U.S. patent application number 10/776797 was filed with the patent office on 2004-11-11 for gene-based lipid hydrolysis therapy for atherosclerosis and related diseases.
Invention is credited to Du, Hong, Grabowski, Gregory.
Application Number | 20040223960 10/776797 |
Document ID | / |
Family ID | 26876237 |
Filed Date | 2004-11-11 |
United States Patent
Application |
20040223960 |
Kind Code |
A1 |
Grabowski, Gregory ; et
al. |
November 11, 2004 |
Gene-based lipid hydrolysis therapy for atherosclerosis and related
diseases
Abstract
The present invention comprises a method to diminish and/or
eliminate atherosclerotic plaques, in mammals, through direct and
indirect treatment of these plaques, in situ, using suitable
substances which are capable of lipid removal, primarily through
hydrolysis, either by a catalytic or stoichiometric process,
wherein the substance targets receptors in and/or on the cell which
lead to uptake into the lysosome. Such substances used to diminish
and/or eliminate atherosclerotic plaques are generally comprised of
lipid hydrolyzing proteins and/or polypeptides.
Inventors: |
Grabowski, Gregory;
(Cincinnati, OH) ; Du, Hong; (Cincinnati,
OH) |
Correspondence
Address: |
FROST BROWN TODD LLC
2200 PNC Center
201 E. Fifth Street
Cincinnati
OH
45202-4182
US
|
Family ID: |
26876237 |
Appl. No.: |
10/776797 |
Filed: |
February 11, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10776797 |
Feb 11, 2004 |
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09775517 |
Feb 2, 2001 |
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60180362 |
Feb 4, 2000 |
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Current U.S.
Class: |
424/94.6 |
Current CPC
Class: |
C12N 9/20 20130101; A23L
33/18 20160801; A61P 9/00 20180101; A61K 9/0019 20130101; A61P 3/06
20180101; C12Y 301/01013 20130101; A61P 43/00 20180101; A61K 48/005
20130101; C12N 2799/04 20130101; A61P 9/10 20180101; A61K 38/465
20130101; A61K 38/40 20130101; C12N 7/00 20130101; A61K 38/18
20130101; A61K 48/00 20130101; C12N 2799/021 20130101 |
Class at
Publication: |
424/094.6 |
International
Class: |
A61K 038/46 |
Claims
1-36 (cancelled)
37) A composition comprising a safe and effective amount of a lipid
hydrolyzing protein or polypeptide and a pharmaceutically
acceptable carrier.
38) The composition of claim 37 wherein the lipid hydrolyzing
protein or polypeptide is the protein lysosomal acid lipase.
39) The composition of claim 37 wherein the lipid hydrolyzing
protein or polypeptide is a protein showing at least 85% sequence
homology to lysosomal acid lipase.
40) The composition of claim 37 wherein said lipid hydrolyzing
protein or polypeptide is a polypeptide possessing similar
biological activity as lysosomal acid lipase.
41) The composition of claim 37 wherein said lipid hydrolyzing
protein or polypeptide is a protein having a Ser.sup.153
residue.
42) The composition of claim 37 wherein said lipid hydrolyzing
protein or polypeptide is a polymorphic variant protein of
lysosomal acid lipase with substitution of amino acid Pro(-6) to
Thr and Gly2 to Arg.
43) The composition of claim 38 wherein the lysosomal acid lipase
has fewer than six N-linked acetylglycosylation residues.
44) The composition of claim 38 wherein the lysosomal acid lipase
has more than six N-linked acetylglycosylation residues.
45) The composition of claim 43 wherein the N-acetylglycosylation
residue is oligosaccharide-terminated.
46) The composition of claim 45 wherein the oligosaccharide
terminating residue is a mannose residue.
47) The composition of claim 44 wherein the N-acetylglycosylation
residue is oligosaccharide-terminated.
48) The composition of claim 47 wherein the oligosaccharide
terminating residue is a mannose residue.
49) A composition comprising a safe and effective amount of
lysosomal acid lipase in a pharmaceutically acceptable carrier.
50) A composition comprising a safe and effective amount of a lipid
hydrolyzing protein showing at least 85% sequence homology to
lysosomal acid lipase in a pharmaceutically acceptable carrier.
51) A method for providing biologically active lipid hydrolyzing
protein or polypeptide, or mixtures thereof, to cells of a mammal
having deficiency in biologically active lipid hydrolyzing protein
or polypeptide, said method comprising administration into cells a
vector comprising and expressing a DNA sequence encoding
biologically active lipid hydrolyzing protein or polypeptide, and
expressing the DNA sequence in said cells to produce biologically
active lipid hydrolyzing protein or polypeptide.
52) The method of claim 51 wherein the cells harboring the vector
secrete the biologically active lipid hydrolyzing protein or
polypeptide which is taken up by other cells deficient in the lipid
hydrolyzing protein or polypeptide.
53) The method of claim 51 wherein the biologically active human
lipid hydrolyzing protein or polypeptide is lysosomal acid
lipase.
54) The method of claim 51 wherein the biologically active human
lipid hydrolyzing protein or polypeptide is a protein having at
least 85% sequence homology to lysosomal acid lipase.
55) The method of claim 51 wherein the biologically active human
lipid hydrolyzing protein or polypeptide is a polymorphic variant
protein of lysosomal acid lipase with substitution of amino acid
Pro(-6) to Thr and Gly2 to Arg.
56) A method for providing biologically active lysosomal acid
lipase to cells of a mammal having deficiency in biologically
active lysosomal acid lipase, said method comprising administration
into cells a vector comprising and expressing a DNA sequence
encoding biologically active lysosomal acid lipase and expressing
the DNA sequence in said cells to produce biologically active
lysosomal acid lipase.
57) The method of claim 56 wherein the cells harboring the vector
secrete biologically active lysosomal acid lipase which is taken up
by other cells deficient in lysosomal acid lipase.
58) The method of claim 56 wherein the vector is a viral
vector.
59) The method of claim 58 wherein the viral vector is selected
from the group consisting of a lentivirus, adenovirus,
adeno-associated virus and virus-like vectors.
60) The method of claim 56 wherein the vector is a plasmid.
61) The method of claim 56 wherein the vector is a lipid
vesicle.
62) A method for providing biologically active lysosomal acid
lipase to cells of a mammal with atherosclerosis, comprising
administration into the cells of said mammal an amount of a vector
comprising and expressing a DNA sequence encoding lysosomal acid
lipase and which is effective to transfect and sustain expression
of biologically active lysosomal acid lipase in cells deficient
therein.
63) The method of claim 62 wherein the expressed lysosomal acid
lipase is secreted from the infected cells and is taken up by other
cells deficient therein.
64) A method for treatment of Wolman's Disease in a mammal
comprising administering to said mammal a safe and effective amount
of lysosomal acid lipase sufficient to treat said condition.
65) A method for treatment of Cholesteryl Ester Storage Disease in
a mammal comprising administering to said mammal a safe and
effective amount of lysosomal acid lipase sufficient to treat said
condition.
66-68 (cancelled)
Description
[0001] This application is based on and claims priority from U.S.
Provisional Patent Application Ser. No. 60/180,362, Gregory A.
Grabowski
[0002] and Hong Du, filed Feb. 4, 2000.
FIELD OF INVENTION
[0003] The present invention relates to the use of lipid dissolving
substances for the treatment and prevention of coronary artery
disease. More specifically, this invention relates to the use of
lipid hydrolyzing proteins and/or polypeptides, such as lysosomal
acid lipase (LAL), for the treatment and prevention of
atherosclerosis in mammals.
BACKGROUND
[0004] The increasing number of patients suffering from
atherosclerosis continues to drive research into cholesterol and
triglyceride metabolism. Through a large number of investigations,
the essentials of the control of cholesterol metabolism have been
elucidated in the past two decades (see FIG. 1). The central system
for the control of cholesterol metabolism requires two sets of
separable pathways: 1) the endogenous pathway and 2) the exogenous
cholesterol-entry pathways. Both of sets of pathways are modulated
by the protein lysosomal acid lipase (LAL) [1]. In the former, the
cell senses the need for endogenous cholesterol synthesis via the
release of transcription factors, Sterol Regulatory Element Binding
Proteins (SREBP 1 and 2), whose precursors are bound to the nuclear
membrane and endoplasmic reticulum. SREBPs up-regulate HMG-CoA
reductase and other enzymes in the endogenous synthesis pathways
[2-5]. This upregulation is derived from the cell's biochemical
feedback mechanism sensing a low level of free cholesterol in the
surrounding media and/or plasma that is derived from the receptor
mediated endocytosis pathway; i.e., the exogenous pathway [6]. Low
density lipoprotein receptors (LDLR) and other plasma membrane
receptors participate in this uptake process. These LDLR-delivered
and other lipoprotein associated lipids are presented to the
lysosome for degradation by LAL. Once a deficient exogenous
cholesterol supply is sensed, SREBP 1 and 2 stimulate the
transcription of a cascade of enzymes leading to the production of
free intracellular cholesterol and fatty acids [7-10]. The cell
then senses the adequacy of free cholesterol levels and, once
exceeded, ACAT (acyl CoA: cholesterol acyltransferase) is directly
activated by free cholesterol and ACAT synthesis is up regulated.
The net effect is to remove free cholesterol by esterification to a
cytoplasmic storage pool of cholesteryl esters that is not
contained within membranes, i.e., non-lysosomal, and to remove free
cholesterol and cholesteryl esters from the cells. Once the cell
senses that sufficient free cholesterol is available, a steady
state pool of free cholesterol is maintained [11].
[0005] Both SREBP 1 and 2 are transcription factors that bind to
Sterol Regulatory Elements (SREs) in the promoter regions of key
genes in cholesterol and fatty acid synthesis. The SREBPs are
activated by a two step proteolytic process that is mediated by
proteases that are activated by free cholesterol sensing elements
in the plasma membrane and, potentially, other components of the
cell [12, 13]. These proteases cleave the endoplasmic recticulum
(ER) resident SREBPs and release their active components which are
then transported to the nucleus. SREBP 2 has a single transcript
whereas the SREBP-1 gene produces two transcripts and proteins,
SREBP-1a and SREBP-1c. These alternative forms of SREBP 1 arise
from the use of transcription start sites resident in alternative
first exons that are then spliced into a common second exon. In
humans, the mRNAs for SREBP-1a/-1c also display alternative
splicing at the 3' end that leads to proteins that differ by 113
amino acids at the C-terminus [14, 15]. All three SREBP members
share the same structural domains indicating their common function
[16]. These domains include: 1) the NH.sub.2-terminal segment of
480 amino acids is a basic helix-loop-helix-leucine zipper-"like"
transcription activator, 2) the middle segment of 80 amino acids
comprises two membrane spanning sequences, and 3) the
carboxy-terminal half of 590 amino acids that functions as a
regulatory domain [17].
[0006] There are at least two pathways for the entrance of external
cholesterol into monocyte/macrophage derived cells [18]:1) the ldlr
and ldlr-related protein systems [19]; and 2) the scavenger
receptor system (e.g., SRA, SR-B and CD36) for lipoprotein bound
cholesteryl esters (CE's) [20-24]. The SR-B1 pathway delivers
cholesteryl esters into the cell via transfer of cholesteryl esters
through SR-B 1 without uptake of HDL [25, 26].
[0007] In the LDL-CE (cholesteryl ester) or -TG (triglyceride)
pathway, the complexes are taken up into cells following
receptor-mediated recognition. The endosomal pathway delivers these
lipids to the lysosomes after uncoupling the LDL-lipid complexes
from the receptor in the late endosomal acidified compartment. Once
the LDL-lipid particle is delivered to the lysosome, the lipids are
liberated, possible after degradation of the LDL particle, via
proteolysis or by simultaneous attack through proteolysis and by
LAL [27]. This derived free cholesterol is then transported out of
the lysosome into the cytosol by one or more proteins resident in,
or at, the lysosomal membrane. Once it exits the lysosome, free
cholesterol moves to the inner surface of the plasma membrane and
directly to the endoplasmic reticulum. Free cholesterol from the
inner surface of the plasma membrane is then transported to the
endoplasmic reticulum and participates in the feedback control of
the endogenous synthetic pathway. Thus, from this simplified
overview of cholesterol and triglyceride metabolism in cells, it is
clear that LAL occupies a central position in the control of
endogenous cholesterol synthesis since, without its activity,
neither free cholesterol nor free fatty acids (FFA) derived from
the LDL pathway can be liberated from the lysosome to control these
critical pathways.
[0008] The importance of LAL in cholesterol and triglyceride
metabolism is underscored by the human phenotypes resulting from
inherited deficiencies of LAL. These two rare diseases, Wolman
Disease and Cholesteryl Ester Storage Disease, are early and late
onset diseases, respectively [28]. Wolman disease results in the
massive accumulation of cholesteryl esters and triglycerides in
lysosomes of a variety of tissues and cells including those of the
liver (hepatocytes and Kupffer cells), spleen, adrenal gland and
epithelium of the small intestine. This leads to a severe phenotype
characterized by hepatosplenomegaly, adrenal calcification, and a
thickened and dilated small intestine. In comparison, cholesteryl
ester storage disease is a much more heterogeneous disease with
onset from early childhood to late adolescence, and even adulthood
with isolated hepatomegaly and/or progressive cirrhosis and
primarily storage of cholesteryl esters.
[0009] The inventor has discovered that additional circumstantial
evidence has implicated lower LAL activities in monocytes and/or
plaques from patients with atherosclerosis or carotid artery
atheromata. This evidence indicates that polymorphic variants could
lead to differential activity of LAL in various tissues and may
predispose to, or be an additional risk factor in, the development
of atherosclerotic disease in humans [29]. In accordance with this
invention, this suggests that supplementation of LAL activity in
cells of pathologic involvement in athero-/arterio-sclerosis may
provide a means to diminish the accumulated, pathologic cholesteryl
esters and triglycerides that are causally related to these
diseases.
SUMMARY OF THE INVENTION
[0010] As described herein, the present invention comprises a
method to diminish and/or eliminate atherosclerotic plaques in
mammals, through direct and indirect treatment of these plaques, in
situ, using proteins and/or polypeptides. These proteins and/or
polypeptides are capable of lipid removal, primarily through
hydrolysis, either by a catalytic or stoichiometric process,
wherein the lipid hydrolyzing protein or polypeptide targets
receptors in and/or on the cell leading to uptake into the
lysosome. Receptor sites are selected from the group consisting of
oligosaccharide recognition receptors and peptide sequence
recognition receptors.
[0011] Generally, compositions used for practicing this invention
include lipid hydrolyzing proteins or polypeptides, and in
particular, the protein lysosomal acid lipase (LAL). However, other
lipid hydrolyzing proteins or polypeptides may also be used, such
as proteins which show at least 85% sequence homology to lysosomal
acid lipase or proteins having a Ser.sup.153 residue. Other
proteins include polymorphic variants of lysosomal acid lipase with
substitution of amino acid Pro(-6) to Thr and Gly2 to Arg and also
polypeptides showing similar biological activity as lysosomal acid
lipase.
[0012] Exogenously produced lipid hydrolyzing proteins or
polypeptides, contained in a pharmaceutically acceptable carrier,
may be administered either orally, parenterally, by injection,
intravenous infusion, inhalation, controlled dosage release or by
intraperitoneal administration in order to diminish and/or
eliminate atherosclerotic plaques. The preferred method of
administration is by intravenous infusion.
[0013] Endogenously produced lipid hydrolyzing proteins and/or
polypeptides may also be used to diminish and/or eliminate
atherosclerotic plaques. Generally, such a method involves
providing a biologically active human lipid hydrolyzing protein or
polypeptide, such as human lysosomal acid lipase, to cells of an
individual having a deficiency in biologically active human lipid
hydrolyzing protein(s) or polypeptide(s). This is accomplished by
in vivo administration into cells competent for the production of
biologically active human lipid hydrolyzing protein or polypeptide,
a vector comprising and expressing a DNA sequence encoding
biologically active human lipid hydrolyzing protein or polypeptide.
The vector used may be a viral vector, including but not limited to
a lentivirus, adenovirus, adeno-associated virus and virus-like
vectors, a plasmid, or a lipid vesicle. The vector is taken up by
the cells competent for the production of biologically active human
lipid hydrolyzing protein or polypeptide. The DNA sequence is
expressed and the biologically active human lipid hydrolyzing
protein or polypeptide is produced. Additionally, the cells
harboring this vector will secrete this biologically active lipid
hydrolyzing protein or polypeptide which is then subsequently taken
up by other cells deficient in the lipid hydrolyzing protein or
polypeptide.
[0014] Other proteins and/or polypeptides which may be used for
endogenous treatment of atherosclerotic plaques includes
biologically active proteins having at least 85% sequence homology
to lysosomal acid lipase, polymorphic variant proteins of lysosomal
acid lipase with substitution of amino acid Pro(-6) to Thr and Gly2
to Arg and polypeptides showing similar biological activity to
lysosomal acid lipase.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1: Schematic of a mammalian cell to illustrate the
pathways for cholesterol and fatty acid incorporation into cellular
metabolism. Two pathways are illustrated: 1) the endogenous
synthesis of cholesterol and fatty acids controlled by the SREBP 1
and 2 systems that sense the level of extralysosomal cellular
cholesterol as modulated by LAL cleavage of cholesteryl esters and
triglycerides in the lysosomes; 2) the exogenous pathway whereby
cholesteryl esters and triglycerides enter the cell via receptor
mediated endocytosis (shown as LDL-CE as an example) for delivery
to the lysosomes inside of the cells. The LDL receptor and several
other scavenger receptors participate in this pathway. LAL controls
the egress of cholesterol and fatty acids from the lysosomes that
enter the cell via this pathway. The liberation of free cholesterol
and/or fatty acids by LAL or other such therapeutic compounds leads
to a direct effect to reduce cholesterol and FFA synthesis in the
cell via the SREBP sensing systems. Reductions in cellular
cholesterol and/or FFA can be achieved by this direct effect and/or
by removal of the free cholesterol and/or FFA from the cell by
transport of cholesterol across the plasma membrane and out of the
cell.
[0016] The abbreviations for cellular components are as follows:
PM=Plasma membrane, ER=endoplasmic reticulum, TGN=trans-Golgi
network, MVB=multivesicular body, EN=endosome, FC=free cholesterol,
FFA free fatty acid, CYTO CE=re-esterified or esterified
non-lysosomal cholesteryl ester (CE), NPC1=site of the Niemann-Pick
C1 defect, LAL=lysosomal acid lipase.
[0017] FIG. 2: Typical gross pathology of LAL untreated (LC2) and
treated (LA2 and LA4) lal-/- mice: [Top] Ventral views showing the
yellow fat-infiltrated lover in a typical (LC2) untreated lal-/-
mouse. In treated (LA2 and LA4) lal-/- mice, the livers had
essentially normal color. [Middle] Gross appearance of liver (top),
spleen (middle) and kidney (bottom) from LC2, LA4 and LA2 mice. The
untreated mouse spleen is lighter than that from the treated mice
spleens. [Bottom] Gross appearance of the small intestine from
untreated LC2 and treated LA4 and LA2 mice. The small intestine of
untreated mouse (LC2) gives a lighter appearance, indicating
build-up of cholesterol and triglycerides. This is in contrast to
the darker intestines shown for the treated mice (LA4 and LA2).
[0018] FIG. 3: Light microscopy of the liver, spleen and small
intestine from LAL untreated (LC2) and treated (LA2) lal-/- mice. H
& E stained sections from liver (3A and 3B), spleen (3E and
3F). Stained frozen sections from liver (3C and 3D). A, C, E,
(left) are from untreated lal-/- mice. B, D, and F (right) are from
LAL treated mice. Treated mice had substantially diminished
macrophage storage cell numbers compared to those in untreated
mice. The staining indicates large accumulations of neutral fat in
livers from untreated mice and their large decrease to near absence
in liver.
[0019] FIG. 4: Representative sections from the aortic valve of
ldlr-/- mice with or without LAL treatment stained with H & E.
(A nd B) Typical foamy cell-rich fatty streaks in 3.5 month old
ldlr-/- mice on HFCD for 2 months. The asterisk indicates a
necrotic zone next to disrupted medial layer. The arrows point to
cholesterol clefts/crystals. The arrow on the right (cholesterol
clefts/crystals) show a coronary artery near the ostium. (C)
Reduced foamy cells in the fatty streaks of the aortic valve of the
LAL treated mice. This was from the most involved LAL treated
mouse. (D) A typical example (3/5) of the normal aortic valves from
LAL treated mice.
DETAILED DESCRIPTION OF THE INVENTION
[0020] Definitions
[0021] The terms "amino acid" or "amino acid sequence," as used
herein, refer to an oligopeptide, peptide, polypeptide, or protein
sequence, or a fragment of any of these, and to naturally occurring
or synthetic molecules. Where "amino acid sequence" is recited
herein to refer to an amino acid sequence of a naturally occurring
protein molecule, "amino acid sequence" and like terms are not
meant to limit the amino acid sequence to the complete native amino
acid sequence associated with the recited protein molecule.
[0022] As used herein, the term "exogenous lipid hydrolyzing
proteins or polypeptides" refers to those produced or manufactured
outside of the body and administered to the body; the term
"endogenous lipid hydrolyzing proteins or polypeptides" refers to
those produced or manufactured inside the body by some type of
device (biologic or other) for delivery to within or to other
organs in the body.
[0023] As used herein, the term "biologically active" refers to a
protein having structural, regulatory, or biochemical functions of
a naturally occurring molecule.
[0024] The term "derivative," as used herein, refers to the
chemical modification of a polypeptide sequence, or a
polynucleotide sequence. Chemical modifications of a polynucleotide
sequence can include, for example, replacement of hydrogen by an
alkyl, acyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains at least one biological function of the
natural molecule. A derivative polypeptide is one modified, for
instance by glycosylation, or any other process which retains at
least one biological function of the polypeptide from which it was
derived.
[0025] The words "insertion" or "addition," as used herein, refer
to changes in an amino acid or nucleotide sequence resulting in the
addition of one or more amino acid residues or nucleotides,
respectively, to the sequence found in the naturally occurring
molecule.
[0026] The phrases "nucleic acid" or "nucleic acid sequence," as
used herein, refer to a nucleotide, oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer
to DNA or RNA of genomic or synthetic origin which may be
single-stranded or double-stranded and may represent the sense or
the antisense strand, or to any DNA-like or RNA-like material. In
this context, "fragments" refers to those nucleic acid sequences
which, when translated, would produce polypeptides retaining some
functional characteristic, e.g., lipase activity, or structural
domain characteristic, of the full-length polypeptide.
[0027] The phrases "percent identity" or "percent homology" refers
to the percentage of sequence similarity found in homologues of a
particular amino acid or nucleic acid sequence when comparing two
or more of the amino acid or nucleic acid sequences.
[0028] The term "atherosclerosis" refers to the pathologic
processes that leads to abnormal accumulation of cholesterol and
cholesteryl esters and related lipids in macrophages, smooth muscle
cell and other types of cells leading to narrowing and/or occlusion
of one or several arteries and arterioles of the body and bodily
organs, including but not limited to, the coronary arteries, aorta,
renal arteries, corotid arteries, and arteries supplying blood to
the limbs and central nervous system. The associated inflammatory
reactions and mediators of this pathologic process also are
included in this definition.
[0029] The term "atherosclerotic plaque" refers to the build up of
cholesterol and triglycerides due to atherosclerosis.
[0030] Discussion
[0031] The consequences of atherosclerosis are a leading cause of
mortality and morbidity. Macrophages that accumulate cholesteryl
esters are known to be a major component contributing to the
build-up of atherosclerotic plaques in coronary arteries as well as
the carotid arteries, the aorta and other peripheral vessels
throughout the body. These cholesteryl esters are derived from the
circulation where they are carried on lipoproteins. The cholesteryl
esters enter cells via low-density lipoprotein receptors (LDL-R)
and other scavenger receptors for oxidized low-density lipoprotein
(LDL) particles. Once internalized, these particles and their
attached cholesteryl esters are delivered to the lysosome for
cleavage to cholesterol by lysosomal acid lipase (LAL).
[0032] Current therapeutic approaches for treating atherosclerosis
include dietary manipulation (low cholesterol diets) and exercise,
cholesterol synthesis inhibitors and surgical coronary artery
by-pass. However, dissatisfaction with the success of these
interventions provides the impetus for continued development of
new/alternative/adjunctive therapies for this major disease
group.
[0033] There are two primary methods employed for the treatment of
atherosclerosis and the dissolution of atherosclerotic plaque. The
first method of treatment is coronary by-pass surgery. This method
is used to treat patients with established, unstable angina and/or
progressive angina. The second method of treatment is chemical
inhibition of hepatic cholesterol synthesis using the class of
drugs termed "statins." This approach inhibits the synthesis of
cholesterol by inhibiting the action of the rate-limiting enzyme,
HMGCoA reductase, in the cholesterol synthetic pathway. Coronary
artery by-pass surgery is effective in diminishing angina attacks
of selected patients and the statins have been shown to
successfully lower plasma cholesterol and diminish the propensity
to develop atherosclerotic plaques. However, neither approach
offers the potential for direct dissolution of existing
atherosclerotic plaques.
[0034] LAL represents the major biochemical pathway of cholesteryl
ester entry into the body, and is subsequently used to modulate
cellular cholesterol biosynthesis. Once LAL liberates cholesterol
from cholesteryl esters, the free cholesterol exits the lysosome
and leads to the sterol regulatory element binding protein (SREBP)
mediated down regulation of cholesterol synthesis. The accumulation
of cholesteryl esters within the macrophages of atherosclerotic
plaques occurs in the presence of normal amounts of LAL. This fact
indicates that the delivery of cholesteryl esters to these cells
exceeds the capacity of normal amounts of LAL to catabolize the
delivered cholesteryl esters that initiate the development of
atherosclerotic plaques. This process disrupts normal cellular
metabolism for the regulation of endogenous cellular cholesterol
synthesis and leads to excess amounts of cholesterol and cellular
cholesteryl ester synthesis via the lack of down regulation of the
SREBP-mediated system of cholesterol synthesis and the acyl CoA:
cholesterol acyltransferase (ACAT) pathway for intracellular
cholesteryl ester synthesis [30].
[0035] Similar events occur in the liver, which is the major organ
in the body responsible for cholesterol biosynthesis and for
maintenance of cholesterol homeostasis. Delivery of LAL to
hepatocytes in excess of normal amounts enhances the egress of free
cholesterol from the lysosome (i.e., increases the flux of
cholesteryl esters through the lysosomal system) that is a major
pathway for the metabolism of such lipids delivered to hepatocytes
from the portal circulation and the diet. The result is an increase
in cholesterol liberated from lysosomes, which subsequently down
modulates hepatic cholesterol synthesis and its supply to the body.
This diminishes the load of cholesterol and cholesteryl esters to
peripheral sites thereby lowering the atherogenic potential.
[0036] The use of a suitable protein or polypeptide, such as LAL or
a homologue of LAL possessing similar biological activity, offers
an alternative means of therapy for atherosclerosis as well as
peripheral vascular disease. LAL functions by preventing the
progression or promoting the regression of atherosclerotic plaque
legions via two mechanisms: 1) by directly entering the lesional
foam cells and enzymatically dissolving the stored cholesteryl
esters as well as tri-, di-, and mono-acylglycerides; and 2) by
indirectly promoting lysosomal egress of free cholesterol and free
fatty acids that could modulate cellular (hepatic, macrophage and
other) lipid synthesis mediated by the SREBP or other pathways.
Patients who suffer from atherosclerosis have a tendency to have
decreased levels of LAL in the atheromatous plaques.
[0037] LAL, a member of the lipase family, is a 372 amino acid
glycoprotein that is trafficked to the lysosome via the mannose
receptor system [31-33]. The cDNA sequence which encodes LAL has
been previously reported [34]. This glycoprotein has six
glycosylation consensus sequences (Asn-X-Ser-/Thr) and three at
Asn.sup.15, Asn.sup.80 and Asn.sup.252 are conserved among members
of the lipase gene family. All members of the lipase gene family
have conserved GXSXG pentapeptide sequences that contain the active
site serine nucleophiles [35-37]. LAL has two such sequences at
residues 97-101 and 151-155 with potential serine nucleophiles at
residues 99 and 153, where a key nucleophile resides at the Ser 153
residue. LAL cleaves cholesteryl esters and triglycerides in vitro
using phospholipid/detergent systems. Ser.sup.153 has been defined
as a part of the Asp-Ser-His catalytic triad common to many
lipases.
[0038] Suitable lipid hydrolyzing substances for use in this
invention include, but are not limited to, glycoproteins such as
LAL, homologues of LAL, wherein the homologues possess at least 85%
sequence homology, due to degeneracy of the genetic code which
encodes for LAL, polypeptides possessing similar biological
activity to LAL and non-peptide derived substances. Also included
are lipid hydrolyzing proteins and polypeptides which contain the
catalytic lipase triad Asp-Ser-His, where the Ser is a Ser.sup.153
residue. Additional substances include polymorphic variants of LAL
in which two of the amino acids are replaced with different amino
acids. An example of such polymorphic variants are prepared by
cloning LAL from normal human liver cDNA library and changing two
nucleotides (C86 to A and G107 to A) which results in substitution
of amino acid Pro(-6) to Thr and Gly2 to Arg in LAL, yielding four
different polymorphic variants of LAL. Additional amino acid
sequences include those capable of lipid hydrolysis, either
catalytic or stoichiometric, wherein the residue 153 of the amino
acid chain is a serine residue.
[0039] Further LAL-derived proteins include those proteins having
the native LAL sequence, but which have more than six N-linked
acetylglycosylation residues or fewer than six N-linked
acetylglycosylation residues. Each glycosylation site has two
N-linked acetylglucosamine residues, which are
oligosaccharide-terminated, where the oligosaccharide-terminating
residue is preferably an .alpha.-mannose residue and where there
are at least three oligosaccharide-terminating residues at each
glycosylation site.
[0040] For the treatment of atherosclerosis, the lipid hydrolyzing
substance targets receptors which lead to uptake into the lysosome.
These receptors include but are not limited to the categories of
oligosaccharide recognition receptors, which includes the mannose
receptor, the mannose-6-phosphate receptor and the category of
peptide sequence recognition receptors, which includes CD 36 and
LDL receptors.
[0041] Methods of Treatment of Atherosclerosis Using Lipid
Hydrolyzing Amino Acid Sequences
[0042] LAL could be used in conjunction with statins to reduce the
level of artherosclerotic plaques. Additionally, LAL could also be
used in conjunction with by-pass surgery for some patients who
develop restenosis and/or to prevent redevelopment of plaques
following surgery. In addition, treatment with therapeutic agents,
such as LAL, can effect beneficial improvements in arteries and/or
arterioles that cannot be accessed by surgical or other such
invasive approaches. Additional advantages of LAL treatment may
include the elimination of the need for surgery in some patients
and supplying a natural product to patients without the attendant
or potential side effects of synthetic chemicals, as is the case
for the statin therapy approach.
[0043] LAL therapy can also be used for the treatment of two rare
human diseases, Wolman Disease and Cholesteryl Ester Storage
Disease. Both of these diseases are due to mutations at the LAL
locus. The former leads to death in the first year of life and the
latter is a prolonged disease with development of cirrhosis of the
liver in later life. Neither disease currently has therapy regimes
available.
[0044] Additional potential therapeutic roles for LAL treatment
include its use in the treatment of fatty liver of pregnancy,
unspecified fatty infiltration of the liver, peripheral
atherosclerotic disease due to secondary diseases such as diabetes
mellitus, carotid stenosis due to atherosclerosis, and similar
disease states.
[0045] The lipid hydrolyzing protein or polypeptide can be used
therapeutically either as an exogenous material or as an endogenous
material. Exogenous lipid hydrolyzing proteins or polypeptides are
those produced or manufactured outside of the body and administered
to the body. Endogenous lipid hydrolyzing proteins or polypeptides
are those produced or manufactured inside the body by some means
(biologic or other) for delivery to within or to other organs in
the body. LAL is present in body tissue. Patients who suffer from
atherosclerosis have a tendency to have decreased levels of LAL in
the atheromatous plaques. In order to achieve such desired results
for both direct and indirect treatment of the plaques, the lipid
hydrolyzing protein or polypeptide targets specific organs via
specific receptors. For example, LAL can target the mannose
receptor systems, or other oligosaccharide specific receptors and
enters macrophages, smooth muscle cells, endothelial cells and
hepatocytes.
[0046] Endogenous Therapy:
[0047] An indirect treatment of plaques involves supplying LAL to
the major organs of cholesterol biosynthesis, primarily the liver.
This leads to a greater net lysosomal throughput of cholesteryl
esters and delivery of free cholesterol to the cytoplasm, where
overall cholesterol synthesis would be diminished. It also results
in a reduction of the endogenous supply of cholesterol from the
liver to peripheral organs, i.e. macrophages in developed or
developing plaques.
[0048] The principles of gene therapy for the production of
therapeutic products within the body include the use of delivery
vehicles (termed vectors) that can be non-pathogenic viral
variants, lipid vesicles (liposomes), carbohydrate and/or other
chemical conjugates of nucleotide sequences encoding the
therapeutic protein or substance. These vectors are introduced into
the body's cells by physical (direct injection), chemical or
cellular receptor mediated uptake. Once within the cells, the
nucleotide sequences can be made to produce the therapeutic
substance within the cellular (episomal) or nuclear (nucleus)
environments. Episomes usually produce the desired product for
limited periods whereas nuclear incorporated nucleotide sequences
can produce the therapeutic product for extended periods including
permanently.
[0049] Such gene therapy approaches are used to produce therapeutic
products for local (i.e., within the cell or organ) or distant
beneficial effects. Both may provide decreases in pathologic
effects and may combine to produce additive and/or synergistic
therapy. For either effect, local or distant, the natural (termed
normal) or altered (mutated) nucleotide sequences may be needed to
enhance beneficial effects. The latter may be needed for targeted
delivery to the specific cellular type involved in the pathology of
the disease. For atherosclerosis distant delivery would be needed
to macrophages (foam cells), smooth muscle cells and other various
cell types within the pathologic lesions, known as atheromata.
Subcellular delivery to the lysosomes may also be necessary and
variants made available or produced for such an approach.
[0050] An approach for the use of lipid removal substances,
particularly lipid hydrolyzing proteins and polypeptides for the
treatment of atherosclerosis and removal of atherosclerotic
plaques, can be achieved by the gene therapy approaches discussed
above. Such approaches provide a source of a biologically active
human lipid hydrolyzing protein or polypeptide for delivery into
the body by biologic or other production systems. This method of
introduction can be achieved by internal or production sources
(biologic or other, gene therapy vectors, liposomes, gene
activation etc.) which lead to the production of biologically
active human lipid hydrolyzing proteins and polypeptides by certain
cells of the body. The source may provide for the local or distant
supply by, for example, direct effects within the cell or by
secretion out of the cells for delivery to other cells of the body,
like those in atheromatous plaques. This includes, but is not
limited to, somatic gene therapy approaches that would allow for
the synthesis and/or otherwise production of the therapeutic
substance in the body. In particular, nucleotide sequences encoding
the functional, lipid hydrolyzing, sequences of the lysosomal acid
lipase incorporated into conjugates, liposomes, viral (i.e.,
lentivirus, adenovirus, adeno-associated virus or other viruses or
such virus-like vectors) vectors for expression of the active
sequences for therapeutic effect. In addition, nucleotide sequences
encompassing the functional components of biologic and therapeutic
interest and residing in the body's cells could be made to produce,
express or otherwise make the requisite compound in therapeutic
amounts. The therapeutic lipid hydrolyzing protein or polypeptide,
thus produced in the body, would lead to a reduction or elimination
of the atheromatous plaques or other lesions of atherosclerotic
plaques.
[0051] Variants and homologous nucleotide or encoded sequences of
human lysosomal acid lipase incorporated for synthesis and/or
production of the active protein/peptide are transiently or
permanently integrated into cells for therapeutic production. The
normal, polymorphic variants, specifically mutated or modified
lysosomal acid lipase sequences may be expressed from the context
of the vectors incorporated into cells for normal and/or
specifically modified function to enhance or otherwise promote
therapeutic effects.
[0052] Such sequences can lead to the in vivo synthesis of the
desired biologically active human lysosomal acid lipase or other
therapeutic proteins within cells after incorporation into cells by
various routes as described above. Once within cells, the
synthesized biologically active human lysosomal acid lipase or
another therapeutic protein hydrolyzes cholesteryl esters and/or
triglycerides within the lysosomes following their targeted
delivery. The resulting release of free cholesterol from the
lysosomes leads to down regulation of the endogenous cholesterol
synthetic pathway via the SREBP controlled systems. Additionally,
human lysosomal acid lipase or other therapeutic human proteins or
polypeptides produced from incorporated nucleotide sequences are
secreted from cells, enter the circulatory system and are taken up
by distant cells via receptor mediated endocytosis or other such
lysosomal delivery systems to the lysosomes of pathologically
involved cells of the atheromatous plaques. Such plaques include
but are not limited to macrophages and smooth muscle cells.
Lysosomal liberation of free cholesterol within such cells has at
least two beneficial effects on atheromatous plaque reduction
and/or elimination: 1) free cholesterol exits from the lysosome and
participates in the SREBP mediated down regulation of endogenous
macrophage or other cell type cholesterol synthesis, and 2) free
cholesterol exits from the lysosome and exits the cell by reverse
cholesterol transport. Both effects are beneficial in reducing the
amount of accumulated cholesteryl esters within lysosomes of foam
cell macrophages and/or other cells of the atheromatous
lesions.
[0053] The gene vectors containing the requisite nucleotide
sequences or other components necessary for therapeutic expression
are introduced into the body's cells by several routes as described
above and also their direct introduction into atheromatous plaque
cells using delivery by angiographic device.
[0054] Endogenous therapy also contemplates the production of a
protein or polypeptide where the cell has been transformed with a
genetic sequence that turns on the naturally occurring gene
encoding the protein, i.e., endogenous gene-activation
techniques.
[0055] Exogenous Therapy:
[0056] A method for the direct treatment of atherosclerotic plaques
involves supplying LAL to the plaques and the macrophages, and
smooth muscles cells therein, so that the cholesteryl esters and/or
triglycerides, which are stored or accumulated within lysosomes of
these cells, are degraded and eliminated. This subsequently results
in the liberation of cholesterol from the lysosomes and a decrease
in endogenous cholesterol synthesis within the foam cells
(macrophages and smooth muscles cells). The net effect is to reduce
the amount of cholesterol accumulating directly in the target site
of pathology and to diminish the size of the plaques and other such
legions in situ.
[0057] It should be noted that the direct and indirect targeting of
the plaques are not mutually exclusive and may be synergistic with
both local and global effects on cholesterol homeostasis and the
diminution of atherogenic potential.
[0058] The lipid hydrolyzing proteins or polypeptides useful in the
present invention for exogenous therapy may be administered by any
suitable means. One skilled in the art will appreciate that many
suitable methods of administering the compound to a host in the
context of the present invention, in particular a mammal, are
available, and, although more than one route may be used to
administer a particular protein or polypeptide, a particular route
of administration may provide a more immediate and more effective
reaction than another route.
[0059] Formulations suitable for administration by inhalation
include aerosol formulations placed into pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen,
and the like. The active agent may be aerosolized with suitable
excipients. For inhalation administration, the composition can be
dissolved or dispersed in liquid form, such as in water or saline,
preferably at a concentration at which the composition is fully
solubilized and at which a suitable dose can be administered within
an inhalable volume.
[0060] Formulations suitable for oral administration include (a)
liquid solutions, such as an effective amount of the compound
dissolved in diluents, such as water or saline, (b) capsules,
sachets or tablets, each containing a predetermined amount of the
active ingredient, as solids or granules, (c) suspensions in an
appropriate liquid, and (d) suitable emulsions. Tablet forms may
include one or more of lactose, mannitol, corn starch, potato
starch, microcrystalline cellulose, acacia, gelatin, colloidal
silicon dioxide, croscarmellose sodium, talc, magnesium stearate,
stearic acid, and other excipients, colorants, diluents, buffering
agents, moistening agents, preservatives, flavoring agents, and
pharmacologically compatible carriers.
[0061] Formulations suitable for intravenous infusion and
intraperitoneal administration, for example, include aqueous and
nonaqueous, isotonic sterile injection solutions, which can contain
anti-oxidants, buffers, bacteriostats, and solutes that render the
formulation isotonic with the blood of the intended recipient, and
aqueous and nonaqueous sterile suspensions that can include
suspending agents, solubilizers, thickening agents, stabilizers,
and preservatives. The formulations can be presented in unit-dose
or multi-dose sealed containers, such as ampules and vials, and can
be stored in a freeze-dried (lyophilized) condition requiring only
the addition of the sterile liquid carriers for example, water, for
injections, immediately prior to use. Extemporaneous injection
solutions and suspensions can be prepared for sterile powders,
granules, and tablets of the kind previously described.
[0062] Parenteral administration, if used, could also be by
injection. Injectables can be prepared in conventional forms,
either as liquid solutions or suspensions, solid forms suitable for
solution or suspension in liquid prior to injection, or as
emulsions. A more recently revised approach for parenteral
administration involves use of a slow release or sustained release
system, such that a constant level of dosage is maintained. See,
e.g., U.S. Pat. No. 3,710,795, Higuchi, issued 1973, which is
incorporated by reference herein.
[0063] The appropriate dosage administered in any given case will,
of course, vary depending upon known factors, such as the
pharmacodynamic characteristics of the particular protein or
polypeptide and its mode and route of administration; the age,
general health, metabolism, weight of the recipient and other
factors which influence response to the compound; the nature and
extent of the atherosclerosis; the kind of concurrent treatment;
the frequency of treatment; and the effect desired.
[0064] A preferred method of treating mammals possessing
atherosclerotic plaque involves introduction of suitable lipid
hydrolyzing protein or polypeptide by intravenous infusion of a
safe and effective amount of a lipid hydrolyzing protein or
polypeptide, so as to cause the diminution and elimination of the
plaque. A safe and effective amount of the lipid hydrolyzing
protein or polypeptide is defined as an amount, which would cause a
decrease in the level of atherosclerotic plaques in a patient while
minimizing undesired side effects. An experienced practioner,
skilled in this invention would have knowledge of the appropriate
dosing ratios. The activity level of the lipid hydrolyzing protein
or polypeptide must also be considered in determining the number of
units to administer to achieve the desired effect. Thus, the
activity level of the lipid hydrolyzing protein or polypeptide
should be sufficient to cause a reduction in atherosclerotic
plaques within a reasonable dosage administered.
EXPERIMENTAL EXAMPLES
Study Design
[0065] This study was designed for age-matched cohorts of lysosomal
acid lipase deficient, lal-/- or low density lipoprotein receptor
deficient, ldlr-/-, mice as an open-label, controlled trial of
treated and untreated mice. A single dose of LAL was used in all
mice. All mice were sacrificed after 30 days of LAL administration.
LAL was given as an i.v. bolus via tail vein every third day for 30
days. The cohorts were divided into equal groups for injections on
alternate days. Injections were begun at 2.0 or 2.5 months of age
for the lal-/- or ldlr-/- mice, respectively. The overall study
design is presented in Table 1. The lal-/- mice received a regular
chow diet throughout the entire study period. LAL dosing was begun
at 2 months of age. The ldlr-/- mice were maintained on a regular
chow diet for 1.5 months and then placed on a high cholesterol diet
(7.5% fat; 1.25% cholesterol). The LAL dosing was started after the
ldlr-/- mice had been on the high fat/high cholesterol diet for 30
days; i.e., at 2.5 months of age. Doses of LAL in the treated
groups were 1.48 U (21 .mu.g; 70 .mu.l) LAL in 1.times.PBS with 2%
human serum albumin and 10 mM of dithiothreitol (DTT). The control
groups received 1.times.PBS with 2% HSA and 10 mM of DTT. The final
cohort was the lal-/-; ldlr-/- combined deficiency.
[0066] The mice avidly consumed the high fat/high cholesterol diet
and tolerated the injections well. All injections (325) were
successful with i.v. administration obtained for all. One ldlr-/-
mouse died just prior to initiating the injections. The high
mortality in the lal-/-; ldlr-/- mice was due to massive small
bowel infarction possibly secondary to vessel blockage from massive
macrophage infiltration of the submucosa and lamina propria. The
data from these latter double homozygotes: are not included
here.
[0067] Samples for plasma lipid determinations and antibody
analyses were obtained at 32 days after the first injection. All
mice were sacrificed 48 h after the final LAL injection.
1TABLE 1 Study Design # of Age* Dosage*** Total Name Genotype Diet
mice (mos.) Injection (U) Injections LC lal-/- chow 5 2 PBS** 0 10
LA, LB lal-/- chow 8 2 LAL 1.48 10 RC ldlr-/- HF/HCh 4 2.5 PBS 0 10
RA, RB ldlr-/- HF/HCh 8 2.5 LAL 1.48 10 LRC lal-/-/ldlr-/- HF/HCh 4
2.5 PBS 0 10 LRA, LRB lal-/-/ldlr-/- HF/HCh 8 2.5 LAL 1.48 10 *The
age refers to that at beginning of injections. **The control
injection was 1 .times. PBS, with 2% HSA and 10 mM DTT. ***Doses
were given every third day to each mouse. 1.48 U = 21 .mu.g.
[0068] Stability of LAL Activity
[0069] The stability of LAL activity at 4.degree. C. was monitored
every 3-4 days for 34 days. The LAL activities remained relatively
stable over this period of time, although rigorous standardization
of the assay remains to be accomplished.
[0070] General Methods
[0071] Animals. The mice were provided care in accordance with
institutional guidelines and all procedures received prior approval
by the IACUC at the Children's Hospital Research Foundation,
Cincinnati, Ohio. The lal-A mice originated from mixed genetic
backgrounds of 129Sv and CF-1. The ldlr-/- mice were purchased from
Jackson Laboratory and were cohorts of C57BL6/J. Mice were housed
in micro-isolation, under 12 h/12 h, dark/light cycles. Water and
food, regular chow diets or HFCD, were available ad libitum. The
mice were genotyped by PCR-based screening of tail DNA.
[0072] Plasma lipid analyses. Blood was collected from the inferior
vena cava (IVC) of mice after they had been anesthetized with 200
.mu.l triple sedative (Ketamine, Acepromazine, and Xylazine).
Plasma was collected after centrifugation (5,000.times.g; 10 min;
4.degree. C.) of blood and stored at -20.degree. C. Total plasma
free cholesterol was determined colorimetrically with a COD-PAP kit
(Wako Chemicals). Total plasma, triglycerides were determined in
plasma samples with a Triglycerides/GB kit (Boehringer Mannheim).
Total plasma cholesterol was determined using a Cholesterol/HP kit
(Boehringer Mannheim).
[0073] Tissue Lipid analyses. Total lipids were extracted from
liver, spleen and small intestine by the Folch method (Folch, J.,
Lees, M., and Sloane-Stanley, G. H. (1957) A simple method for the
isolation and purification of total lipids from animal tissue. J.
Biol. Chem., 226, 497-505). Triglyceride concentrations were
measured using chemical analysis developed by Biggs. Briefly, both
standards and samples in chloroform were evaporated under vacuum.
The lipids were resuspended into the following reagents in order:
0.5 ml of isopropanol, 4.5 ml of H.sub.2O: isopropanol: 40 mM
H.sub.2SO.sub.4 (0.5:3.0:1.0) and 2.0 ml of Heptane, and mixed by
vigorous agitation at each step. The tubes were left to biphase
(.about.5 minutes). In a set of new tubes, 80 mg of florisil was
added and 1.0 ml of the upper phase from each sample was
transferred into tubes that contained florisil and mixed by
agitation. Then, 0.2 ml of this upper phase was transferred to a
new set of tubes and 28 mM sodium alkoxide (2.0 ml) was added and
mixed carefully. The tubes were incubated at 60.degree. C. for 5
min. Sodium metaperiodate (3 mM, 1 ml) was added to each tube and
mixed well. The tubes were left to oxidize for 45 minutes. Finally,
1.0 ml of 73 mM acetyl acetone was added to each tube and incubated
at 60.degree. C. for 20 min. The tubes were cooled at room
temperature (.about.25 min), read at 410 nm on a Beckman DU640
spectrophotometer.
[0074] Total tissue cholesterol concentrations were measured using
the O-phthalaldehyde. Briefly, cholesterol standards and Folch
extracted samples were evaporated under N.sub.2. O-phthalaldehyde
(3 ml, Sigma) was added to each cholesterol standard and tissue
sample and mixed. Concentrated sulfuric acid (1.5 ml) was added
slowly and, then, mixed and cooled for 5-10 min, and read at 550 nm
in a Beckman DU640 spectrophotometer.
[0075] Western blot analysis and LAL activity assay: Immunoblots
were conducted with anti-LAL antiserum as described. LAL activities
were estimated with the fluorogenic substrate, 4-MU-oleate (4-MUO).
All assays were conducted in duplicate. Assays were linear within
the time frame used and less than 10% of substrates were
cleaved.
[0076] Histological Analyses. Light microscopic examinations of the
livers, spleen, intestine, adrenal glands, kidneys, heart, lung,
thymus, pancreas, and brain were performed. The sections were
stained with hematoxylin/eosin (paraffin embedded) or Oil
red-O(ORO) (frozen sections) for light microscopic analysis.
[0077] Immunohistochemical staining. Immunohistochemical analyses
were with paraffin-embedded liver sections and were performed with
rabbit anti-LAL antibody. The endogenous peroxidase activity was
saturated by incubation in methanol containing 0.5% H.sub.2O.sub.2
for 10 min.
[0078] The primary antibody (1:200) was incubated at 40.degree. C.
for overnight. The sections were then washed with 1.times.PBS three
times (5 min per wash), incubated with alkaline
phosphatase-conjugated IgG as secondary antibody for 30 min at room
temperature, and washed with 1.times.PBS for 5 min. The signal was
detected using VECTASTAIN ABC-AP kit (Vector) and counter stained
with Nuclear Fast Red.
[0079] LAL uptake studies in J774E and J774A. I macrophage
cultures: J774E and J774A.1 cells were maintained in DMEM medium
with 60 .mu.M of 6-Thioguanine or in DMEM medium, respectively,
supplemented with 10% fetal calf serum, penicillin and streptomycin
(37.degree. C.; 5% CO.sub.2). For the uptake studies, cells were
seeded at 2.times.10.sup.5 per well one day before adding LAL or
Ceredase. At designated post-incubation times, cells were washed
with 1.times.PBS twice, collected with a rubber policeman, and
centrifuged (12,000 rpm, 1 min.) at room temperature. The
intracellular proteins were extracted by cell lysis with 1%
taurocholate/1% Triton X-100, frozen/thawed five times (dry ice and
37.degree. C. water bath), and centrifuged (12,000 rpm, 10 min.) at
4.degree. C. The protein extracts were analyzed by Western
blot.
[0080] For immunofluorescence staining, cells (1.5.times.10.sup.5)
were seeded on chamber slide, incubated with LAL for 5, 18 or 24
hrs, washed with PBS twice, and fixed with 2% Paraformaldehyde for
1 hr. Immunofluoresence staining was performed.
[0081] Results
[0082] 1) Reduction of lipid storage in liver, spleen, and small
intestine of lal-/- mice following LAL treatment.
[0083] a. Phenotypic and Gross Pathologic Changes (FIG. 2): In
lal-/- mice, treatment with LAL resulted in significant correction
of lipid storage phenotypes in various organs. At 3 months of age,
untreated lal-/- mice developed a yellow/white creamy color to the
liver and significant hepatosplenomegaly was present. In
comparison, the LAL treated mice had livers and spleens with much
more normal colors. The normal livers in age matched controls were
about 5% of body weight whereas the livers were 14% in the
untreated lal-/- mice. LAL administration decreased this by about
30% (p=0.0029). The splenic weights were similar in the untreated
and treated lal-/- mice (p=0.5044). However, the color of the
spleen reverted to near normal in the treated group. The small
intestine in untreated lal-/- mice was yellow in the duodenum and
creamy white in the jejunum. In the treated group, the small
intestine partially reverted to a normal color.
[0084] b. Histologic Evaluation: H & E or Oil-Red-O staining of
liver, spleen and small intestine from untreated and treated mice
showed clear differences. In liver, the LAL treated lal-/- mice had
reductions in the size and number of lipid filled Kupffer cells
(see FIGS. 3A and B). Hepatocytes have less lipid storage than
Kupffer cells in untreated lal-/- mice and this hepatocyte storage
appeared unchanged in the treated group. Using Oil-Red-O staining
for neutral lipids, a significant difference between the livers of
the treated and untreated mice was apparent (see FIGS. 3C and D).
In the spleen, the treated group showed a reduction in lipid
storage cells compared to those present in untreated mice. In the
small intestine, the Oil-Red-O staining of LAL treated and
untreated mice showed substantial differences. The sections of
intestine from untreated mice were full of Oil-Red-O staining cells
(macrophages) in lamina propria while comparable sections from
treated mice were almost completely negative for Oil-Red-O
staining. The aortic arches, aortic base and valves, and coronary
arteries of lal-/- mice, treated or untreated, were essentially
normal throughout the study.
[0085] c. Immunohistochemistry: Immunohistologic analyses of liver
with anti-LAL (E. Coli produced recombinant hLAL) showed
predominantly dark staining (positive) of the sinusoidal lining
cells. Some antigen could be detected in the storage cells, but
this signal was at a low level due to the very large dilution space
presented by these cells. The samples of liver were obtained 30
min. after injection. The uninjected lal-/- mice had undetectable
lal.
[0086] d. Biochemical Findings: Tissue cholesterol (both free and
esterified) and triglycerides from liver, spleen and small
intestine were determined by chemical analyses. Compared to age
matched wild-type mice, the lal-/- mice have elevated cholesteryl
esters and triglycerides in several tissues. The average total
cholesteryl ester per organ at 3.5 months of age was increased
31-fold in liver and 19-fold in spleen compared to wild-type. LAL
administration to such mice was associated with reductions of total
cholesterol by 47% in total liver (267.22.+-.8.22 mg vs.
144.23.+-.7.99 mg; p=0.0003, n=3) and by 69% in total spleen
(8.73.+-.0.43 mg vs. 2.63.+-.0.50 mg, p=0.0008, n=3). Similar
decreases of triglycerides also were observed: 58% in total liver
(26.52.+-.17.93 mg vs. 39.79.+-.6.38 mg, p=0.047, n=4) and 45% in
total spleen (8.23.+-.0.68 mg vs. 4.55.+-.1.26 mg, p=0.042, n=4).
Although no change in the concentration of cholesterol in small
intestine was observed (p=0.67), the triglyceride concentration of
the treated group was 65% reduced (49.52.+-.2.40 .mu.g/mg vs.
17.09.+-.4.8 .mu.g/mg, p=0.042, n=4).
[0087] e. Summary
[0088] Limited treatment of lal-/- mice with LAL (10 injections in
30 days, 1.48 U/dose) led to gross, histologic and biochemical
corrections of cholesterol and triglyceride levels in treated
mice.
[0089] 2. Plasma Chemistries and Lipid Levels in lal-/- and ldlr-/-
Mice.
[0090] No differences in plasma glucose levels were observed in
treated or untreated lal-/- or lal-/- mice although ldlr-/- mice
have higher plasma glucose levels than wild type or lal-/- mice.
The lal-/- and ldlr-/- mice had increased plasma non-esterified
fatty acids (NEFA) levels compared to the wild-type controls (162%
and 227%, respectively). LAL administration was associated with
increases of the NEFA by 32.6% in lal-/- mice and 24.5% in lal-/-
mice. Plasma triglycerides levels decreased in treated lal-/- mice,
but were unchanged in ldlr-/- mice. The HFCD produced
hypercholesterolemia in lal-/- mice. The plasma free cholesterol
concentration increased 22-fold and plasma cholesteryl ester
concentration increased 13.8-fold compared to wild-type mice. The
LAL treated ldlr-/- mice had decreases in plasma free cholesterol
of 18.2% (p=0.0894) and in cholesteryl esters of 26.7% (P=0.0025).
The free cholesterol and cholesterol ester levels were unchanged in
treated lal-/- mice.
[0091] 3. Histologic and Biochemical Effects of LAL Administration
in ldlr-/- Mice.
[0092] a. Gross Anatomic and Histologic Studies
[0093] The visceral organs of these mice appeared normal. Whole
mounts of the aortic arches were prepared from ldlr-/- mice and
examined by transillumination. At 3.5 months, all (3/3) untreated
ldlr-/- mice had extensive lesions of the arch and take-offs of the
major vessels, i.e., brachiocephalic arteries. Although not
quantitatively determined, LAL administration appeared to have
little effect on these lesions in treated ldlr-/- mice.
[0094] To evaluate the coronary artery lesions, the hearts of
treated and untreated ldlr-/- mice were sequentially sectioned and
analyzed. Four lal-/- mice were untreated. One of these was found
dead just before the LAL administration began (at age of 2.5
months). Eight mice received LAL and all survived for the entire
study period. The results are summarized in Table 2. All untreated
ldlr-/- mice had severe plaque lesions in aortic valve and ostia of
the coronary arteries (see FIGS. 4A and B). Of the aortic valves
examined in the treated group, two had mild to moderate (++), one
had very mild (+), and two had no accumulation of foam cells (see
FIG. 4C). The aortic valves from three treated mice were not
examined histologically since they had been removed for the whole
mount aortic arch studies.
[0095] The coronary lesions in the untreated group were extensive
and multifocal. All had heavy infiltration of the coronary ostia by
macrophages with plaques extending a considerable distance in the
coronary arteries. Also, individual isolated and scattered plaques
were found throughout the first third of the coronary arteries. In
one case, the main branch of the left coronary was completely
obliterated with an advanced lesion containing cholesterol crystals
and apparent inflammatory. In comparison, 7/8 of the treated
ldlr-/- mice had normal coronary vessels (see Table 2). One LAL
treated ldlr-/- mouse had foamy cells in one small intramuscular
coronary vessel. The other coronary arteries in this mouse were
normal. This particular mouse (RA1) also had mild-moderate lesions
of the aortic valve.
[0096] To obtain a more quantitative assessment of the coronary
artery lesions in ldlr-/- mice, sequential H&E sections
(total=210; 10 .mu.m) of the heart were examined in an untreated
mouse (RC2) and in one treated mouse (RB2). RC2 had multiple
plaques in coronary arteries whereas RB2 had completely normal
coronary arteries.
2TABLE 2 Effect of LAL on the Aortic Valves and Coronary Arteries
of Idlr -/- Mice LAL Untreated Mice Designation Aortic Valve Lesion
Coronary Artery Lesions RC2 ++++ ++++ RC3 ++++ +++ RC4 ++++ ++++
LAL Treated Mice Designation Aortic Valve Lesion Coronary Artery
Lesions RA1 ++ + RA2 + - RA3 + - RA4 ++ - RB1 - - RB2 - - RB3 ND -
RB4 ND - ++++ = severe lesions; +++ = moderate, ++ = mild-moderate;
+ = mild; - = no lesions; ND = Not done due to aortic arch removal
for whole mounts
[0097] These results show a major selective effect of a single
fixed dose level of LAL on the presence of aortic valvular and
coronary artery foam cell and progressive atherogenic lesions.
[0098] b. Biochemical Studies:
[0099] Plasma lipid results are reported above for the ldlr-/-
treated and untreated groups. Liver and splenic cholesterol and
triglyceride levels were increased over wild-type mice in the
untreated ldlr-/- group. No significant effects were observed on
the total cholesterol in liver (p=0.8816) and spleen (p=0.1061), or
cholesterol concentration (0.0927) in the small intestine. The
triglycerides were reduced 65.1% in total liver (91.54.+-.1.98 mg
vs. 59.60.+-.6.86 mg; p=0.002), and 53.3% in total spleen
(3.24.+-.0.39 mg vs. 1.73.+-.0.33 mg; p=0.0183). The concentration
of triglycerides in small intestine also was reduced 43%
(41.74.+-.3.69 .mu.g/mg vs. 23.79.+-.2.08 .mu.g/mg p=0.001).
[0100] c. Antibody Studies:
[0101] Serum was obtained at sacrifice from each mouse of each
genotype and used in Western analyses. Prep #3 (2.65 ng/well) was
used as antigen. Serum was used at 1:100 dilutions. All mice
exposed to 10 injections of LAL gave positive western signals. The
positive bands co-migrated with the LAL detected with rabbit
anti-LAL. With one mouse serum positive signals were achieved with
1:100 to 1:6400 dilutions using Prep #3. Additional studies were
conducted to determine the reactivity of these mouse sera to LAL or
unglycosylated LAL produced in E. coli. Using 2.65 ng of antigen,
the unglycosylated LAL gave very low to absent signals with all but
one mouse serum. These results indicate that the antibody's
specificity is directed more toward the oligosaccharides than the
LAL protein in these conformations.
[0102] Summary of Data
[0103] The data from the ldlr-/- data show clear and dramatic
effects of LAL administration on the presence of aortic valvular
and coronary artery plaques and foam cells. All of the lesions were
greatly diminished or absent in the treated mice compared to very
severe lesions in the untreated cohort. The changes in hepatic,
splenic and intestinal triglycerides indicate a direct effect of
the LAL in these organs.
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