 68 which holds an immobilized enzyme,
such as horseradish peroxidase coated ~eads 70. Hydrogen
peroxide 72 is introduced to the bottom of the container
resulting in the formation of oxidized lipoproteins 74 in
the blood which exits from the top of the container. The
oxidized lipoprotein-containing blood 74 is stored in
container 75 and then introduced to the patient 76 when
treatment of a disease state is indicated.
There is an additional method of oxidating
lipoproteins, in accordance with the present invention,
which consists of adding a photosensitizer to a lipoprotein
solution and then irradiating the solution with ultraviolet
li~ht. It has been discovered, that by irradiating -
8-methoxypsoralen, radical intermediates may be produced
resulting in generation of hydroxyl radicals. In turn,
these radicals cause oxidation of the lipoproteins which
have a therapeutic effect on the diseased cells. Thus,
blood is removed from the patient, 8-methoxypsoralen added
to the blood and the sample then irradiated with ultraviolet
light thereby causing oxidation of the lipoproteins in the
blood.
Hematoporphyrins, used in the same manner as the
8-methoxypsoralen described above, are also known to cause
death of cancer cells in irradiated blood. In accordance
with the present invention, 8-methoxypsoralen as well as
,
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., - . . .
W09t/0~36 PCTt~S90/05679
19 2Q~7~6~
hematoporphyrins are believed to ef~ect peroxidation of
lipoproteins.
There is yet another method for effecting oxidation of
lipoproteins according to the present invention. This
method consists of the addition of a chemotherapeutic agent
to a lipoprotein-containing solution.
Adriamycin (Ad) and other molecules of its class have
long been used as chemotherapeutic agents effective against
certain types of cancer. They also have been suspected of
generating free-radicals. The mechanism for such action is
unknown, but two possible mechanisms are shown below:
1) AdH2 + ~~~> AdH ~ +
AdH I 0~+ ---> Ad + O +
20 + 2~ ---> H O2 +
2~2 enzyme > 20~ 2
2) AdH2 + 2 ~~~> AdH + o~
AdH_ O 3+ > Ad + O + H
20 + + 2~e ---> 2Fe2+ 2+ 20
2Fe2 + 2H2o ---> 2Fe3 + 20~ + 20H
Other metal ions can substitute for Pe3 . In addition,
mltomycin-D which is a molecule in the same class, can-be
used interchangeably with adriamycin as a chemotherapeutic
agent. Thus, in accordance with the present invention, it
is believed that adriamycin a~d mitomycin-D generate free
radicals which in turn cause oxidation of lipoproteins.
In a separate patent application, filed on even date
with the present application, Eric T. Fossel teaches that
- . . -, . ~ . -
. . . .
'' '
;, . , :` , ': ~' .
W O 91/05536 PCr/US90/05679
20673S4 20
oxidized lipoproteins may be helpful in fightingdisease states, such as cancer, malaria and viral infections
such as acquired immunode~iciency syndrome. These disease
states are characterized by diseased cells with an increased
number of lipoprotein receptors or an enhanced ability to
take up lipoproteins. When low density lipoproteins are
oxidized, they have a cytotoxic effect which preferentially
kills diseased cells which have an enhanced ability to
take-up lipoproteins. As part of its response to disease
such as cancer, a human host oxidizes lipoproteins
circulating in the blood. The diseased cells have been found
to be more susceptible than healthy cells to the cytotoxic
ef~ect of oxidized lipoproteins. Thus, therapy with
oxidized lipoproteins is believed to have the effect of
killing the diseased cells.
FIG. 10A illustrates how the cytotoxicity of
peroxidized low density lipoproteins helps fight cancer in
humans. In nature, a cancer cell 30 is sensèd by a
macrophage 32 which secretes tumor necrosis factor (TNF) 34.
The TNF 34 induces polymorphonuclear neutr~phils (PMN) 31 to
undergo a res~iratory burst to release superoxide 2 The
superoxide causes the formation of hydroxyl free-radicals,
OH which in turn oxidize low density lipoproteins 33;to
peroxidized low density lipoproteins (p-LDL) 35 while being
converted lnto hydrox~de ions, OH. The p-LDL 35 then exert
their cytotoxic effect on malignant cells 36 leading to cell
death. The malignant cells 36 killed by p~LDL 35 may the
. , ~ , :.: , ,. .. , . .. ., ., ., ::~ . ,. ,: ,, ., , : , ,
WO9lt05536 PCT/US90/05679
21 20~73S~
same or different than the originally sensed tumor cell
30.
FIG. 10B illustrates the method o~ the present the
invention. Low density lipoproteins 33 will be converted
directly to peroxidized low density lipoprotein (p-LDL) by
exposure to a peroxidizing agent 40. In one embodiment, the
agent 40 is ditertiarybutyl peroxide. In another
embodiment, the agent 40 is peroxidase together with a
peroxide. The malignant cell 36 is killed by exposure to
p-LDL.
The present invention is further illustrated by the
following non-limiking examples.
Exam~le 1
An oxidized low density lipoprotein composition is
prepared from low density lipoproteins obtained from Sigma
Chemical, catalog no. L2139 or prepared by standard methods
from fresh human or animal plasma. Lindren FT, Silvers A,
Jutagir R, Layshot L, Bradley DD. Li~ids 1977i 12:278-282
and Lindren FT, Adamson GL, Jensen LC, Wood PD. LiDids 1975;
10:750-756. Oxidation of the low density lipoprotein is
carried out usin~ either soluble horsexadish peroxidase
(Enzyme Commlssion Classification No. 1.11.1.7) or
immobilized horseradish peroxidase as a catalyst. The
immobilized enzyme has the advantage that it can be removed
WO91/05536 PCT/US90/05679
20~73~ 22
from the solution of oxidized low density lipoprotein
before use. Each ml of low density lipoprotein solution
(most preferably 5 mg. protein per ml) is diluted with an
equal volume of Dulbecco's phosphate buffered saline and the
peroxidase of either form is added to a level of
approximately 800-1000 units per ml of solution. Following
this, hydrogen peroxide, H2O2, most preferably 0.1 ml of 3%
hydrogen peroxide, is added per ml of low density
lipoprotein solution. The solution is maintained at room
temperature and 0.1 ml of peroxide solution per ml low
density lipoprotein solution is added each hour for two
hours. The peroxidation level is measured by the ratio of
the intensity of the resonances at 128 and 130 ppm in the
solution's carbon-13 NMR spectrum. A lower ratio indicates
a reduction of the amount of polyunsaturated fatty acid side
chains in the lipoprotein lipids. The 128/130 ppm ratio is
typically greater than 0.9 before peroxidation and between
0.7 and 0.85 after peroxidation.
Exam~le 2
.
Peroxidized low density lipoproteins are prepared by
treating human low density lipoprotein, most preferably 5mg
of protein/ml, (as described in Example 1) with horseradish
peroxidase Type II, most preferably 2 mg/ml. This is
followed by the addition of approximately 70 to 200 ~ liters
of hydrogen peroxide, most preferably 3% hydrogen peroxide
in one or ~wo equal ali~uots, the second addition being made
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. : . ., , . :............................ . .
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WO91/05536 PCT/US90~0567
23 2~ S 7g~4
several hours after the ~irst addition. The
peroxidation level is measured by the ratio of the intensity
of the resonances at 128 and 130 ppm in the solutionls
carbon-13 NMR spectrum as described in Example 1.
This technique can be employed with any
lipoprotein. It is particularlY desirable to peroxidize low
density lipoproteins.
FIG. 7 shows the olefinic region of a spectrum
from a normal plasma sample. The ratio of the peak at
128-129 to the peak at 130-131 is near one. FIGS. 8A & B
show the olefinic region of 125.8 MHz proton decoupled C-13
spectra from normal human plasma and the same plasma
following the addition of peroxidase, most preferably 2mg/ml
and 3 aliquots of 3% hydrogen peroxide (100 ~ l/ml) at
hourly intervals. The 128J130 ratio was substantially
decreased following treatment of the plasma, indicating that
peroxidation has occurred.
Exam~le 3
Oxidized lipoproteins were prepared by reacting
8-methoxypsoralen was reacted with LDL. LDL was isolated
from fresh human plasma and then dial~lzed overnight to
remove EDTA. Lipid concentration was monitored using C-13
NMR spectroscopy and the LDL preparation was then stored at
4 C and used within two weeks. The photosensitized ~ -
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WOgl/0~36 PCT/US90/05679
2 ~ 67 3 6~ 24
.: . .
reactions were carried out using 540 ~ l (4-5 mg protein/ml)
and 60 ~l of 8-methoxypsoralen stock solution. The final
concentration of 8-methoxypsoralen was 200 ng/ml. The
reaction was allowed to equilibrate for 30 minutes with
stirring before irradiation at 27-30 C. Six Sylvania
FR15T12 P WA lamps irradiated the samples for up to six
hours and any reduction in volume was replenished every 30
minutes during irradiation. C-13 NMR spectra of all samples
were obtained before and after the addition of
8-methoxypsoralen and the irradiation. These spectra were
used to monitor the photosensitized reactions of the lipids
of LDL and characterize the chemical changes induced by
these reactions.
The control plasma showed no change in the 128/130
ppm C-13 ratio following 30 minutes of ultraviolet A
irradiation (the ratio was 0.98 before and 0.97 afterwards).
However, the 8-methoxypsoralen containing plasma showed a
reduction of the ratio from a pre-irradiation va~ue of 0.98
to 0.77 following 30 minutes of light treatment. Thus, the
results show that free-radical induced oxidation appears to
occur and may be responsible for the therapeutic effect
observed by Edelson et al.
Exam~le 4
Oxidized lipoproteins were prepared by the addition of
adriamycin to six aliquots of normal plasma and bubbling
. ' ., ' ' . , ' '` ' . , .. . ' .
W O 91/05536 P ~ /US90/05679
2~3~4
intermittently with 95% 2 5% CO2. An equal amount of
adriamycin was also added to six additional aliquots of
plasma and bubbled similarlY with 95% N2: 5% CO2. The ratio
of the 128/130 ppm resonances changed as shown in the Table
below. The lower ratio, as compared with the control sample,
indicates that peroxidation has occurred. Adriamycin
mediated lipid peroxidation occurred in the presence of
oxygen but not in its absence.
128/130 ~m
intensitY
Control plasma 0.96 +/- 0.06 (n = 6)
Control plasma + Adriamycin + 2 .72 +/- 0.09 (n = 6)
Control plasma + Adriamycin + N2 0 94 +/- 0.05 (n = 6)
Exam~le S
Ditertiarybutyl peroxide is administered to a person by
i.v. (intravenous) injection. The level of oxidized
lipoprotein is monitored by nuclear magnetic resonance and
the ditertiarybutyl peroxide dose adjusted accordingly.
The patient's blood oxygen supply may also be augmented
by inhalation of elemental oxygen or by i.v. injection of
perfluorocarbon fluosal before admi~istering the
ditertiarybutyl peroxide.
- . , .. ~ .
-
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W09t/05536 PCT/US-0/05679
2`~673~4 26
Additionally, the patient's supply of lipoproteins may
be augmented by intravenous in;ection of lipoproteins
enriched with triglycerides, phospholipids, or cholesterol
esters before administering the ditertiarybutyl peroxide.
The ditertiarybutyl peroxide of this procedure may be
replaced with any of the following in its proper dose:
riboflavin, peroxidase, lipoxidase, or other flavins,
peroxides, organic peroxides or oxidases.
Exam~le 6
An atrioventricular shunt or arterial bypass is
attached to a person. An extracorporeal peroxidizlng module
is attached to the AV shunt or arterial bypass. It has an
inlet fluid connection from a pump which introduces hydrogen
peroxide into the module which contains peroxidase or
lipoxidase which peroxidizes the plasma lipoproteins in the
presence of the hydrogen peroxide.
A supply of blood is secured. The blood suppl~ source
may be a donor, a blood bank, or any o~her blood supply
source.
: ~ .
The lipoproteins of the blood supply are then oxidized
by adding an oxidant to the blood, thus producing of
oxidized lipoproteins.
The oxygen available in the blood may be further
.
.. . ..
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,
WO91/05536 2 a~ r ~ ~S90/05679
,;, .....
27
increased by adding elemental oxygen or perfluorocarbon
fluosal to the blood.
The lipoprotein content of the blood can also be
augmented by adding lipoproteins enriched with
triglycerides, phospholipids, or cholesterol esters.
While the foregoing invention has been described with
reference to its preferred embodiments, various alterations
and modifications will occur to those skilled in the art.
All such alternations and modifications are intended to fall
within the scope of the appended claims.
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