U.S. patent application number 11/401206 was filed with the patent office on 2006-09-14 for lactoferrin in the reduction of circulating cholesterol, vascular inflammation, atherosclerosis and cardiovascular disease.
Invention is credited to Jose Engelmayer, Peter Glynn, Atul Varadhachary, Yenyun Wang.
Application Number | 20060205634 11/401206 |
Document ID | / |
Family ID | 32474601 |
Filed Date | 2006-09-14 |
United States Patent
Application |
20060205634 |
Kind Code |
A1 |
Varadhachary; Atul ; et
al. |
September 14, 2006 |
Lactoferrin in the reduction of circulating cholesterol, vascular
inflammation, atherosclerosis and cardiovascular disease
Abstract
The present invention relates to methods of using lactoferrin
(LF) to reduce circulating levels of cholesterol and vascular
inflammation, in order to treat, prevent or reduce the incidence of
atherosclerosis and cardiovascular disease.
Inventors: |
Varadhachary; Atul;
(Houston, TX) ; Glynn; Peter; (Houston, TX)
; Wang; Yenyun; (Houston, TX) ; Engelmayer;
Jose; (Houston, TX) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
1301 MCKINNEY
SUITE 5100
HOUSTON
TX
77010-3095
US
|
Family ID: |
32474601 |
Appl. No.: |
11/401206 |
Filed: |
April 10, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10728275 |
Dec 4, 2003 |
7026295 |
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11401206 |
Apr 10, 2006 |
|
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60430867 |
Dec 4, 2002 |
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60498337 |
Aug 27, 2003 |
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Current U.S.
Class: |
514/1.9 ;
514/16.4; 514/2.5; 514/7.4 |
Current CPC
Class: |
A61K 38/40 20130101;
A61K 38/40 20130101; A61K 31/785 20130101; A61K 31/785 20130101;
A61K 45/06 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
514/006 |
International
Class: |
A61K 38/40 20060101
A61K038/40 |
Claims
1-31. (canceled)
32. A method of modulating atherosclerosis in a subject comprising
the step of administering to said subject an effective amount of a
lactoferrin composition to modulate atherosclerosis in said
subject.
33. The method of claim 32, wherein modulating is reducing the
incidence of atherosclerosis in said subject.
34. The method of claim 32, wherein modulating is reducing the
severity of atherosclerosis in said subject.
35. The method of claim 32, wherein said lactoferrin composition
reduces levels of circulating total cholesterol, low density
lipoproteins (LDL), very low density lipoproteins (VLDL), or
triglycerides in said subject.
36. The method of claim 32, wherein said lactoferrin composition
increases the levels of circulating high density lipoproteins (HDL)
in said subject.
37. The method of claim 32, wherein said lactoferrin composition
reduces the levels of vascular inflammation in said subject.
38. The method of claim 32, wherein said lactoferrin composition
reduces circulating C-reactive protein (CRP) in said subject.
39. The method of claim 32, wherein said lactoferrin composition
reduces the proliferation of vascular smooth muscle cells in said
subject.
40. The method of claim 32, wherein said lactoferrin composition
reduces the vascular spasm or vascular hyper-reactivity in said
subject.
41. The method of claim 32, wherein said lactoferrin composition
promotes endothelial integrity or healing in said subject.
42. The method of claim 32, wherein said lactoferrin composition is
dispersed in a pharmaceutically acceptable carrier.
43. The method of claim 32, wherein said lactoferrin is mammalian
lactoferrin.
44. The method of claim 43, wherein said lactoferrin is human or
bovine.
45. The method of claim 32, wherein said lactoferrin is recombinant
lactoferrin.
46. The method of claim 32, wherein said lactoferrin composition
comprises an N-terminal lactoferrin variant.
47. The method of claim 46, wherein the N-terminal lactoferrin
variant lacks at least the N-terminal glycine residue.
48. The method of claim 47, wherein said N-terminal lactoferrin
variant comprises at least 1% to at least 50% of the lactoferrin
composition.
49. The method of claim 32, wherein said lactoferrin composition is
administered parenterally.
50. The method of claim 49, wherein parenterally is subcutaneously,
intramuscularly, intraperitoneally, intravenously, intraarterially,
intramyocardially, transendocardially, transepicardially, or
intrathecally.
51. The method of claim 32, wherein said lactoferrin composition is
administered orally.
52. The method of claim 51 further comprising administering an
antacid in conjunction with said lactoferrin composition.
53. The method of claim 51 further comprising administering the
lactoferrin in a delayed release formulation.
54. The method of claim 53 where the lactoferrin release occurs in
the small intestine.
55. The method of claim 53 where the lactoferrin release occurs in
the large intestine.
56. The method of claim 32, wherein the amount of the lactoferrin
that is administered is about 1 ng to about 20 g per day.
57. The method of claim 32, wherein the amount of the lactoferrin
that is administered is about 0.1 g to about 5 g per day.
58. The method of claim 32, wherein said lactoferrin composition
reduces the production or activity of pro-inflammatory
cytokines.
59. The method of claim 32 further comprising administering a
lactoferrin composition in combination with an anti-cholesterol
agent or an anti-inflammatory agent.
60. The method of claim 59, wherein the anti-cholesterol agent is
selected from the group consisting of cholesterol absorption
inhibitors, bile acid sequestrants, nicotinic acid, fibric acids
and HMG-coA reductase inhibitors.
61. The method of claim 60, wherein the bile acid sequestrants are
selected from the group consisting of cholestryramine, cholestipol
and colesevalam.
62. The method of claim 60, wherein the fibric acids are selected
from the group consisting of gemfibrozil, fenofibrate and
clofibrate.
63. The method of claim 60, wherein the HMG-coA reductase
inhibitors are selected from the group consisting of lovastatin,
pravastatin, simvastatin, fluvastatin, atorvastatin and
cerivastatin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/728,275 filed Dec. 4, 2003 which claims the benefit of U.S.
Provisional Applications 60/498,337 filed Aug. 27, 2003 and
60/430,867 filed Dec. 4, 2002, both of which are incorporated
herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to methods of using
lactoferrin (LF) to reduce circulating levels of cholesterol and
vascular inflammation, in order to treat, prevent or reduce the
incidence of atherosclerosis and cardiovascular disease. More
particularly, the present invention relates to methods of reducing
circulating levels of cholesterol and vascular inflammation by
administering a composition of lactoferrin.
BACKGROUND OF THE INVENTION
[0003] According to current estimates, 61,800,000 people in America
have one or more forms of cardiovascular disease. These diseases
claimed 958,775 lives in 1999 (40.1 percent of all deaths).
Atherosclerosis is a leading form of cardiovascular disease, which
involves the slow build-up of fatty plaques on the arterial wall.
This build-up can damage the vascular endothelium causing
inflammation, a narrowing of the arteries and potential arterial
blockages that can result in heart attacks. Atherosclerosis is a
complex disease that starts in childhood and often progresses when
people grow older. In some people it progresses rapidly, even in
their third decade. Elevated levels of cholesterol, in particular
LDL (low-density lipoprotein), and triglycerides in the blood have
been associated with the development of fatty plaques, which can
lead to generalized vascular damage, atherosclerosis and eventually
heart attack. Atherosclerosis and cardiac disease is also
associated with increased cardiovascular inflammation, specifically
as measured by levels of circulating C-reactive protein (CRP).
[0004] One key strategy for reducing the risk of atherosclerosis
has been to lower the levels of cholesterol in the blood.
Cholesterol levels in many people can be controlled by diet, but
for many patients diet changes alone are insufficient to reduce
high cholesterol. In recent years, cholesterol lowering drugs such
as Zocor.RTM. (simvastatin) and Lipitor.RTM. (atorvastatin) have
been increasingly prescribed to help patients lower their
cholesterol levels. These drugs however, are not equally effective
in all patients and frequently are associated with significant
adverse side effects. A second key emerging strategy is the
reduction of CRP, an important indicator of vascular inflammation
and independently associated with increased risk of cardiovascular
disease. Thus, safer and more effective treatments for lowering
cholesterol and for reducing the vascular inflammation associated
with atherosclerosis are of great potential value.
[0005] Lactoferrin is a single chain metal binding glycoprotein.
Many cell types, such as monocytes, macrophages, lymphocytes, and
brush-border cells in the intestine, are known to have lactoferrin
receptors. Lactoferrin is found mainly in external secretions of
mucosal epithelia such as breast milk, saliva, tears, bile, and
pancreatic fluid and has a wide array of functions related to host
primary defense mechanisms. For example, lactoferrin has been
reported to activate natural killer (NK) cells, induce colony
stimulating activity, activate polymorphonuclear neutrophils (PMN),
regulate granulopoeisis, enhance antibody-dependent cell
cytotoxicity, stimulate lymphokine-activated killer (LAK) cell
activity, and potentiate macrophage toxicity.
[0006] Recombinant human lactoferrin has previously been described
as being purified after expression in a variety of prokaryotic and
eukaryotic organisms including aspergillus (U.S. Pat. No.
6,080,559), cattle (U.S. Pat. No. 5,919,913), rice, corn,
Sacharomcyes (U.S. Pat. No. 6,228,614) and Pichia pastoris (U.S.
Pat. No. 6,455,687, 6,277,817, 6,066,469). Also described are
expression systems for the expression of full-length human
lactoferrins (e.g., U.S. Pat. No. 6,100,054). In all cases, part of
the teaching is expression of the full length cDNA and purification
of the intact protein whose N-terminal, after processing of the
leader peptide, is the amino acid glycine. Nuijens et al. (U.S.
Pat. No. 6,333,311) separately describe variants of human
lactoferrin but their focus is limited to deletion or substitution
of arginine residues found in the N-terminal domain of
lactoferrin.
[0007] The present invention is the first to use a lactoferrin
composition as a means of reducing cholesterol and cardiovascular
inflammation and for treating or reducing atherosclerosis and
cardiovascular disease.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention is directed to a method for modulating
circulating levels of cholesterol and reducing the vascular
inflammation associated with atherosclerosis and cardiovascular
disease. The method of treatment involves administration of a
lactoferrin composition.
[0009] The lactoferrin composition, which is dispersed in a
pharmaceutically acceptable carrier, comprises lactoferrin or an
N-terminal lactoferrin variant in which at least the N-terminal
glycine residue is truncated or substituted. The lactoferrin is
mammalian lactoferrin, more particularly, the lactoferrin is human
or bovine. Yet further, the lactoferrin is recombinant lactoferrin.
N-terminal lactoferrin variants include variants that at least lack
the N-terminal glycine residue or contain a substitution at the
N-terminal glycine residue. The substitution can comprise
substituting a natural or artificial amino acid residue for the
N-terminal glycine residue. For example, the substitution can
comprise substituting a positive amino acid residue or a negative
amino acid residue for the N-terminal glycine residue or
substituting a neutral amino acid residue other than glycine for
the N-terminal glycine residue. Other N-terminal lactoferrin
variants include lactoferrin lacking one or more N-terminal
residues or having one or more substitutions in the N-terminal. In
specific embodiments, the N-terminal lactoferrin variant comprises
at least 1% of the lactoferrin composition, at least 5% of the
lactoferrin composition, at least 10% of the lactoferrin
composition, at least 25% of the lactoferrin composition, at least
50% of the lactoferrin composition or any range in between.
[0010] The amount of the lactoferrin that is administered is about
1 ng to about 20 g per day, more preferably, the amount is about
0.1 g to about 5 g per day. More particularly, the composition is a
solution, capsule or a tablet having a lactoferrin concentration of
about 0. 1% to about 100%.
[0011] In further embodiments, a metal chelator dispersed in a
pharmaceutically acceptable carrier can also be administered with
the lactoferrin composition. Preferred metal chelator include, but
are not limited to ethylenediaminetetraacetic acid (EDTA) or
[ethylenebis(oxyethylenenitrilo)] tetraacetic acid (EGTA). More
preferably, the metal chelator is EDTA. The amount of EDTA that is
administered is about 1 ng to about 1 g per day.
[0012] An embodiment of the present invention is a method of
treating a cardiovascular disease comprising the step of
administering to a subject a lactoferrin composition in an
effective amount to provide an improvement in the cardiovascular
disease, for example, atherosclerosis. The lactoferrin composition
reduces the levels of circulating total cholesterol, low density
lipoproteins (LDL), or very low density lipoproteins (VLDL). Still
further, the lactoferrin composition increases the levels of
circulating high density lipoproteins (HDL). In addition to
modulating levels of cholesterol, the lactoferrin composition
reduces the levels of vascular inflammation, circulating C-reactive
protein (CRP), triglycerides, or proliferation of vascular smooth
muscle cells. The lactoferrin composition may also reduce vascular
spasms or vascular hyper-reactivity or promote endothelial
integrity or healing. In further embodiments, the lactoferrin
composition reduces the production or activity of pro-inflammatory
cytokines.
[0013] The lactoferrin composition of the present invention can be
administered parenterally, for example, subcutaneously,
intramuscularly, intraperitoneally, intravenously, intraarterially,
intramyocardially, transendocardially, transepicardially, or
intrathecally.
[0014] In a further embodiment, the lactoferrin composition is
administered orally. For oral administration, an antacid in
combination with the lactoferrin composition can be administered.
The lactoferrin can be formulated in a delayed release formulation.
Still further, the lactoferrin composition can be formulated
wherein release occurs in the small intestine or in the large
intestine.
[0015] Another embodiment of the present invention is a method of
modulating atherosclerosis in a subject comprising the step of
administering to the subject a lactoferrin composition in an
effective amount to modulate atherosclerosis in the subject.
Modulating is reducing the incidence of atherosclerosis or reducing
the severity of atherosclerosis. In further embodiments, the
lactoferrin composition can be administered in combination with an
anti-cholesterol agent or an anti-inflammatory agent. The
anti-cholesterol agent is selected from the group consisting of
cholesterol absorption inhibitors, bile acid sequestrants
(cholestryramine, cholestipol and colesevalam), nicotinic acid,
fibric acids (gemfibrozil, fenofibrate and clofibrate) and HMG-coA
reductase inhibitors (lovastatin, pravastatin, simvastatin,
fluvastatin, atorvastatin and cerivastatin).
[0016] Another embodiment is a method of preventing a
cardiovascular disease in a subject at risk for developing a
cardiovascular disease comprising the step of administering to the
subject a lactoferrin composition in an amount sufficient to result
in prophylaxis of the cardiovascular disease in the subject. The
cardiovascular disease is atherosclerosis.
[0017] Still further, another embodiment is a method of reducing
the risk of cardiovascular disease in a subject at risk for
developing a cardiovascular disease comprising the step of
administering to the subject a lactoferrin composition in an
effective amount to result in a reduction of risk of the
cardiovascular disease in the subject. The cardiovascular disease
is atherosclerosis.
[0018] The foregoing has outlined rather broadly the features and
technical advantages of the present invention in order that the
detailed description of the invention that follows may be better
understood. Additional features and advantages of the invention
will be described hereinafter which form the subject of the claims
of the invention. It should be appreciated that the conception and
specific embodiment disclosed may be readily utilized as a basis
for modifying or designing other structures for carrying out the
same purposes of the present invention. It should also be realized
that such equivalent constructions do not depart from the invention
as set forth in the appended claims. The novel features which are
believed to be characteristic of the invention, both as to its
organization and method of operation, together with further objects
and advantages will be better understood from the following
description when considered in connection with the accompanying
figures. It is to be expressly understood, however, that each of
the figures is provided for the purpose of illustration and
description only and is not intended as a definition of the limits
of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] For a more complete understanding of the present invention,
reference is now made to the following descriptions taken in
conjunction with the accompanying drawings.
[0020] FIG. 1 shows the net reduction of total serum cholesterol in
subjects receiving oral rhLF for seven days with respect to
patients receiving placebo for seven days.
[0021] FIG. 2 shows the reduction of C-reactive protein in subjects
receiving oral rhLF for seven days.
[0022] FIG. 3A and FIG. 3B show the effect of rhLF on
hyperlipidemia in mice. FIG. 3A shows the effect of rhLF on HDL,
LDL and total cholesterol levels. FIG. 3B shows the effect on
HDL/LDL ratio.
[0023] FIG. 4 shows a dose effect of rhLF on hyperlipidemia in
mice.
[0024] FIG. 5 shows the effect of oral rhLF with lovastatin on
hyperlipidemia in mice.
DETAILED DESCRIPTION OF THE INVENTION
[0025] It is readily apparent to one skilled in the art that
various embodiments and modifications can be made to the invention
disclosed in this Application without departing from the scope and
spirit of the invention.
[0026] As used herein, the use of the word "a" or "an" when used in
conjunction with the term "comprising" in the claims and/or the
specification may mean "one," but it is also consistent with the
meaning of "one or more," "at least one," and "one or more than
one." Still further, the terms "having", "including", "containing"
and "comprising" are interchangeable and one of skill in the art is
cognizant that these terms are open ended terms.
[0027] The term "atherosclerosis" as used herein includes a form of
arteriosclerosis characterized by a combination of changes in the
intima of arteries, such changes include, but are not limited to
accumulation of lipids, complex carbohydrates, blood and blood
products, fibrous tissue and calcium deposits. Yet further,
atherosclerotic plaques can be characterized into at least two
areas. One type is characterized by prominent proliferation of
cells with small accumulations of lipids. The second type consists
mostly of intracellular and extracellular lipid accumulation and a
small amount of cellular proliferation.
[0028] The term "cardiovascular disease or disorder" as used herein
refers to disease and disorders related to the cardiovascular or
circulatory system. Cardiovascular disease and/or disorders
include, but are not limited to, diseases and/or disorders of the
pericardium, heart valves (e.g., incompetent valves, stenosed
valves, rheumatic heart disease, mitral valve prolapse, aortic
regurgitation), myocardium (e.g., coronary artery disease,
myocardial infarction, heart failure, ischemic heart disease,
angina) blood vessels (e.g., hypertension, arteriosclerosis,
aneurysm) or veins (e.g., varicose veins, hemorrhoids). Yet
further, one skill in the art recognizes that cardiovascular
diseases and/or disorders can result from congenital defects,
genetic defects, environmental influences (e.g., dietary
influences, lifestyle, stress, etc.), and other defects or
influences.
[0029] The term "chemokine" as used herein refers to small
cytokines that are involved in the migration and activation of
cells, for example phagocytic cells and lymphocytes. One of skill
in the art realizes that chemokines play a central role in
inflammatory and immune response processes.
[0030] The term "cholesterol" as used herein refers to the
monohydric alcohol form, which is a white, powdery substance that
is found in all animal cells and in animal-based foods (not in
plants). Cholesterol is an essential nutrient necessary for many
functions, including the following: repairing cell membranes,
manufacturing vitamin D on the skin's surface, production of
hormones, such as estrogen and testosterone, and possibly helping
cell connections in the brain that are important for learning and
memory.
[0031] The term "chylomicron" as used herein refers to the largest
in size and lowest in density of the triglyceride carrying
lipoproteins.
[0032] The term "cytokine" as used herein refers to proteins that
are made by cells that affect the behavior of other cells, for
example stimulate or inhibit cell proliferation. For example,
cytokines that are made by lymphocytes are often called lymphokines
or interleukins. One of skill in the art realizes that the term
cytokine is a generic term used in the literature to refer to
proteins that are made by cells that can effect the behavior of
other cells.
[0033] The term "effective amount" or "therapeutically effective
amount" are interchangeable as used herein and refer to an amount
that results in an improvement or remediation of the symptoms of
the disease or condition.
[0034] The term "high-density lipoprotein" or "HDL" as used herein
is the smallest and most dense type of cholesterol-carrying
lipoprotein and is often referred to as the "good" cholesterol.
[0035] The term "intermediate density lipoprotein" or "IDL" as used
herein refers to a triglyceride-carrying lipoprotein.
[0036] The term "lactoferrin" or "LF" as used herein refers to
native or recombinant lactoferrin. Native lactoferrin can be
obtained by purification from mammalian milk or colostrum or from
other natural sources. Recombinant lactoferrin (rLF) can be made by
recombinant expression or direct production in genetically altered
animals, plants, fungi, bacteria, or other prokaryotic or
eukaryotic species, or through chemical synthesis.
[0037] The term "lactoferrin composition" as used herein refers to
a composition having lactoferrin, a portion or part of lactoferrin,
an N-terminal lactoferrin variant, or a combination thereof.
[0038] The term "lipid" as used herein refers to the building
blocks of any of the fats or fatty substances found in animals and
plants, which are characterized by their insolubility in water and
solubility in fat solvents such as alcohol, ether and chloroform.
Lipids include fats (e.g., esters of fatty acids and glycerol);
lipoids (e.g., phospholipids, cerebrosides, waxes) and sterols
(e.g., cholesterol).
[0039] The term "lipoproteins" as used herein are protein spheres
that transport cholesterol, triglyceride, or other lipid molecules
through the bloodstream. Lipoproteins are categorized into five
types according to size and density. They can be further defined by
whether they carry cholesterol [the two smaller lipoproteins (HDL
and LDL)] or triglycerides [the three largest lipoproteins (IDL,
VLDL, and chylomicrons)].
[0040] The term "low density lipoprotein" or "LDL" as used herein
is a type of cholesterol-carrying lipoprotein which is often called
the "bad" cholesterol.
[0041] The term "N-terminal lactoferrin variant" as used herein
refers to lactoferrin wherein at least the N-terminal glycine has
been truncated and/or substituted. N-terminal lactoferrin variants
also include, but are not limited to deletion and/or substitution
of one or more N-terminal amino acid residues, for example 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 N-terminal amino
acid residues, etc. Thus, N-terminal lactoferrin variants comprise
at least deletions or truncations and/or substitutions of 1 to 16
N-terminal amino acid residues. The deletion and/or substitution of
at least the N-terminal glycine of lactoferrin mediates the same
biological effects as full-length lactoferrin and/or may enhance
lactoferrin's biological activity, for example by stimulating the
production of various cytokines (e.g., IL-18, MIP-3.alpha., GM-CSF
or IFN-.gamma.) by inhibiting various cytokines, (e.g., IL-2, IL-4,
IL-5, IL-10, or TNF-.alpha.) by improving a cardiovascular disease,
e.g., atherosclerosis, or the parameters relating to cardiovascular
disease including circulating levels of total cholesterol, HDL,
LDL, VLDL, trigylcerides and C-reactive protein (CRP).
[0042] The term "metal chelator" as used herein refers to a
compound which binds metal. Metal chelators that can be used in the
present invention include the divalent metal chelators, for
example, ethylenediaminetetraacetic acid (EDTA), [ethylenebis
(oxyethylenenitrilo)] tetraacetic acid (EGTA),
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA),
hydroxyethlene triamine diacetic acid, (HEDTA) or salts
thereof.
[0043] The term "oral administration" as used herein includes oral,
buccal, enteral or intragastic administration.
[0044] The term "parenteral administration" as used herein includes
any form of administration in which the compound is absorbed into
the subject without involving absorption via the intestines.
Exemplary parenteral administrations that are used in the present
invention include, but are not limited to intramuscular,
intravenous, intraperitoneal, intraocular, subcutaneous or
intraarticular administration. Yet further, parenteral
administration also includes administration into a surgical
field.
[0045] The term "pharmaceutically acceptable carrier" as used
herein includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
vectors or cells of the present invention, its use in therapeutic
compositions is contemplated. Supplementary active ingredients also
can be incorporated into the compositions.
[0046] The term "preventing" as used herein refers to minimizing,
reducing or suppressing the risk of developing a disease state or
parameters relating to the disease state or progression or other
abnormal or deleterious conditions.
[0047] The term "statin" as used herein includes compounds that are
HMG-CoA reductase inhibitors, for example, but not limited to
simvastatin (Zocor.RTM.) and atorvastatin (Lipitor.RTM.). Thus, as
used herein the terms "statin" and "HMG-CoA reductase inhibitor"
are interchangeable.
[0048] The term "subject" as used herein, is taken to mean any
mammalian subject to which a lactoferrin composition is
administered according to the methods described herein. Thus, a
skilled artisan realizes that a mammalian subject, includes, but is
not limited to humans, monkeys, horses, pigs, cows, dogs, cats,
rats and mice. In a specific preferred embodiment, the methods of
the present invention are employed to treat a human subject. In
more preferred embodiments, the subject has signs or indicators of
atherosclerosis. These signs or indicators include, for example,
the development of cholesterol plaques in the arteries and
calcification, the extent of which can be determined by Sudan IV
staining, or the development of foam cells in an artery or arterial
spasm. Atherosclerosis also is characterized by a narrowing of the
arteries detected by, for example, coronary angioplasty, ultrasound
and ultrafast CT. In further embodiments, the subject is at risk of
developing a cardiovascular disease. Thus, the subject may or may
not be cognizant of their disease state or potential disease state
and may or may not be aware that they are need of treatment
(therapeutic treatment or prophylactic treatment).
[0049] The term "topical administration" as used herein includes,
but is not limited to topical, dermal, or epidermal.
[0050] The term "total cholesterol" as used herein refers to the
sum of three kinds of lipids: high-density lipoprotein (HDL),
low-density lipoprotein (LDL), and triglycerides. Levels of serum
total cholesterol of >200 mg/dl are levels that are an
indicating risk factor for atherosclerosis and cardiovascular
disease.
[0051] The term "triglycerides" as used herein are composed of
fatty acid molecules and are the basic chemicals contained in fats
in both animals and plants.
[0052] The term "treating" and "treatment" as used herein refers to
administering to a subject a therapeutically effective amount of a
recombinant human lactoferrin composition so that the subject has
an improvement in a cardiovascular disease or the parameters
relating to cardiovascular disease including circulating levels of
total cholesterol, HDL, LDL, VLDL, trigylcerides and C-reactive
protein (CRP). The improvement is any observable or measurable
improvement. Thus, one of skill in the art realizes that a
treatment may improve the patient condition, but may not be a
complete cure of the disease.
[0053] The term "very low density lipoprotein" or "VLDL" as used
herein refers to a triglyceride carrying lipoprotein.
A. LACTOFERRIN
[0054] The lactoferrin used according to the present invention can
be obtained through isolation and purification from natural
sources, for example, but not limited to mammalian milk. The
lactoferrin is preferably mammalian lactoferrin, such as bovine or
human lactoferrin. In preferred embodiments, the lactoferrin is
produced recombinantly using genetic engineering techniques well
known and used in the art, such as recombinant expression or direct
production in genetically altered animals, plants or eukaryotes, or
chemical synthesis. See, for example, U.S. Pat. Nos. 5,571,896;
5,571,697 and 5,571,691, which are herein incorporated by
reference.
[0055] In certain aspects, the present invention provides
lactoferrin variants having enhanced biological activities over
natural LF and or rLF, e.g., the ability to stimulate and/or
inhibit cytokines or chemokines. In particular, the invention
provides variants of lactoferrin from which at least the N-terminal
glycine residue has been substituted and/or truncated. The
N-terminal lactoferrin variants may occur naturally or may be
modified by the substitution or deletion of one or more amino
acids.
[0056] The deletional variants can be produced by proteolysis of
lactoferrin and/or expression of a polynucleotide encoding a
truncated lactoferrin as described in U.S. Pat. No. 6,333,311,
which is incorporated herein by reference.
[0057] Substitutional variants or replacement variants typically
contain the exchange of one amino acid for another at one or more
sites within the protein. Substitutions can be conservative, that
is, one amino acid is replaced with one of similar shape and
charge. Conservative substitutions are well known in the art and
include, for example, the changes of: alanine to serine; arginine
to lysine; asparagine to glutamine or histidine; aspartate to
glutamate; cysteine to serine; glutamine to asparagine; glutamate
to aspartate; glycine to proline; histidine to asparagine or
glutamine; isoleucine to leucine or valine; leucine to valine or
isoleucine; lysine to arginine; methionine to leucine or
isoleucine; phenylalanine to tyrosine, leucine or methionine;
serine to threonine; threonine to serine; tryptophan to tyrosine;
tyrosine to tryptophan or phenylalanine; and valine to isoleucine
or leucine.
[0058] In making such changes, the hydropathic index of amino acids
may be considered. The importance of the hydropathic amino acid
index in conferring interactive biologic function on a protein is
generally understood in the art (Kyte and Doolittle, 1982). It is
accepted that the relative hydropathic character of the amino acid
contributes to the secondary structure of the resultant protein,
which in turn defines the interaction of the protein with other
molecules, for example, enzymes, substrates, receptors, DNA,
antibodies, antigens, and the like.
[0059] Each amino acid has been assigned a hydropathic index on the
basis of their hydrophobicity and charge characteristics (Kyte and
Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
[0060] It is known in the art that certain amino acids may be
substituted by other amino acids having a similar hydropathic index
or score and still result in a protein with similar biological
activity, e.g., still obtain a biological functionally equivalent
protein. In making such changes, the substitution of amino acids
whose hydropathic indices are within .+-.2 is preferred, those that
are within .+-.1 are particularly preferred, and those within
.+-.0.5 are even more particularly preferred.
[0061] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by
reference, states that the greatest local average hydrophilicity of
a protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with a biological property of the protein. As
detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity
values have been assigned to amino acid residues: arginine (+3.0);
lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine
(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine
(-0.4); proline (-0.5.+-.1); alanine (-0.5); histidine -0.5);
cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5);
tryptophan (-3.4).
[0062] Still further, it is understood that an amino acid can be
substituted for another having a similar hydrophilicity value and
still obtains a biologically equivalent and immunologically
equivalent protein. In such changes, the substitution of amino
acids whose hydrophilicity values are within .+-.2 is preferred,
those that are within .+-.1 are particularly preferred, and those
within .+-.0.5 are even more particularly preferred.
[0063] Thus, in the present invention, substitutional variants or
replacement can be produced using standard mutagenesis techniques,
for example, site-directed mutagenesis as disclosed in U.S. Pat.
Nos. 5,220,007; 5,284,760; 5,354,670; 5,366,878; 5,389,514;
5,635,377; 5,789,166, and 6,333,311, which are incorporated herein
by reference. It is envisioned that at least the N-terminal glycine
amino acid residue can be replaced or substituted with any of the
twenty natural occurring amino acids, for example a positively
charged amino acid (arginine, lysine, or histidine), a neutral
amino acid (alanine, asparagine, cysteine, glutamine, glycine,
isoleucine, leucine, methionine, phenylaline, proline, serine,
threonine, tryptophan, tyrosine, valine) and/or a negatively
charged amino acid (aspartic acid or glutamic acid). Still further,
it is contemplated that any amino acid residue within the range of
N1 to N16 can be replaced or substituted. It is envisioned that at
least up to 16 of the N-terminal amino acids residues can be
replaced or substituted as long as the protein retains it
biological and/or functional activity, which is stimulating the
production of various cytokines, (e.g., IL-18, MIP-3.alpha., GM-CSF
or IFN-.gamma.) by inhibiting various cytokines, (e.g., IL-2, IL-4,
IL-5, IL-10, and TNF-.alpha.) by improving a cardiovascular
disease, e.g., atherosclerosis, or the parameters relating to
cardiovascular disease including circulating levels of total
cholesterol, HDL, LDL, VLDL, trigylcerides and C-reactive protein
(CRP). Thus, the N-terminal lactoferrin variants of the present
invention are considered functional equivalents of lactoferrin.
[0064] In terms of functional equivalents, it is well understood by
the skilled artisan that, inherent in the definition of a
"biologically functional equivalent" protein is the concept that
there is a limit to the number of changes that may be made within a
defined portion of the molecule while retaining a molecule with an
acceptable level of equivalent biological activity and/or enhancing
the biological activity of the lactoferrin molecule. Biologically
functional equivalents are thus defined herein as those proteins in
which selected amino acids (or codons) may be substituted.
Functional activity is defined as the ability of lactoferrin to
stimulate or inhibit various cytokines or chemokines and/or by
improving a cardiovascular disease, e.g., atherosclerosis, or the
parameters relating to cardiovascular disease including circulating
levels of total cholesterol, HDL, LDL, VLDL, trigylcerides and
C-reactive protein (CRP).
[0065] Still further, the N-terminal amino acid residues can be
substituted with a modified and/or unusual amino acids. A table of
exemplary, but not limiting, modified and/or unusual amino acids is
provided herein below. TABLE-US-00001 TABLE 1 Modified and/or
Unusual Amino Acids Abbr. Amino Acid Abbr. Amino Acid Aad
2-Aminoadipic acid EtAsn N-Ethylasparagine BAad 3-Aminoadipic acid
Hyl Hydroxylysine BAla beta-alanine, beta-Amino- AHyl
allo-Hydroxylysine propionic acid Abu 2-Aminobutyric acid 3Hyp
3-Hydroxyproline 4Abu 4-Aminobutyric acid, 4Hyp 4-Hydroxyproline
piperidinic acid Acp 6-Aminocaproic acid Ide Isodesmosine Ahe
2-Aminoheptanoic acid Aile allo-Isoleucine Aib 2-Aminoisobutyric
acid MeGly N-Methylglycine, sarcosine BAib 3-Aminoisobutyric acid
MeIle N-Methylisoleucine Apm 2-Aminopimelic acid MeLys
6-N-Methyllysine Dbu 2,4-Diaminobutyric acid MeVal N-Methylvaline
Des Desmosine Nva Norvaline Dpm 2,2'-Diaminopimelic acid Nle
Norleucine Dpr 2,3-Diaminopropionic acid Orn Ornithine EtGly
N-Ethylglycine
[0066] The presence and the relative proportion N-terminal
lactoferrin variants (deletions and/or substitutions) in a
preparation of lactoferrin (lactoferrin composition) may be done by
determination of the N-terminal amino acid sequence by the process
of Edman degradation using standard methods. A relative proportion
of N-terminal lactoferrin variant comprise at least 1% of the
lactoferrin composition, at least 5% of the lactoferrin
composition, at least 10% of the lactoferrin composition, at least
25% of the lactoferrin composition, at least 50% of the lactoferrin
composition or any range in between.
[0067] In this method, the protein is reacted with
phenylisothiocyanate (PITC), which reacts with the amino acid
residue at the amino terminus under basic conditions to form a
phenylthiocarbamyl derivative (PTC-protein). Trifluoroacetic acid
then cleaves off the first amino acid as its anilinothialinone
derivative (ATZ-amino acid) and leaves the new amino terminus for
the next degradation cycle.
[0068] The percentage of N-terminal lactoferrin variant may also be
done more precisely by using a Dansylation reaction. Briefly,
protein is dansylated using Dansyl chloride reacted with the
protein in alkaline conditions (pH 10). Following the Dansylation,
the reaction mixtures are dried to pellets, then completely
hydrolyzed in 6N HCl. The proportion of N-terminal amino acids are
identified by RP HPLC using an in-line fluorometer in comparison
with standards made up of known dansylated amino acids.
B. PHARMACEUTICAL COMPOSITIONS
[0069] The present invention is drawn to a composition comprising
lactoferrin that is dispersed in a pharmaceutical carrier, which is
used to treat cardiovascular disease. The lactoferrin that is
contained in the composition of the present invention comprises
lactoferrin or an N-terminal lactoferrin variant in which at least
the N-1 terminal glycine residue is truncated or substituted. More
specifically, the N-terminal lactoferrin variant comprises at least
1% of the composition, at least 5% of the composition, at least 10%
of the composition, at least 25% of the composition, at least 50%
of the composition or any range in between.
[0070] Yet further, the composition comprises lactoferrin in
combination with a metal chelator dispersed in a pharmaceutical
carrier. Thus, the present invention is drawn to a lactoferrin
composition with or without a metal chelator that is dispersed in a
pharmaceutical carrier. One of skill in the art understands that
both compositions (e.g., lactoferrin alone or lactoferrin in
combination with a metal chelator) are within the scope of the
present invention and can be used interchangeably depending upon
the type of response that is desired. It is envisioned that the
addition of a metal chelator to the lactoferrin composition
enhances the sequestering of metal ions and thus strengthens the
immune system or enhances the effect of lactoferrin.
[0071] Metal chelators that can be used in combination with
lactoferrin, include the divalent metal chelators, for example,
ethylenediaminetetraacetic acid (EDTA),
[ethylenebis(oxyethylenenitrilo)] tetraacetic acid (EGTA),
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA),
hydroxyethlene triamine diacetic acid, (HEDTA) or any salts
thereof. More preferably, EDTA is used in combination with
lactoferrin.
[0072] Further in accordance with the present invention, the
composition of the present invention suitable for administration is
provided in a pharmaceutically acceptable carrier with or without
an inert diluent. The carrier should be assimilable and includes
liquid, semi-solid, e.g., pastes, or solid carriers. Except insofar
as any conventional media, agent, diluent or carrier is detrimental
to the recipient or to the therapeutic effectiveness of a the
composition contained therein, its use in administrable composition
for use in practicing the methods of the present invention is
appropriate. Examples of carriers or diluents include fats, oils,
water, saline solutions, lipids, liposomes, resins, binders,
fillers and the like, or combinations thereof.
[0073] In accordance with the present invention, the composition is
combined with the carrier in any convenient and practical manner,
e.g., by solution, suspension, emulsification, admixture,
encapsulation, absorption and the like. Such procedures are routine
for those skilled in the art.
[0074] In a specific embodiment of the present invention, the
composition is combined or mixed thoroughly with a semi-solid or
solid carrier. The mixing can be carried out in any convenient
manner such as grinding. Stabilizing agents can be also added in
the mixing process in order to protect the composition from loss of
therapeutic activity, e.g., denaturation in the stomach. Examples
of stabilizers for use in an the composition include buffers, amino
acids such as glycine and lysine, carbohydrates such as dextrose,
mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol,
mannitol, etc., proteolytic enzyme inhibitors, and the like. Yet
further, it is envisioned that divalent metal chelators, for
example EDTA, can also be used to stabilize the composition of the
present invention. More preferably, for an orally administered
composition, the stabilizer can also include antagonists to the
secretion of stomach acids.
[0075] The composition for oral administration which is combined
with a semi-solid or solid carrier can be further formulated into
hard or soft shell gelatin capsules, tablets, or pills. More
preferably, gelatin capsules, tablets, or pills are enterically
coated. Enteric coatings prevent denaturation of the composition in
the stomach or upper bowel where the pH is acidic. See, e.g., U.S.
Pat. No. 5,629,001. Upon reaching the small intestines, the basic
pH therein dissolves the coating and permits the lactoferrin
composition to be released and absorbed by specialized cells, e.g.,
epithelial enterocytes and Peyer's patch M cells.
[0076] In another embodiment, a powdered composition is combined
with a liquid carrier such as, e.g., water or a saline solution,
with or without a stabilizing agent.
[0077] The compositions of the present invention may be formulated
in a neutral or salt form. Pharmaceutically-acceptable salts
include the acid addition salts (formed with the free amino groups
of the protein) and which are formed with inorganic acids such as,
for example, hydrochloric or phosphoric acids, or such organic
acids as acetic, oxalic, tartaric, mandelic, and the like. Salts
formed with the free carboxyl groups can also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the
like.
[0078] Sterile injectable solutions are prepared by incorporating
the lactoferrin in the required amount in the appropriate solvent
with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0079] Further, the composition for topical administration which is
combined with a semi-solid carrier can be further formulated into a
gel ointment. A preferred carrier for the formation of a gel
ointment is a gel polymer. Preferred polymers that are used to
manufacture a gel composition of the present invention include, but
are not limited to carbopol, carboxymethyl-cellulose, and pluronic
polymers. Gel polymers prevent denaturation of the composition in
the open skin by serum proteases.
[0080] The amount of lactoferrin in the present invention may vary
from about 1 ng to about 100 g of lactoferrin, more preferably 1 ng
to 20 g per day, most preferably 1 .mu.g to 5 g. In preferred
embodiments, the composition of the present invention comprises a
lactoferrin concentration of about 0.1% to about 100%. The
lactoferrin composition may comprise lactoferrin or an N-terminal
lactoferrin variant in which at least the N-1 terminal glycine
residue is truncated and/or substituted.
[0081] More preferably, the composition of the present invention
also contains metal chelators, for example, but not limited to
ethylenediaminetetraacetic acid (EDTA), [ethylenebis
(oxyethylenenitrilo)]tetraacetic acid (EGTA),
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA),
hydroxyethlene triamine diacetic acid, (HEDTA) or salts thereof.
The amount of the metal chelator in the composition may vary from
about 1 ng to about 1 g. A preferred metal chelator is EDTA.
[0082] Upon formulation, solutions are administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective to result in an improvement or
remediation of the symptoms. The formulations are easily
administered in a variety of dosage forms such as ingestible
solutions, drug release capsules and the like. Some variation in
dosage can occur depending on the condition of the subject being
treated. The person responsible for administration can, in any
event, determine the appropriate dose for the individual
subject.
C. TREATMENT OR PROPHYLAXIS OF CARDIOVASCULAR DISEASE
[0083] In accordance with the present invention, the composition
provided in any of the above-described pharmaceutical carriers is
administered to a subject who has experienced or is at risk of
developing cardiovascular disease. Risk factors include, but are
not limited to elevated levels of cholesterol or CRP. One of skill
in the art can determine the patients who would potentially benefit
from a therapeutic agent that would reduce circulating levels of
total cholesterol or triglycerides or cardiovascular inflammation.
One of skill in the art can determine the therapeutically effective
amount of the composition to be administered to a subject based
upon several considerations, such as local effects,
pharmacodynamics, absorption, metabolism, method of delivery, age,
weight, disease severity and response to the therapy.
[0084] Oral administration of the composition includes oral,
buccal, enteral or intragastric administration. It is also
envisioned that the composition may be used as a food additive. For
example, the composition is sprinkled on food or added to a liquid
prior to ingestion.
[0085] In addition to oral administration, the lactoferrin
composition can also be administered parenterally, which includes,
but is not limited to intradermal, subcutaneous, intramuscular,
intraperitoneal, intravenous, intraarterial, intramyocardial,
transendocardial, transepicardial, intrathecal, and infusion
techniques.
[0086] Cardiovascular diseases and/or disorders include, but are
not limited to, diseases and/or disorders of the pericardium, heart
valves (e.g., incompetent valves, stenosed valves, Rheumatic heart
disease, mitral valve prolapse, aortic regurgitation), myocardium
(e.g., coronary artery disease, myocardial infarction, heart
failure, ischemic heart disease, angina) blood vessels (e.g.,
hypertension, arteriosclerosis, aneurysm) or veins (e.g., varicose
veins, hemorrhoids). In specific embodiments, the cardiovascular
disease is atherosclerosis.
[0087] In specific embodiments of the present invention, the
lactoferrin composition is administered to a subject suffering from
or at risk for developing atherosclerosis. Thus, it is envisioned
that the lactoferrin composition modulates or reduces the severity
and/or incidence of atherosclerosis.
[0088] Prophylactic treatment can be administered to those subjects
at risk for developing atherosclerosis. One risk factor is an
atherogenic lipoprotein profile. For example, a ratio of serum
cholesterol to high density lipoproteins of above 5:1 indicates a
higher than average risk of developing atherosclerosis. Other
factors indicating increased risk for atherosclerosis include a
serum cholesterol level of above 240 mg/dl; a high density
lipoprotein level below about 35 mg/dl; and a low density
lipoprotein level above about 160 mg/dl.
[0089] Another embodiment includes treating a human subject with an
elevated level of circulating total cholesterol or CRP according to
the then medically established guidelines. It is contemplated that
the lactoferrin composition of the present invention reduces or
attenuates the levels of circulating total cholesterol, low density
lipoproteins or very low density lipoproteins. It is contemplated
that the lactoferrin composition of the present invention can
interfere with how cholesterol enters the circulation either via
absorption from food (exogenous pathway) or synthesis by the liver
(endogenous pathway).
[0090] In further embodiments, the composition is administered in
conjunction with an antacid. Thus, an antacid is administered prior
or substantially simultaneously with or after oral administration
of the composition. The administration of an antacid just prior or
immediately following the administration of the composition may
help to reduce the degree of inactivation of the lactoferrin in the
digestive tract. Examples of appropriate antacids include, but are
not limited to, sodium bicarbonate, magnesium oxide, magnesium
hydroxide, calcium carbonate, magnesium trisilicate, magnesium
carbonate and alumin hydroxide gel.
[0091] In a preferred embodiment of the present invention, the
composition is administered in an effective amount to decrease,
reduce, inhibit or abrogate cardiovascular disease. Thus, a subject
is administered a therapeutically effective amount of a lactoferrin
composition so that the subject has an improvement in the
parameters relating to cardiovascular disease including circulating
levels of total cholesterol, HDL, LDL, VLDL, trigylcerides and
C-reactive protein (CRP). The amount of lactoferrin in the
composition may vary from about 1 ng to about 20 g. Preferably, the
composition that is orally administered contains the range of 0.5 g
to 5 g of lactoferrin per day.
[0092] The improvement is any observable or measurable improvement.
Thus, one of skill in the art realizes that a treatment may improve
the patient condition, but may not be a complete cure of the
disease. In certain aspects, the composition is administered in an
effective amount to decrease, reduce, inhibit or abrogate excess
amounts of cholesterol levels in circulation. A subject requires
treatment for cholesterol levels based upon any of the following
situations: LDL of 160 mg/ml or greater; LDL of 130-159 mg/ml and
also have two or more cardiovascular risk factors; LDL of 100 mg/ml
or greater in subjects with coronary heart disease (CHD);
triglycerides of 200 mg/dl or higher; total cholesterol of 240
mg/dl or higher or HDL of less than 40 mg/dl. Thus, after
administration of lactoferrin, if any of the above conditions
improve, then the amount of lactoferrin is considered an effective
amount to decrease, reduce, inhibit or abrogate cholesterol levels
in the circulation.
[0093] Another embodiment is a method of reducing vascular
inflammation by administering the lactoferrin composition of the
present invention. Vascular inflammation can be tied to a number of
the underlying processes contributing to atherosclerosis which
include endothelial dysfunction, vascular proliferation and matrix
alteration. Recent studies have emphasized the involvement of
inflammation in mediating all stages of atherosclerosis. Vascular
inflammation is thought to be a consequence of damage to the
vascular endothelium and may also involve the proliferation of
vascular smooth muscle cells (vsmcs). One precursor of lesion
development in humans may be focal accumulation of vsmcs within the
intima. In early atherosclerosis, vsmcs may contribute to the
development of the atheroma through the production of
pro-inflammatory mediators such as monocyte chemoattractant protein
1 and vascular cell adhesion molecule, and through the synthesis of
matrix molecules required for the retention of lipoproteins.
Inflammation of the vascular endothelium and proliferation of vsmcs
may also impact the stability of the plaque through the formation
of a firm fibrous cap. Indeed, in lipid-laden lesions in which the
fibrous cap is thin and weak, there is evidence of vsmc apoptosis,
especially at the "shoulder" region, associated with inflammation.
In addition, the local inflammatory milieu can induce expression of
collagenase and inhibit expression of proteolytic inhibitors, thus
rendering the fibrous cap weak and susceptible to rupture.
Lactoferrin, having known anti-inflammatory properties, may thus
serve to inhibit the underlying processes associated with the
development of atherosclerosis.
[0094] In further embodiments, the lactoferrin composition may also
reduce vascular spasms or vascular hyper-reactivity. Vascular
spasms are a sudden, brief tightening of a blood vessel, which can
temporarily reduce blood flow to tissues supplied by that
vessel.
[0095] Still further, the lactoferrin composition may also promote
endothelial integrity or healing. Endothelia are the layer of cells
lining the blood vessels. Endothelial dysfunction most commonly
refers to impairment of endothelium-dependent vasodilation and
widespread abnormalities in endothelial integrity and homeostasis.
It is believed that HDLs help maintain endothelial integrity,
facilitate vascular relaxation, inhibit blood cell adhesion to
vascular endothelium, reduce platelet aggregability and
coagulation, and may favor fibrinolysis. The integrity or
completeness of the endothelia lining of the vessels is important
to preventing/treating the development of plaques and
atherosclerosis. Thus, it is envisioned that the lactoferrin
composition of the present invention will promote or modulate
endothelial integrity or healing.
[0096] Yet further, another embodiment is a method of preventing a
cardiovascular disease in a subject at risk for developing a
cardiovascular disease comprising the step of administering to the
subject a lactoferrin composition in an amount sufficient to result
in prophylaxis of the cardiovascular disease in the subject. In
preferred embodiments, the cardiovascular disease is
atherosclerosis. It is envisioned that the lactoferrin composition
not only possess therapeutic benefits for those subjects suffering
from cardiovascular diseases, but also possess prophylactic
properties for those subjects at risk for developing cardiovascular
disease. A subject at risk may or may not be cognizant of their
disease state or potential disease state and may or may not be
aware that they are need of treatment.
[0097] Thus, prophylatically, it is envisioned that the lactoferrin
composition can reduce any of the following: the levels of
circulating total cholesterol, low density lipoproteins (LDL), very
low density lipoproteins (VLDL), levels of vascular inflammation,
circulating C-reactive protein (CRP), triglycerides, and the
proliferation of vascular smooth muscle cells in the subject. Yet
further, the lactoferrin composition may also increase the levels
of circulating high density lipoproteins (HDL).
[0098] Still yet, a further embodiment is a method of enhancing
immune response in a subject comprising the step of administering
to the subject the composition of the present invention. It is
envisioned that the immune response, whether local, systemic or
mucosal, is enhanced by lactoferrin stimulating cytokines and/or
chemokines. Exemplary cytokines include interleukin-18 and GM-CSF
in the gastrointestinal tract, which are known to enhance immune
cells or stimulate production of immune cells. For example,
interleukin-18 enhances natural killer cells or T lymphocytes. In
specific embodiments, interleukin-18 (IL-18) enhances CD4+, CD8+
and CD3+cells. It is known by those of skill in the art that IL-18
is a Th.sub.1 cytokine that acts in synergy with interleukin-12 and
interleukin-2 in the stimulation of lymphocyte IFN-gamma
production. Other cytokines or chemokines may also be enhanced for
example, but not limited to IL-12, IL-1b, MIP-3.alpha.,
MIP-1.alpha. or IFN-.gamma.. Other cytokines or enzymes may be
inhibited for example, but not limited to IL-2, IL-4, IL-5, IL-10,
TNF-.alpha., or matrix metalloproteinases. It is further
contemplated that lactoferrin inhibits the production of
TNF-.alpha., which inhibits cells involved in inflammation. It is
also envisioned that lactoferrin stimulates interleukin-18 and a
Th.sub.1 response following oral administration, which inhibits
pro-inflammatory cytokines, e.g., IL-4, IL-5, IL-6, IL-8 and
TNF-.alpha..
[0099] The lactoferrin composition of the present invention can
also result in inhibition of a cytokine or chemokine. The cytokines
include, but are not limited to interleukin-2 (IL-2), interleukin-4
(IL-4), interleukin-5 (IL-5), interleukin-10 (IL-10), and tumor
necrosis factor alpha (TNF-.alpha.). Still further, the lactoferrin
composition can also inhibit the production of matrix
metalloproteinases (MMPs).
[0100] In further embodiments, cytokines, for example,
interleukin-18 or granulocyte/macrophage colony-stimulating factor,
can stimulate the production or activity of immune cells. The
immune cells include, but are not limited to T lymphocytes, natural
killer cells, NK-T cells, macrophages, dendritic cells, and
polymorphonuclear cells. More specifically, the polymorphonuclear
cells are neutrophils and the T lymphocytes are selected from the
group consisting of CD4+, CD8+ and CD3+T cells.
[0101] In further embodiments, the composition of the present
invention also contains metal chelators, for example, but not
limited to ethylenediaminetetraacetic acid (EDTA), [ethylenebis
(oxyethylenenitrilo)]tetraacetic acid (EGTA),
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA),
hydroxyethlene triamine diacetic acid, (HEDTA) or salts
thereof.
[0102] Treatment regimens may vary as well, and often depend on the
health and age of the patient. Obviously, certain types of disease
will require more aggressive treatment, while at the same time,
certain patients cannot tolerate more taxing regimens. The
clinician will be best suited to make such decisions based on the
known efficacy and toxicity (if any) of the therapeutic
formulations.
[0103] In specific embodiments, the composition is given in a
single dose or multiple doses. The single dose may be administered
daily, or multiple times a day, or multiple times a week, or
monthly or multiple times a month. In a further embodiment, the
lactoferrin is given in a series of doses. The series of doses may
be administered daily, or multiple times a day, weekly, or multiple
times a week, or monthly, or multiple times a month.
D. COMBINATION TREATMENTS
[0104] In order to increase the effectiveness of the composition,
it may be desirable to combine these compositions and methods of
the invention with a known agent effective in the treatment or
prevention of cardiovascular disease or disorder, for example known
agents to treat or prevent atherosclerosis. In some embodiments, it
is contemplated that a conventional therapy or agent, including but
not limited to, a pharmacological therapeutic agent, a surgical
therapeutic agent (e.g., a surgical procedure) or a combination
thereof, may be combined with the composition of the present
invention.
[0105] The composition of the present invention may precede, be
co-current with and/or follow the other agent(s) by intervals
ranging from minutes to weeks. In embodiments where the composition
of the present invention, and other agent(s) are applied separately
to a cell, tissue or organism, one would generally ensure that a
significant period of time did not expire between the time of each
delivery, such that the composition and agent(s) would still be
able to exert an advantageously combined effect on the cell, tissue
or organism.
[0106] Various combination regimens of the composition and one or
more agents are employed. One of skill in the art is aware that the
composition of the present invention and agents can be administered
in any order or combination. In other aspects, one or more agents
may be administered substantially simultaneously, or within about
minutes to hours to days to weeks and any range derivable therein,
prior to and/or after administering the composition.
[0107] Administration of the composition to a cell, tissue or
organism may follow general protocols for the administration of
cardiovascular therapeutics, taking into account the toxicity, if
any. It is expected that the treatment cycles would be repeated as
necessary. In particular embodiments, it is contemplated that
various additional agents may be applied in any combination with
the present invention.
[0108] A. Pharmacological Therapeutic Agents
[0109] Pharmacological therapeutic agents and methods of
administration, dosages, etc. are well known to those of skill in
the art (see for example, the "Physicians Desk Reference", Goodman
& Gilman's "The Pharmacological Basis of Therapeutics",
"Remington's Pharmaceutical Sciences", and "The Merck Index,
Eleventh Edition", incorporated herein by reference in relevant
parts), and may be combined with the invention in light of the
disclosures herein. Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person
responsible for administration will, in any event, determine the
appropriate dose for the individual subject, and such individual
determinations are within the skill of those of ordinary skill in
the art.
[0110] Non-limiting examples of a pharmacological therapeutic agent
that may be used in the present invention include an
antihyperlipoproteinemic agent, an antiarteriosclerotic agent, an
anti-cholesterol agent, an anti-inflammatory agent, an
antithrombotic/fibrinolytic agent, a blood coagulant, an
antiarrhythmic agent, an antihypertensive agent, or a vasopressor.
In certain aspects of the present invention, anti-cholesterolemic
agents are used in combination with the composition of the present
invention. Anti-cholesterol agents include but are not limited to
HMG-CoA Reductase inhibitors, cholesterol absorption inhibitors,
bile acid sequestrants, nicotinic acid and derivatives thereof,
fibric acid and derivatives thereof. HMG-CoA Reductase inhibitors
include statins, for example, but not limited to atorvastatin
calcium (Lipitor.RTM.), cerivastatin sodium (Baycol.RTM.),
fluvastatin sodium (Lescol.RTM.), lovastatin (Advicor.RTM.),
pravastatin sodium (Pravachol.RTM.), and simvastatin (Zocor.RTM.).
Agents known to reduce the absorption of ingested cholesterol
include, for example, ezetimibe (Zetia.RTM.). Bile acid
sequestrants include, but are not limited to cholestryramine,
cholestipol and colesevalam. Other anti-cholesterol agents include
fibric acids and derivatives thereof (e.g., gemfibrozil,
fenofibrate and clofibrate); nicotinic acids and derivatives
thereof (e.g., nician, lovastatin) and agents that extend the
release of nicotinic acid, for example niaspan. Anti-inflammatory
agents include, but are not limited to non-sterodial
anti-inflammatory agents (e.g., naproxen, ibuprofen, celeoxib) and
sterodial anti-inflammatory agents (e.g., glucocorticoids).
[0111] B. Surgical Therapeutic Agents
[0112] In certain aspects, a therapeutic agent may comprise a
surgery of some type, which includes, for example, preventative,
diagnostic or staging, curative and palliative surgery. Surgery,
and in particular a curative surgery, may be used in conjunction
with other therapies, such as the present invention and one or more
other agents.
[0113] Such surgical therapeutic agents for cardiovascular diseases
and disorders are well known to those of skill in the art, and may
comprise, but are not limited to, performing surgery on an
organism, providing a cardiovascular mechanical prostheses,
angioplasty, coronary artery reperfusion, catheter ablation,
providing an implantable cardioverter defibrillator to the subject,
mechanical circulatory support or a combination thereof.
Non-limiting examples of a mechanical circulatory support that may
be used in the present invention comprise an intra-aortic balloon
counterpulsation, left ventricular assist device or combination
thereof.
E. EXAMPLES
[0114] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Cholesterol Reduction by Recombinant Human Lactoferrin (rhLF)
[0115] Healthy adult human volunteers were administered a liquid
formulation of either rhLF or placebo for seven days. Fasting serum
was collected at baseline (prior to administration of rhLF) and at
the end of study (Day 10) and levels of total cholesterol
determined. All subjects were housed in an in-patient setting for
the duration of the study and received similar diets.
[0116] In this clinical trial, human subjects treated with placebo
drug experienced a rise in serum cholesterol. RhLF treated subjects
in a similar setting and receiving a similar diet, experienced a
reduction in total cholesterol. FIG. 1 shows that administration of
rhLF for just seven days resulted in 14% reduction in total
cholesterol (P<0.05).
Example 2
Reduction of C-Reactive Protein (CRP) by Recombinant Human
Lactoferrin (rhLF)
[0117] Healthy adult human volunteers were administered a liquid
formulation of rhLF or placebo for seven days. RhLF was
administered for a total of seven days. Serum was collected on Day
1 and Day 7 of rhLF administration and assayed for CRP using a high
sensitive assay.
[0118] A total of six subjects had Day 1 CRP levels that were
measurable by the high sensitive assay (>0.07 mg/dL). Five out
of the six subjects showed a reduction in CRP with the sixth
patient showing no change. As shown in FIG. 2, the six evaluable
subjects showed an average of 49% reduction in CRP levels
(P<0.05) as well as a 17% reduction in their cardiovascular
risk.
Example 3
RhLF Effect on Hyperlipidemia in Mice
[0119] The effect of oral rhLF was tested in a mouse model of
hyperlipidemia induced by the mice being fed a high cholesterol-fat
diet for 14 days (2 g lard, 8 g coconut oil, 1 g cholesterol, 0.3 g
cholic acid per 100 g of feed and 88.7 g standard chow). The
hyperlipidemic mice were administered either placebo vehicle or
rhLF (1000 mg/kg) twice a day for seven days. Twenty-four hours
after the last dose, serum was obtained from individual fasting
animals and assayed for total cholesterol, HDL and LDL. The rhLF
treated mice showed a trend toward decrease in total cholesterol
(16%) and LDL cholesterol (23%) and an increase in HDL cholesterol
(15%). The HDL/LDL ratio was increased by 48.5% and was
statistically significant (FIG. 3).
Example 4
Dose Effects of RhLF on Hyperlipidemia in Mice
[0120] Mice were rendered hypercholesteremic by administration of a
high cholesterol diet for fourteen days. Animals received rhLF
(32.5, 150 or 500 mg/kg) or placebo administered orally twice a day
for 14 consecutive days. Twenty-four hours after the last dose of
drug, fasting animals were sacrificed and serum total cholesterol
(Total, high density lipoprotein (HDL), low density lipoprotein
(LDL) and Total/HDL) ratio were determined.
[0121] High cholesterol diet used to induce hyperlipidemia
consisted of 2 g lard, 8 g coconut oil, 1 g cholesterol, 0.3 g
cholic acid per 100 g of feed and 88.7 g standard chow.
[0122] At the doses tested, a beneficial effect from rhLF was
observed after 14 days treatment. No dose dependence was observed.
As shown in FIG. 4, all doses of rhLF decreased both total
cholesterol and LDL-cholesterol relataive to placebo treated
animals. A statistically significant decrease in LDL-cholesterol
was observed (25% decrease, p<0.05) when all the rhLF treated
animals were compared to the placebo treated animals. There was
also a decrease in total cholesterol (19% reduction; p=0.0594).
Example 5
Effect of Oral rhLF with Lovastatin on Hyperlipidemia in Mice
[0123] The ability of oral rhLF to potentiate the effect of
lovastatin in the induced hyperlipidemia model was also tested.
Hyperlipidemic mice were treated with lovastatin (15 mg/kg) alone
or in combination with rhLF (500 mg/kg b.i.d.) for seven days. Mice
treated with lovastatin plus lactoferrin had astatistically
significant (p<0.05) reduction in both Total Cholesterol and
LDL-Cholesterol relative to the lovastatin treated mice (18% and
25% respectively) and a 13% increase in HDL cholesterol (FIG.
5).
Example 6
Reduction of Systemic Inflammation in Murine Models
[0124] Mice and rats with elevated systemic or cardiovascular
inflammation are administered placebo or rhLF for 7, 14 or 28 days
and markers of inflammation including CRP are assayed.
Example 7
Reduction of Cholesterol with rhLF therapy
[0125] Human patients with elevated levels of cholesterol (>200
mg/dL) are administered rhLF or placebo for 14, 28 and 90 days.
Fasting serum is assayed for total cholesterol, HDL, LDL, VLDL, and
triglycerides.
Example 8
Reduction of CRP with rhLF therapy
[0126] Human patients with elevated levels of CRP are administered
rhLF or placebo for 14, 28 and 90 days. Serum is assayed for CRP
and other markers of inflammation.
Example 9
Dose Ranging Study of rhLF in the Reduction of Cholesterol and
CRP
[0127] Human patients with elevated levels of cholesterol (>200
mg/dL) are given placebo or ascending doses of rhLF for 30, 90 and
180 days. Fasting serum is assayed for total cholesterol, HDL, LDL,
VLDL, triglycerides and CRP.
Example 10
Reduction of Cholesterol and CRP with rhLF in Combination
Therapy
[0128] Human patients with elevated levels of cholesterol (>200
mg/dL) are administered an approved cholesterol reducing drug
either alone or in combination with rhLF for 30, 90 and 180 days.
Cholesterol reducing drugs tested include Lipitor.RTM.
(atorvastatin), Zocor.RTM. (simvastatin), and Zetia.RTM.
(ezetimibe). Fasting serum is assayed for total cholesterol, HDL,
LDL, VLDL, triglycerides and CRP.
Example 11
Reduction of Cardiovascular Incidence with rhLF Alone or in
Combination Therapy
[0129] Human patients considered at an elevated risk for
cardiovascular accidents (including stroke and heart attacks) are
treated with rhLF alone, approved drugs alone or a combination of
rhLF and an approved drug. Fasting serum is assayed for total
cholesterol, HDL, LDL, VLDL, triglycerides and CRP. Incidence and
severity of stroke and heart attacks and incidence of mortality are
also measured.
REFERENCES CITED
[0130] All patents and publications mentioned in the specifications
are indicative of the levels of those skilled in the art to which
the invention pertains. All patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference. [0131] U.S. Pat. No. 5,571,691 [0132]
U.S. Pat. No. 5,571,697 [0133] U.S. Pat. No. 5,571,896 [0134] U.S.
Pat. No. 5,629,001 [0135] U.S. Pat. No. 6,080,559 [0136] U.S. Pat.
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6,333,311
[0143] Although the present invention and its advantages have been
described in detail, it should be understood that various changes,
substitutions and alterations can be made herein without departing
from the invention as defined by the appended claims. Moreover, the
scope of the present application is not intended to be limited to
the particular embodiments of the process, machine, manufacture,
composition of matter, means, methods and steps described in the
specification. As one will readily appreciate from the disclosure,
processes, machines, manufacture, compositions of matter, means,
methods, or steps, presently existing or later to be developed that
perform substantially the same function or achieve substantially
the same result as the corresponding embodiments described herein
may be utilized. Accordingly, the appended claims are intended to
include within their scope such processes, machines, manufacture,
compositions of matter, means, methods, or steps.
* * * * *