U.S. patent application number 16/223429 was filed with the patent office on 2019-04-25 for method of treating acute coronary syndromes.
The applicant listed for this patent is BIOrest Ltd.. Invention is credited to Haim D. DANENBERG, Elazer R. EDELMAN, Gershon GOLOMB, Yoram RICHTER.
Application Number | 20190117678 16/223429 |
Document ID | / |
Family ID | 33540317 |
Filed Date | 2019-04-25 |
United States Patent
Application |
20190117678 |
Kind Code |
A1 |
RICHTER; Yoram ; et
al. |
April 25, 2019 |
Method of Treating Acute Coronary Syndromes
Abstract
The present invention relates to methods and compositions
designed for the treatment or management of acute coronary
syndromes, particularly, unstable angina and acute myocardial
infarction. The methods of the invention comprise the
administration of an effective amount of a formulation containing
one or more therapeutic agents which specifically decreases or
inhibits the activity of phagocytic cells and/or eliminates or
diminishes the amount of phagocytic cells including, but not
limited to, macrophages and monocytes. The formulations are
specifically targeted to phagocytic cells. The invention also
provides pharmaceutical compositions of formulations containing one
or more therapeutic agents of the invention for administration to
subjects currently suffering from or having recently suffered an
acute coronary syndrome such as unstable angina and acute
myocardial infarction.
Inventors: |
RICHTER; Yoram; (Ramat
Hasharon, IL) ; EDELMAN; Elazer R.; (Brookline,
MA) ; GOLOMB; Gershon; (Efrat, IL) ;
DANENBERG; Haim D.; (Mevaseret Zion, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIOrest Ltd. |
Tel Aviv |
|
IL |
|
|
Family ID: |
33540317 |
Appl. No.: |
16/223429 |
Filed: |
December 18, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15791816 |
Oct 24, 2017 |
10213446 |
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16223429 |
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15336395 |
Oct 27, 2016 |
9827254 |
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15791816 |
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10871488 |
Jun 18, 2004 |
9498488 |
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15336395 |
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10607623 |
Jun 27, 2003 |
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10871488 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 9/10 20180101; A61K
9/127 20130101; A61K 31/663 20130101; A61P 9/00 20180101; A61K 9/51
20130101; A61K 31/66 20130101; A61P 43/00 20180101; A61K 31/675
20130101 |
International
Class: |
A61K 31/663 20060101
A61K031/663; A61K 31/66 20060101 A61K031/66; A61K 31/675 20060101
A61K031/675; A61K 9/127 20060101 A61K009/127; A61K 9/51 20060101
A61K009/51 |
Claims
1. A method of treating acute myocardial infarction comprising
administering an effective amount of a formulation to a patient in
need thereof, said formulation comprising a bisphosphonate which is
encapsulated in a particle, embedded in a particle, or in a
particulate, said particle or particulate having a size of 0.05 to
1.0 microns, wherein said bisphosphonate is phagocytosed and acts
intracellularly, thereby decreasing phagocytic cell activity.
2. A method of treating acute myocardial infarction comprising
administering an effective amount of a formulation to a patient in
need thereof, said formulation comprising a bisphosphonate which is
encapsulated in a particle, embedded in a particle, or in a
particulate, said particle or particulate having a size of 0.05 to
1.0 microns, wherein said bisphosphonate is phagocytosed and acts
intracellularly, thereby decreasing phagocytic cell numbers.
3. A method of treating acute myocardial infarction comprising
administering an effective amount of a formulation to a patient in
need thereof, said formulation comprising a bisphosphonate which is
encapsulated in a particle, embedded in a particle, or in a
particulate, said particle or particulate having a size of 0.05 to
1.0 microns and said formulation inhibiting macrophages or
monocytes.
4. A method of treating acute myocardial infarction comprising
administering an effective amount of a formulation to a patient in
need thereof, said formulation comprising a bisphosphonate
encapsulated in a liposome, said liposome having a size of 0.05 to
1.0 microns, and said bisphosphonate acting intracellularly,
thereby decreasing phagocytic cell activity.
5. The method of any one of claims 1-4, wherein said acute
myocardial infarction is caused by at least one of a vascular
injury and thrombosis in a coronary vessel, thereby causing
occlusion of the coronary vessel.
6. The method of any one of claims 1-4, wherein said formulation
causes minimization of the group consisting of: an infarct size, an
amount of myocardial necrosis, and combination thereof.
7. The method of any one of claims 1-4, wherein said formulation is
administered to a patient at increased risk of acute myocardial
infarction.
8. The method of any one of claims 1-4, wherein said bisphosphonate
comprises a compound having formula (I): ##STR00003## wherein
R.sub.1 is H, OH or halogen group; and R.sub.2 is halogen; linear
or branched C.sub.1-C.sub.10 alkyl or C.sub.2-C.sub.10 alkenyl,
optionally substituted by heteroaryl or heterocyclyl
C.sub.1-C.sub.10 alkylamino or C.sub.3-C.sub.8 cycloalkylamino;
--NHY where Y is hydrogen, C.sub.3-C.sub.8 cycloalkyl, aryl or
heteroaryl; or --SZ, where Z is chlorosubstituted phenyl or
pyridinyl.
9. The method of any one of claims 1-4, wherein said bisphosphonate
is selected from the group consisting of clodronate, etidronate,
tiludronate, pamidronate, alendronate and risendronate.
10. The method of any one of claims 1-4, wherein multiple
therapeutic agents are contained in the formulation, and wherein at
least one of the therapeutic agents comprises a bisphosphonate.
11. The method of any one of claims 1-3, wherein said
bisphosphonate is encapsulated in a liposome.
12. The method of any one of claims 1-3, wherein said
bisphosphonate is embedded in a carrier selected from the group
consisting of microparticles, nanoparticles, microspheres, and
nanospheres.
13. The method of any one of claims 1-3, wherein said
bisphosphonate is formulated as a particulate selected from the
group consisting of aggregates, flocculates, colloids, polymer
chains, insoluble salts and insoluble complexes.
14. The method of any one of claims 1-3, wherein said
bisphosphonate is encapsulated in a particle and said particle
comprises cholesterol.
15. The method of claim 4, wherein said liposome comprises
cholesterol.
16. The method of any one of claims 1-4, wherein said formulation
is administered to the patient during the acute myocardial
infarction.
17. The method of any one of claims 1-4, wherein said formulation
is administered to the patient at least one of prior to and after
onset of the acute myocardial infarction.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 15/791,816, filed Oct. 24, 2017, which is a continuation of
U.S. application Ser. No. 15/336,395 filed Oct. 27, 2016, issued as
U.S. Pat. No. 9,827,254 on Nov. 28, 2017, which is a continuation
of U.S. application Ser. No. 10/871,488 filed Jun. 18, 2004, issued
as U.S. Pat. No. 9,498,488 on Nov. 22, 2016, which is a
continuation-in-part of U.S. application Ser. No. 10/607,623 filed
Jun. 27, 2003, which is incorporated by reference herein in its
entirety.
1. FIELD OF INVENTION
[0002] The present invention relates to methods and compositions
designed for the treatment or management of acute coronary
syndromes, particularly, unstable angina and acute myocardial
infarction. The methods of the invention comprise the
administration of an effective amount of a formulation containing
one or more therapeutic agents which specifically decreases or
inhibits the activity of and/or eliminates or diminishes the amount
of phagocytic cells including, but not limited to, macrophages and
monocytes.
2. BACKGROUND OF THE INVENTION
[0003] Coronary artery disease is a leading cause of death in
industrialized countries. In the United States, 50-60% of heart
attacks occur in people without documented coronary artery disease.
A chief contributor to the pathology of the disease is the
formation of atherosclerotic plaques. Atherosclerotic plaques are
thickened areas in vessel walls which result from an accumulation
of cholesterol, proliferating smooth muscle cells, and inflammatory
cells.
Atherosclerotic Plaques
[0004] In general, an atherosclerotic plaque consists of a raised
focal point within the intima having a central core of
extra-cellular lipids covered by a fibrous cap. The core within the
plaque contains crystalline cholesterol, cholesterol esters,
phospholipids, cellular degradation products and collagen remnants.
The fibrous cap separates the core of the plaque from the lumen of
the blood vessel or artery and is comprised mainly of connective
tissues that are a dense, fibrous extracellular matrix made up of
collagens, elastins, proteoglycans and other extracellular matrix
materials. The fibrous cap varies in thickness, number of smooth
muscle cells and macrophages, and collagen content. (Vallabhajosula
et aI., 1997, J. Nucl. Med. 38(11): 1788-1796).
[0005] Atherosclerotic plaques can be characterized as active and
prone to rupture ("vulnerable or high-risk plaques") or inactive
and relatively stable ("stable plaques"). A vulnerable, high-risk
or rupture-prone plaque is characterized by an abundance of
inflammatory cells (such as macrophages), a thin fibrous cap, and a
large lipid core. The size of the lipid pool within the
atherosclerotic plaque and the thickness of the overlying fibrous
cap are important characteristics predicting the stability of the
plaque. The edge of the fibrous cap (the shoulder region) is a
location of high stress and predisposed to rupture, in part, due to
the accumulation of inflammatory cells (such as macrophages) in the
area and their secretion of enzymes that cause degradation of the
material that makes up the fibrous cap (Moreno et al, Circulation.,
1994, 90:775-8; van der Wal et al., 1994, Circulation 89:36-44.;
Jander et al, 1998, Stroke, 29:1625-1630) which can lead to rupture
of the plaque.
[0006] Rupture of the lipid-laden plaque exposes the highly
thrombogenic core and the sub-endothelial vascular smooth muscle
cell component of the arterial wall to circulating blood. Platelet
activation, adhesion and aggregation follow this almost
immediately. Platelet adhesion and activation results in the
release of coagulation factors and the initiation of the
coagulation cascade. The released growth factors, specifically
platelet-derived growth factor (PDGF) stimulate the proliferation
and migration of vascular smooth muscle cells. Proliferation and
migration of vascular smooth muscle cells can lead to plaque
remodeling and increased vascular stenosis, or interact with the
platelets leading to enhanced thrombogenesis (Pasterkamp et al.,
2000, J. Clin. Basic Cardiol. 3:81-86). The resulting thrombosis
caused by the vulnerable plaque can cause unstable angina, acute
myocardial infarction, stroke, acute deterioration in peripheral
artery disease, or sudden coronary death.
Unstable Angina
[0007] The heart requires oxygen-rich blood to function. The right
and left coronary arteries branch from the aorta and carry
oxygenated blood to the tissues of the heart. When the coronary
arteries fail to deliver an adequate amount of oxygen-rich blood (a
condition called hypoxia) to the heart, chest pain, pressure, or
discomfort, commonly known as angina, result. If this situation is
prolonged, oxygen depravation can damage the heart muscle itself (a
situation known as ischemia) either reversibly or irreversibly.
[0008] Angina is classified broadly as stable or unstable,
depending on its severity and pattern of occurrence. Stable angina
occurs when increased physical activity (e.g., hurrying across a
street or climbing a long flight of stairs) raises the demand for
oxygen-rich blood. Due to a possible multitude of factors (the most
common of which is one or more occluded coronary arteries), the
supply created by the coronary blood flow cannot meet this
increased demand and hypoxia results. Unstable angina is understood
as anginal pain that occurs with lesser degrees of exertion,
increasing frequency, or at rest (i.e., without exertion). Unstable
angina that occurs at rest represents the condition in its most
serious form. It usually is caused by the formation of a blood clot
in a coronary artery at the site of a ruptured plaque and, if left
untreated, it may result in a heart attack and irreversible damage
to the heart.
[0009] Unstable angina is likely due to the partial rupture of a
vulnerable plaque that has become unstable. The plaque's partial
rupture causes a thrombus to develop, but does not completely
occlude the artery. Endogenous clot-fighting mechanisms serve to
break up the clot but, over time, the plaque continues to rupture
and the clotting episodes repeat. Although this patient may not
have yet suffered a myocardial infarction, he or she is at high
risk of doing so (e.g., if the unstable plaque completely ruptures
or if the endogenous clot fighting mechanisms cannot eliminate the
clot before total occlusion of the artery). Disrupted fibrous caps
taken post mortem from patients with unstable angina are often more
heavily infiltrated with macrophages at the plaque rupture site
than plaque from cases of stable angina.
Acute Myocardial Infarction
[0010] Acute myocardial infarction ("AMI") refers to a common
clinical condition that leads to necrosis of myocardial tissue.
This condition is well known in the art and is characterized by the
occurrence of pain (in most cases precordial), characteristic
electrocardiographic changes, and an increase in plasma levels of
intracellular enzymes (such as creatinine phosphokinase and
.alpha.-hydroxybutyrate dehydrogenase) or cardiac proteins (such as
components of the troponin complex, and myoglobin) released by the
necrotic cardiac tissue. AMI may be accompanied by hypotension,
circulatory failure, pulmonary edema and arrhythmia. In most cases,
but not exclusively, AMI results from vascular injury and
thrombosis in the coronary vessels, which causes these vessels to
become occluded with subsequent impaired blood flow to the
jeopardized myocardium (Fuster et al., 1992, New Engl. J. Med.,
326:242-310). In most cases, the time of the occlusion of the
coronary vessel can be estimated from the medical history, the
course of plasma levels of intracellular heart muscle enzymes and
electrocardiographic changes.
[0011] The initiating event of many myocardial infarctions (heart
attacks) is rupture of an atherosclerotic plaque. Such rupture may
result in formation of a thrombus or blood clot in the coronary
artery which supplies the infarct zone. The infarct zone or area,
as it is commonly referred to, is an area of necrosis which results
from an obstruction of blood circulation. The thrombus formed is
composed of a combination of fibrin and blood cells. The location,
degree and duration of the occlusion caused by the clot determine
the mass of the infarct zone and the extent of damage. Ultimately,
the extent of myocardial damage caused by the coronary occlusion
depends upon the "territory" supplied by the affected vessel, the
degree of occlusion of the vessel, the amount of blood supplied by
collateral vessels to the affected tissue, and the demand for
oxygen of the myocardium whose blood supply has suddenly been
limited (Pasternak and Braunwald, 1994, Acute Myocardial
Infarction, Harrison's Principles of Internal Medicine, 13.sup.th
Ed., pgs.1066-77).
Macrophages and the Inflammatory Response
[0012] Macrophages are involved in the cause and/or pathology of
some coronary syndromes. Macrophage secretion of proteolytic
proteins that degrade the fibrous caps of plaques decrease cap
thickness as well as increase additional macrophage infiltration
thus contributing to plaque instability. Therefore macrophages are
considered to have a central role in plaque rupture and their
presence in large concentrations is considered predictive to such
rupture. Indeed, erosion and/or disruption of the fibrous cap of
atherosclerotic plaques is known to modulate arterial thrombus
formation, leading to the onset of acute ischemic events. It is
clear that rupture at the site of a vulnerable atherosclerotic
plaque is the most frequent cause of acute coronary syndromes, such
as unstable angina, myocardial infarction or sudden death.
[0013] Inflammation has been related both to the pathogenesis of
acute myocardial infarctions and to the healing and repair
following AMI. Myocardial ischemia prompts an inflammatory
response. In addition, reperfusion, the mainstay of current acute
therapy of AMI, also enhances inflammation. Reperfusion involves
the rapid dissolution of the occluding thrombus and the restoration
of blood flow to the area of the heart which has had its blood
supply cut off The presence of inflammatory cells in the ischemic
myocardial tissues has traditionally been believed to represent the
pathophysiological response to injury. However, experimental
studies have shown that while crucial to healing, the influx of
inflammatory cells into tissues, specifically macrophages which are
phagocytic cells, results in tissue injury beyond that caused by
ischemia alone.
[0014] Macrophages and other leukocytes infiltrate the myocardium
soon after ischemia ensues. Macrophages secrete several cytokines,
which stimulate fibroblast proliferation. However, the activated
macrophages also secrete cytokines and other mediators that promote
myocardial damage. Accordingly, the influx of macrophages into the
myocardium increases myocardial necrosis and expands the zone of
infarct. Thus, although the acute phase of inflammation is a
necessary response for the healing process, persistent activation
is in fact harmful to the infarct area as well as the area
surrounding it, the so-called `peri-infarct zone`.
[0015] The inflammatory response that follows myocardial ischemia
is critical in determining the severity of the resultant damage
caused by the activated macrophages. Plasma levels of inflammatory
chemotactic factors (macrophage chemoattractant protein-1 (MCP-1),
macrophage inflammatory protein-1 alpha (MIP-1 alpha), have been
shown to correlate with subsequent heart failure and left
ventricular dysfunction (see, for example, Parissis, et al., 2002,
J. Interferon Cytokine Res., 22(2):223-9). Peripheral monocytosis
(an elevated number of monocytes) at two and three days after AMI
is associated with left ventricular dysfunction and left
ventricular aneurysm, suggesting a possible role of monocytes in
the development of left ventricular remodeling after reperfused AMI
(Maekawa, Y. et al., 2002, J. Am. Coll. Cardiol., 39(2):241-6).
Left ventricular remodeling after acute myocardial infarction is
the process of infarct expansions followed by progressive left
ventricular dilation and is associated with an adverse clinical
outcome. Furthermore, plasma levels of macrophage chemoattractant
protein-1 (MCP-1) are elevated in patients with acute myocardial
infarction. MCP-1 is induced by myocardial ischemia/reperfusion
injury and neutralization of this chemokine significantly reduced
infarct size.
[0016] Suppression of the inflammatory response by nonspecific
anti-inflammatory composites after coronary occlusion, in several
coronary occlusion/reperfusion models, was shown to reduce the
infarct area (See, for example, Squadrito, et al., 1997, Eur. J.
Pharmacol.; 335:185-92; Libby, et al., 1973, J. Clin. Invest.,
3:599-607; Spath, et al., 1974, Circ. Res., 35: 44-51). However,
these nonspecific regimens are associated with adverse effects,
such as interference with scar formation and healing, and, in some
patients, the development of aneurysm and rupture of the
ventricular wall. As such, these regimens are precluded from
clinical use. However, animal models that have a decreased ability
to suppress macrophage function due to a deficiency in the
anti-inflammatory cytokine interleukin-10 were shown to suffer from
increased infarct size and myocardial necrosis in a coronary
occlusion model (Yang, Z. et al., 2000, Circulation,
101:1019-1026.)
[0017] One object of the present invention is the identification of
therapeutic agents capable of blocking the accumulation of and/or
the biological function including secretion of factors from
phagocytic cells (particularly macrophages and monocytes) in the
patient suffering from an acute coronary syndrome (particularly
unstable angina or and acute myocardial infarction).
[0018] Another object of the invention is the development of
methods for treating an acute coronary syndrome (particularly
unstable angina or and acute myocardial infarction) as well as
stabilizing the plaques associated with these syndromes.
3. SUMMARY OF THE INVENTION
[0019] The present invention relates to methods and compositions
designed for the treatment or management of acute coronary
syndromes, particularly, unstable angina and acute myocardial
infarction. The methods of the invention comprise the
administration of an effective amount of a formulation containing
one or more therapeutic agents which specifically inhibits the
activity of and/or diminishes the amount of phagocytic cells
including, but not limited to, macrophages and monocytes.
Administration of a formulation containing one or more therapeutic
agents according to the invention acts as an acute, treatment aimed
at stabilizing the patient's coronary syndrome condition. In one
embodiment, a formulation containing one or more therapeutic agents
is administered to a patient suffering from unstable angina to
stabilize a vulnerable or unstable plaque. In another embodiment, a
formulation is administered to a patient currently suffering or
recently having suffered an acute myocardial infarction to minimize
infarct size and myocardial necrosis.
[0020] In preferred embodiments, the formulation specifically
targets phagocytic cells. Because phagocytic cells possess the
unique ability of phagocytosis, in these embodiments, the
formulations are prepared such that they comprise particles of such
properties as to enter into a cell primarily or exclusively via
phagocytosis. The formulation may comprise an encapsulated
therapeutic agent, an embedded therapeutic agent, or a particulate
therapeutic agent. Once phagocytosed, the therapeutic agent is
released from the formulation into the targeted phagocytic cells,
e.g., macrophages and monocytes, and inhibits the function of
and/or destroys the phagocytic cell.
[0021] In one embodiment, the present invention relates to a method
of treating an acute coronary syndrome by administering to an
individual in need thereof an effective amount of a formulation
comprising an encapsulated therapeutic agent. The therapeutic agent
is encapsulated in a suitable carrier of a specific dimension. The
formulation specifically targets phagocytic cells by virtue of its
properties, such as, for example, size or charge, which allow the
formulation to be taken-up primarily or exclusively by
phagocytosis. Once the formulation is taken-up by the phagocytic
cell, the encapsulated therapeutic agent is released and the agent
is able to inhibit the activity of and/or destroy the phagocytic
cell.
[0022] In another embodiment, the present invention relates to a
method of treating an acute coronary syndrome by administering to
an individual in need thereof an effective amount of a formulation
comprising an embedded therapeutic agent. The therapeutic agent is
embedded in a suitable carrier of a specific dimension. The
formulation specifically targets phagocytic cells by virtue of its
properties, such as, for example, size and/or charge, which allow
the formulation to be taken-up primarily or exclusively by
phagocytosis. Once inside the phagocytic cells the embedded
therapeutic agent is released and the agent is able to inhibit the
activity of and/or destroy the phagocytic cell.
[0023] In another embodiment, the present invention relates to a
method of treating an acute coronary syndrome by administering to
an individual in need thereof an effective amount of a formulation
comprising a particulate therapeutic agent. The therapeutic agent
is made into particulates of a specific dimension. The formulation
specifically targets phagocytic cells by virtue of the
particulate's properties, such as, for example, size and/or charge,
which allow the formulation to be taken-up primarily or exclusively
by phagocytosis. Once inside the phagocytic cells the particulate
therapeutic agent is able to inhibit the activity of and/or destroy
the phagocytic cell.
[0024] The present invention also relates to a method of
stabilizing plaques associated with an acute coronary syndrome by
administering to an individual in need thereof an effective amount
of a formulation comprising an encapsulated, embedded, or
particulate therapeutic agent.
[0025] In a further embodiment, the present invention includes a
pharmaceutical composition for administration to subjects currently
suffering from or having recently suffered an acute coronary
syndrome such as unstable angina and acute myocardial infarction
comprising a formulation selected from the group consisting of an
encapsulated therapeutic agent, an embedded therapeutic agent, and
a particulate therapeutic agent together with a pharmaceutically
acceptable vehicle, carrier, stabilizer or diluent for the
treatment of an acute coronary syndrome.
[0026] The formulation of present invention is preferably in the
size range of 0.03-1.0 microns. However, depending on the type of
agent and/or the carrier used, the more preferred ranges include,
but are not limited to, 0.07-0.5 microns, 0.1-0.3 microns and 0.1
to 0.18.
4. BRIEF DESCRIPTION OF THE FIGURES
[0027] FIG. 1 illustrates the effect of liposomal alendronate
treatment on the size of infarct area after transient coronary
artery occlusion in rabbits. The size of the infarct zone was
calculated as the area of the infarcted zone as a % of the left
ventricular area supplied by the occluded artery and thus at risk
for subsequent infarction. Data are expressed as mean.+-.SD, with
n=4/group and a p value of p<0.05.
[0028] FIGS. 2A-2B illustrate the effect of liposomal alendronate
treatment on myocardial morphology after reversible coronary
occlusion in rabbits. Control rabbits (A) have distorted myocardial
morphology while rabbits treated with liposomal alendronate (B)
have a more normal myocardial morphology.
[0029] FIGS. 3A-3B illustrate the reduction in macrophage
infiltration following treatment with liposomal alendronate after
reversible coronary occlusion in rabbits. Control rabbits (A) show
increased RAM11+macrophage accumulation in the zone of infarct as
compared to rabbits treated with liposomal alendronate (B).
5. DETAILED DESCRIPTION OF THE INVENTION
[0030] Phagocytic cells, particularly macrophages and monocytes,
are involved in the cause and/or pathology of some coronary
syndromes. Macrophages/monocytes degrade the fibrous caps of
plaques through the secretion of various substances that not only
decrease cap thickness, but also serve to recruit additional
macrophages/monocytes to the area. Degradation of the fibrous cap
leads to exposure to blood of the lipid core of the plaque as well
as initiation of the clotting cascade which culminates in a
thrombus. The thrombus may partially occlude the lumen leading to
unstable angina or it may completely occlude the lumen thus causing
an acute mycardial infarction. Once an acute myocardial infarction
occurs, macrophages/monocytes are recruited to the damaged
myocardial tissue and secrete cytokines and other mediators that
promote myocardial damage thus resulting in tissue injury beyond
that caused by ischemia alone and increases myocardial necrosis
which expands the zone of infarct. Although a complete and chronic
incapacitation and/or ablation of phagocytic cells is not
desirable, such a decrease in phagocytic cell activity and/or
presence is desirable in the short term during or after an acute
coronary syndrome to stabilize the patient and/or reduce the damage
of the coronary syndrome.
[0031] The present invention relates to methods and compositions
designed to decrease or inhibit the activity of and/or eliminate or
diminish the amount of phagocytic cells (including, but not limited
to, macrophages and monocytes) for an acute, short term period
during or following an acute coronary syndrome for the treatment or
management of the acute coronary syndrome (including, but not
limited to, unstable angina and acute myocardial infarction). The
methods of the invention comprise the administration of an
effective amount of a formulation containing one or more
therapeutic agents which specifically decreases or inhibits the
activity of and/or eliminates or diminishes the amount of
phagocytic cells (including, but not limited to, macrophages and
monocytes) in a patient. Administration of a formulation containing
one or more therapeutic agents is contemplated as an acute, short
term treatment aimed at stabilization of the patient and/or
minimization of the immediate and long term damage from the acute
coronary syndrome. In one embodiment, a formulation containing one
or more therapeutic agents are administered to a patient suffering
from unstable angina to stabilize a vulnerable or unstable plaque
and decrease the immediate threat of an acute myocardial
infarction. In another embodiment, one or more therapeutic agents
are administered to a patient currently suffering or recently
having suffered an acute myocardial infarction to minimize the
infarct size and myocardial necrosis.
[0032] The formulations used in the methods of the invention
specifically decrease or inhibit the activity of phagocytic cells
and/or eliminate or diminish the amount of phagocytic cells in a
patient. Specificity of the formulation is due to the ability of
the composition to affect only particular cell types (e.g.,
macrophages and/or monocytes). In preferred embodiments,
specificity of the formulation for phagocytic cells is due to the
physiochemical properties ,e.g. size or charge, of the formulation
such that it can only or primarily be internalized by phagocytosis.
Once phagocytosed and intracellular, the therapeutic agent inhibits
or decreases the activity of the phagocytic cell and/or destroys
the phagocytic cell. Although not intending to be bound by any
particular mechanism of action, the therapeutic agents of the
formulation are released upon becoming intracellular before
disabling an/or destroying the phagocytic cell.
[0033] The formulation of the present invention, e.g., the
encapsulated therapeutic agent, embedded therapeutic agent or the
particulate therapeutic agent, suppresses the inflammatory response
by transiently depleting and/or inactivating cells that are
important triggers in the inflammatory response, namely macrophages
and/or monocytes. The encapsulated agent, embedded agent and/or
particulate agent are taken-up, by way of phagocytosis, by the
macrophages and monocytes. In contrast, non-phagocytic cells are
incapable of taking up the formulation due to the large dimension
and/or other physiochemical properties of the formulation.
[0034] The term "phagocytosis" as used herein refers to a preferred
means of entry into a phagocytic cell and is well understood in the
art. However, the term should be understood to also encompass other
forms of endocytosis which may also accomplish the same effect. In
particular, it is understood that pinocytosis, receptor-mediated
endocytosis and other cellular means for absorbing/internalizing
material from outside the cell are also encompassed by the methods
and compositions of the present invention.
[0035] The invention also provides pharmaceutical compositions
comprising one or more therapeutic agents of the invention for
administration subjects currently suffering from or recently having
suffered an acute coronary syndrome such as unstable angina and
acute myocardial infarction.
5.1 Therapeutic Agents
[0036] The therapeutic agents used in the formulations and in the
methods of the invention specifically decrease or inhibit the
activity of phagocytic cells and/or eliminate or diminish the
amount of phagocytic cells in a patient, by virtue of the
physiochemical properties, such as size or charge, of the
formulation. The therapeutic agent may be an intra-cellular
inhibitor, deactivator, toxin, arresting substance and/or
cytostatic/cytotoxic substance that, once inside a phagocytic cell
such as a macrophage or monocyte, inhibits, destroys, arrests,
modifies and/or alters the phagocytic cell such that it can no
longer function normally and/or survive.
[0037] As used herein, the term "therapeutic agents" refers to
molecules which either make up the formulation or form a part of
the formulation and provide the inactivating/toxic potency to the
formulation., e.g., inhibits or decreases phagocytic cell activity
and/or eliminates or decreases the amount of phagocytic cells.
Compounds that can be therapeutic agents include, but are not
limited to, inorganic or organic compounds; or a small molecule
(less than 500 daltons) or a large molecule, including, but not
limited to, inorganic or organic compounds; proteinaceous
molecules, including, but not limited to, peptide, polypeptide,
protein, post-translationally modified protein, antibodies etc.; or
a nucleic acid molecule, including, but not limited to,
double-stranded DNA, single-stranded DNA, double-stranded RNA,
single-stranded RNA, or triple helix nucleic acid molecules.
Compounds can be natural products derived from any known organism
(including, but not limited to, animals, plants, bacteria, fungi,
protista, or viruses) or from a library of synthetic molecules.
Therapeutic agents can be monomeric as well as polymeric
compounds.
[0038] In preferred embodiments where the preferred therapeutic
agent may be a bisphosphonate or analog thereof. The term
"bisphosphonate" as used herein, denotes both geminal and
non-geminal bisphosphonates. In a specific embodiment, the
bisphosphonate has the following formula (I):
##STR00001##
wherein R.sub.1 is H, OH or a halogen atom; and R.sub.2 is halogen;
linear or branched C.sub.1-C.sub.10 alkyl or C.sub.2-C.sub.10
alkenyl optionally substituted by heteroaryl or heterocyclyl
C.sub.1-C.sub.10 alkylamino or C.sub.3-C.sub.8 cycloalkylamino
where the amino may be a primary, secondary or tertiary; --NHY
where Y is hydrogen, C.sub.3-C.sub.8 cycloalkyl, aryl or
heteroaryl; or R.sub.2 is --SZ where Z is chlorosubstituted phenyl
or pyridinyl.
[0039] In a more specific embodiment, the bisphosphoate is
alendronate or an analog thereof. In such an embodiment, the
alendronate has the following formula (II):
##STR00002##
[0040] In other specific embodiments, additional bisphosphonates
can be used in the methods of the invention. Examples of other
bisphosphonates include, but are not limited to, clodronate,
tiludronate,
3-(N,N-dimethylamino)-1-hydroxypropane-1,1-diphosphonic acid, e.g.
dimethyl-APD; 1-hydroxy-ethylidene-1,1-bisphosphonic acid, e.g.
etidronate;
1-hydroxy-3(methylpentylamino)-propylidene-bisphosphonic acid,
(ibandronic acid), e.g. ibandronate; 6-amino-
1-hydroxyhexane-1,1-diphosphonic acid, e.g. amino-hexyl-BP;
3-(N-methyl-N-pentylamino)-1-hydroxypropane-1,1-diphosphonic acid,
e.g. methyl-pentyl-APD;
1-hydroxy-2-(imidazol-1-yl)ethane-1,1-diphosphonic acid, e.g.
zoledronic acid; 1-hydroxy-2-(3-pyridyl)ethane-1,1-diphosphonic
acid (risedronic acid), e.g. risedronate;
3-[N-(2-phenylthioethyl)-N-methylamino]-1-hydroxypropane-1,1-bishosphonic
acid; 1-hydroxy-3-(pyrrolidin-1-yl)propane-1,1-bisphosphonic acid,
1-(N-phenylaminothiocarbonyl)methane-1,1-diphosphonic acid, e.g. FR
78844 (Fujisawa);
5-benzoyl-3,4-dihydro-2H-pyrazole-3,3-diphosphonic acid tetraethyl
ester, e.g. U81581 (Upjohn); and
1-hydroxy-2-(imidazo[1,2-a]pyridin-3-yl)ethane-1,1-diphosphonic
acid, e.g. YM 529, or analogs thereof.
[0041] Other formulations containing therapeutic agents include,
but are not limited to, gallium, gold, selenium, gadolinium,
silica, mithramycin, paclitaxel, sirolimus, everolimus, and other
similar analogs thereof. Generally, chemotherapeutic agents, such
as, for example, 5-fluorouracil, cisplatinum, alkylating agents and
other anti-proliferation or anti-inflammatory compounds, such as,
for example, steroids, aspirin and non-steroidal anti-inflammatory
drugs may also be used in a formulation.
[0042] The present invention is meant to encompass the
administration of one or more formulations to manage or treat an
acute coronary syndrome. More than one formulation can be
administered in combination to the patient. The term "in
combination" is not limited to the administration of the
formulation at exactly the same time, but rather it is meant that
the formulations are administered to a patient in a sequence and
within a time interval such that they can act together to provide
an increased benefit than if they were administered otherwise. For
example, each formulation may be administered at the same time or
sequentially in any order at different points in time; however, if
not administered at the same time, they should be administered
sufficiently close in time so as to provide the desired therapeutic
effect. Each formulation can be administered separately, in any
appropriate form and by any suitable route which effectively
transports the therapeutic agent to the appropriate or desirable
site of action. Preferred modes of administration include
intravenous (IV) and intra-arterial (IA). Other suitable modes of
administration include intramuscular (IM), subcutaneous (SC), and
intraperitonal (IP) and oral (PO). Such administration may be bolus
injections or infusions. Another mode of administration may be by
perivascular delivery. The formulation may be administered directly
or after dilution. Combinations of any of the above routes of
administration may also be used in accordance with the
invention.
[0043] In various embodiments, the formulations are administered
less than 1 hour apart, at about 1 hour apart, at about 1 hour to
about 2 hours apart, at about 2 hours to about 3 hours apart, at
about 3 hours to about 4 hours apart, at about 4 hours to about 5
hours apart, at about 5 hours to about 6 hours apart, at about 6
hours to about 7 hours apart, at about 7 hours to about 8 hours
apart, at about 8 hours to about 9 hours apart, at about 9 hours to
about 10 hours apart, at about 10 hours to about 11 hours apart, at
about 11 hours to about 12 hours apart, no more than 24 hours apart
or no more than 48 hours apart. In one embodiment two or more
formulations are administered concurrently or within the same
patient visit.
5.1.1 Identification of Therapeutic Agents
[0044] The invention provides methods of screening for compounds
that can be used as a therapeutic agent. Although not intending to
be bound by a particular mechanism of action, a compound that is a
therapeutic agent for use in the methods of the invention can, once
targeted to the phagocytic cell by the physiochemical properties of
the formulation itself, i) inhibit phagocytic cell activity, ii)
decrease phagocytic cell activity, iii) eliminate phagocytic cells
from circulation and/or from the area affected by the acute
coronary syndrome, and/or iv) decrease the number of phagocytic
cells in circulation and/or in the area affected by the acute
coronary syndrome.
[0045] The methods of screening for therapeutic agents generally
involve incubating a candidate compound with phagocytic cells
either in vitro or in vivo and then assaying for an alteration
(e.g., decrease) in phagocytic cell activity or longevity thereby
identifying a compound that is a therapeutic agent for use in the
present invention. Any method known in the art can be used to assay
phagocytic cell activity or longevity. In one embodiment,
phagocytic cell activity is assayed by the level of cell activation
in response to an activating stimulus. For example,
macrophage/monocyte activation can be assayed by quantifying the
levels of chemotactic factors such as macrophage chemoattractant
protein-1 (MCP-1) and macrophage inflammatory protein-1 alpha
(MIP-1 alpha) as well as other substances produced by macrophages
such as interleukin 1 beta (IL-1.beta.) and tissue necrosis factor
alpha (TNF-.alpha.). In another embodiment, phagocytic cell
longevity is assayed. For example, cell proliferation can be
assayed by measuring .sup.3H-thymidine incorporation, by direct
cell count, by detecting changes in transcriptional activity of
known genes such as proto-oncogenes (e.g., fos, myc) or cell cycle
markers; or by trypan blue staining. Any method known in the art
can be used to assay for levels of mRNA transcripts (e.g., by
northern blots, RT-PCR, Q-PCR, etc.) or protein levels (e.g.,
ELISA, western blots, etc.).
[0046] In one embodiment, a compound that decreases the activity of
a phagocytic cell is identified by:
[0047] a) contacting a phagocytic cell with a first compound and a
second compound, said first compound being a compound which
activates said phagocytic cell and said second compound being a
candidate compound; and
[0048] b) determining the level of activation in said contacted
phagocytic cell, wherein a decrease in activation in said contacted
cell as compared to the level of activation in a phagocytic cell
contacted with said first compound in the absence of said second
(i.e., a control cell) indicates that said second compound
decreases the activity of a phagocytic cell.
[0049] In another embodiment, a compound that decreases the amount
of phagocytic cells is identified by:
[0050] a) contacting a phagocytic cell with a compound; and
[0051] b) determining the viability of said contacted phagocytic
cell, wherein a decrease in viability in said contacted cell as
compared to the viability of a phagocytic cell not contacted with
said compound (i.e., a control cell) indicates that said compound
decreases the amount of phagocytic cells.
[0052] In other embodiments, candidate compounds are assayed for
their ability to alter phagocytic cell activity or longevity in a
manner that is substantially similar to or better than compounds
known to alter phagocytic cell activity or longevity in a
therapeutically desirable way. As used herein "substantially
similar to" refers to an agent having similar action on a
phagocytic cell as an exemplified agent, i.e., an agent that
inhibits the activity, function, motility, and/or depletion of
phagocytic cells.
[0053] Additionally, candidate compounds can be used in animal
models of acute coronary syndromes to assess their ability to be
used in the methods of the invention. In one embodiment, a rabbit
AMI model can be used (see e.g., Section 6.1).
5.2 Formulation of Therapeutic Agents
[0054] Formulations containing one or more therapeutic agents can
be prepared so that the size of the formulation is large enough to
only or primarily be internalized by phagocytosis, thus imparting
specificity to phagocytic cells. Although non-phagocytic cells may
be affected by such a formulation should it become intracellular,
there is no mechanism for a non-phagocytic cell to internalize a
formulation prepared in this manner. Formulations imparting
extrinsic specificity to one or more therapeutic agents are
preferably in the size range of 0.03-1.0 microns, more preferably
0.07-0.5 microns, more preferably 0.1-0.3 microns, and more
preferably 0.1 to 0.18 microns.
[0055] Any method known in the art can be used to incorporate a
therapeutic agent into a formulation such that it can only or
primarily be internalized via phagocytosis. Formulations of
therapeutic agents may sequester the therapeutic agents for a
sufficient time to enhance delivery of the agent to the target
site. Furthermore, formulations of therapeutic agents may discharge
the therapeutic agent from the particles when they are within the
target cell (e.g., the phagocytic cell) at the target site.
[0056] In one embodiment, the therapeutic agent is encapsulated in
a carrier (i.e., encapsulating agent) of desired properties. In a
specific embodiment, the encapsulating agent is a liposome. The
liposomes may be prepared by any of the methods known in the art
(see, e.g., Monkkonen, J. et al., 1994, J. Drug Target, 2:299-308;
Monkkonen, J. et al., 1993, Calcif. Tissue Int., 53:139-145; Lasic
D D., Liposomes Technology Inc., Elsevier, 1993, 63-105.(chapter
3); Winterhalter M, Lasic D D, Chem Phys Lipids, 1993 Sep;
64(1-3):35-43). The liposomes may be positively charged, neutral
or, more preferably, negatively charged. The liposomes may be a
single lipid layer or may be multilamellar. Suitable liposomes in
accordance with the invention are preferably non-toxic liposomes
such as, for example, those prepared from phosphatidyl-choline
phosphoglycerol, and cholesterol. The diameter of the liposomes
used preferably ranges from 0.03-1.0 .mu.m. However, other size
ranges suitable for phagocytosis by phagocytic cells may also be
used.
[0057] In another embodiment, the therapeutic agent is embedded in
a carrier (i.e., embedding agent) of desired properties. A
therapeutic agent which is embedded includes those therapeutic
agents that are embedded, enclosed, and/or adsorbed within a
carrier, dispersed in the carrier matrix, adsorbed or linked on the
carrier surface, or a combination of any of these forms. In
specific embodiments, the embedding agent (or carrier) is a
microparticle, nanoparticle, nanosphere, microsphere, microcapsule,
or nanocapsule (see e.g., M. Donbrow in: Microencapsulation and
Nanoparticles in Medicine and Pharmacy, CRC Press, Boca Raton,
Fla., 347, 1991). The term carrier includes both polymeric and
non-polymeric preparations. In a specific embodiment, the embedding
agent is a nanoparticle. Preferably, nanoparticles are 0.03-1.0
microns in diameter and can be spherical, non-spherical, or
polymeric particles. The therapeutic agent may be embedded in the
nanoparticle, dispersed uniformly or non-uniformly in the polymer
matrix, adsorbed on the surface, or in combination of any of these
forms. In a preferred embodiment, the polymer used for fabricating
nanoparticles is biocompatible and biodegradable, such as
poly(DL-lactide-co-glycolide) polymer (PLGA). However, additional
polymers which may be used for fabricating the nanoparticles
include, but are not limited to, PLA (polylactic acid), and their
copolymers, polyanhydrides, polyalkyl-cyanoacrylates (such as
polyisobutylcyanoacrylate), polyethyleneglycols, polyethyleneoxides
and their derivatives, chitosan, albumin, gelatin and the like.
[0058] In another embodiment, the therapeutic agent is in
particulate form, the particles each being of desired properties. A
particulate therapeutic agent form includes any insoluble suspended
or dispersed particulate form of the therapeutic agent which is not
encapsulated, entrapped or absorbed within a carrier. A therapeutic
agent which is in particulate form includes those therapeutic
agents that are suspended or dispersed colloids, aggregates,
flocculates, insoluble salts, insoluble complexes, and polymeric
chains of an agent. Such particulates are insoluble in the fluid in
which they are stored/administered (e.g., saline or water) as well
as the fluid in which they provide their therapeutic effect (e.g.,
blood or serum). Typically, "insoluble" refers to a solubility of
one (1) part of a particulate therapeutic agent in more than
ten-thousand (10,000) parts of a solvent. Any method known in the
art to make particulates or aggregates can be used. Preferably,
particulates are 0.03-1.0 microns in diameter and can be any
particular shape.
5.2.1 Determination of Particle Size
[0059] Formulations containing therapeutic agents are preferably
prepared such that the size of the formulation is large enough to
only or primarily be internalized by phagocytosis, that is,
preferably larger than 0.03 microns. In preferred embodiments, such
formulations are 0.03-1.0 microns, more preferably 0.07-0.5
microns, more preferably 0.1-0.3 microns, and most preferably 0.1
to 0.18 microns. Any method known in the art can be used to
determine the size of the formulation before administration to a
patient in need thereof. For example, a Nicomp Submicron Particle
Sizer (model 370, Nicomp, Santa Barbara, Calif.) utilizing laser
light scattering can be used.
5.3 Administration of Tile Formulation
[0060] Effective amounts of the formulations are contemplated as
short term, acute therapy and are not meant for chronic
administration. Time period of treatment is preferably such that it
produces inhibition/depletion of phagocytic cells for a period that
is less than a month, preferably less than two weeks, most
preferably up to one week. Empirically, one can determine this by
administering the compound to an individual in need thereof (or an
animal model of such an individual) and monitoring the level of
inhibition/depletion at different time points. One may also
correlate the time of inhibition with the appropriate desired
clinical effect, e.g. reduction in the acute risk of plaque
rupture.
5.4 Characterization of Therapeutic Utility
[0061] The term "effective amount" denotes an amount of a
particular formulation which is effective in achieving the desired
therapeutic result, namely inhibited or decreased phagocytic cell
activity and/or elimination or reduction in the amount of
phagocytic cells. In one embodiment, the desired therapeutic result
of inhibiting or decreasing phagocytic cell activity and/or
eliminating or reducing in the amount of phagocytic cells
stabilizes a vulnerable or unstable plaque in a patient suffering
from unstable angina. In another embodiment, the desired
therapeutic result of inhibiting or decreasing phagocytic cell
activity and/or eliminating or reducing in the amount of phagocytic
cells minimizes the infarct size and/or the amount of myocardial
necrosis in a patient having suffered an acute myocardial
infarction.
[0062] Toxicity and efficacy of the therapeutic methods of the
instant invention can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., for
determining the LD.sub.50 (the dose lethal to 50% of the
population), the No Observable Adverse Effect Level (NOAEL) and the
ED.sub.50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index and it can be expressed as the ratio
LD.sub.50/ED.sub.50 or NOAEL/ED.sub.50. Formulations that exhibit
large therapeutic indices are preferred. While formulations that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that targets the agents of such
formulations to the site of affected tissue in order to minimize
potential damage to unaffected cells and, thereby, reduce side
effects.
[0063] The data obtained from the cell culture assays and animal
studies can be used in determining a range of dosage of the
formulation for use in humans. The dosage of such formulations lies
preferably within a range of circulating concentrations that
include the ED.sub.50 with little or no toxicity. The dosage may
vary within this range depending upon the dosage form employed and
the route of administration utilized. For any formulation used in
the method of the invention, the effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound that achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may
be measured, for example, by high performance liquid
chromatography.
[0064] The protocols and compositions of the invention are
preferably tested in vitro, and then in vivo, for the desired
therapeutic activity, prior to use in humans One example, of such
an in vitro assay is an in vitro cell culture assay in phagocytic
cells which are grown in culture, and exposed to or otherwise
administered to cells, and observed for an effect of this assay
upon the cells, e.g., inhibited or decreased activity and/or
complete or partial cell death. The phagocytic cells may be
obtained from an established cell line or recently isolated from an
individual as a primary cell line. Many assays standard in the art
can be used to measure the activity of the formulation on the
phagocytic cells; for example, macrophage/monocyte activation can
be assayed by quantitating the levels of chemotactic factors such
as macrophage chemoattractant protein-1 (MCP-1), interleukin 1 beta
(IL-.beta.), tissue necrosis factor alpha (TNF-.alpha.) and
macrophage inflammatory protein-1 alpha (MIP-1 alpha). Many assays
standard in the art can be used to assess survival and/or growth of
the phagocytic cells; for example, cell proliferation can be
assayed by measuring .sup.3H-thymidine incorporation, by direct
cell count, by detecting changes in transcriptional activity of
known genes such as proto-oncogenes (e.g., fos, myc) or cell cycle
markers; cell viability can be assessed by trypan blue
staining.
[0065] Selection of the preferred effective dose can be determined
(e.g., via clinical trials) by a skilled artisan based upon the
consideration of several factors known to one of ordinary skill in
the art. Such factors include the acute coronary syndrome to be
managed or treated, the symptoms involved, the patient's body mass,
the patient's immune status and other factors known to the skilled
artisan to reflect the accuracy of administered pharmaceutical
compositions.
5.5 Pharmaceutical Compositions and Routes of Administration
[0066] Formulations comprising one or more therapeutic agents for
use in the methods of the invention may be in numerous forms,
depending on the various factors specific for each patient (e.g.,
the severity and type of disorder, age, body weight, response, and
the past medical history of the patient), the number and type of
therapeutic agents in the formulation, the type of formulation
(e.g., encapsulated, embedded, particulate, etc.), the form of the
composition (e.g., in liquid, semi-liquid or solid form), and/or
the route of administration (e.g., oral, intravenous,
intramuscular, intra-arterial, intramedullary, intrathecal,
intraventricular, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical, sublingual, vaginal, or rectal
means). Pharmaceutical carriers, vehicles, excipients, or diluents
may be included in the compositions of the invention including, but
not limited to, water, saline solutions, buffered saline solutions,
oils (e.g., petroleum, animal, vegetable or synthetic oils),
starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,
chalk, silica gel, sodium stearate, glycerol monostearate, talc,
sodium chloride, dried skim milk, glycerol, propylene, glycol,
ethanol, dextrose and the like. The composition, if desired, can
also contain minor amounts of wetting or emulsifying agents, or pH
buffering agents. These compositions can take the form of
solutions, suspensions, emulsion, tablets, pills, capsules,
powders, sustained-release formulations and the like.
[0067] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances, which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. In addition, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acid esters, such as
ethyloleate or triglycerides, or liposomes. Optionally, the
suspension may also contain suitable stabilizers or agents which
increase the solubility of the compounds to allow for the
preparation of highly concentrated solutions.
[0068] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic,
and the like. Salts tend to be more soluble in aqueous solvents, or
other protonic solvents, than are the corresponding free base
forms.
[0069] Pharmaceutical compositions can be administered systemically
or locally, e.g., near the site of pathology of an acute coronary
syndrome. Additionally, systemic administration is meant to
encompass administration that can target to a particular area or
tissue type of interest.
[0070] Pharmaceutical compositions are preferably administered
immediately at the onset of the first symptoms of actual plaque
rupture; such as, for example, chest pain, pain that radiates to
the shoulder, arm, teeth, jaw, abdomen or back or shortness of
breath or cough, lightheadedness, fainting, nausea, vomiting,
sweating or anxiety associated with a plaque rupture. Other
symptoms will be apparent to the skilled artisan and medical
doctor, and may be signals to administer the instant pharmaceutical
composition. Alternatively and/or additionally, the pharmaceutical
compositions may be administered just after onset of symptoms, for
example, within minutes of symptom onset. Alternatively and/or
additionally, the compositions may be administered within 1 hour,
or about 2 hours, or about 3 hours or about 4 hours, or about 5
hours or about 6 hours, up to within 1-3 days after onset of
symptoms.
[0071] In another regime, pharmaceutical compositions are
administered to a patient with an increased risk of plaque rupture.
For example, the compositions of the invention may be administered
to a patient prior to a procedure which increases the risk of
plaque rupture, such as, for example, an angioplasty procedure. It
may be preferred to administer the composition up to 3 days before
such a procedure. Also preferred, administration may be 1-6 hours
before the procedure or within 1 hour of the procedure or less than
1 hour before or even within minutes of the procedure. The skilled
person can readily determine the appropriate timing of
administration depending on various physiological factors, specific
to the individual patient, such as, for example, weight, medical
history and genetic predisposition, as well as various factors
which influence the anticipated risk of plaque rupture such as
complexity of the procedure to be performed.
[0072] The contents of all published articles, books, reference
manuals and abstracts cited herein, are hereby incorporated by
reference in their entirety to more fully describe the state of the
art to which the invention pertains.
[0073] As various changes can be made in the above-described
subject matter without departing from the scope and spirit of the
present invention, it is intended that all subject matter contained
in the above description, or defined in the appended claims, be
interpreted as descriptive and illustrative of the present
invention. Modifications and variations of the present invention
are possible in light of the above teachings.
6. EXAMPLES
[0074] The following examples as set forth herein are meant to
illustrate and exemplify the various aspects of carrying out the
present invention and are not intended to limit the invention in
any way.
6.1 Effect of Liposomal Alendronate on the Size of the Zone of
Infarct
[0075] The effects of treatment with encapsulated bisphosphonates
on the zone of infarct were studied in a rabbit AMI model.
Liposomal Alendronate, approx. 0.150 .mu.m in diameter was made
using the following outline: [0076] a. Dissolve lipids, DSPC, DSPG
and cholesterol in 1/1 ethanol/tert-butanol. [0077] b. Dilute
solvent into buffer containing Alendronate to generate large
multilamellar vesicles (MLVs). [0078] c. Extrude MLVs through 200
nm polycarbonate filters to generate large unilamellar 150.+-.20 nm
vesicles (LUVs). [0079] d. Ultra-filtrate LUVs to remove
un-encapsulated alendronate. [0080] e. Sterile filter
[0081] Eight New Zealand White male rabbits, 2.5-3.5 kg B. W., were
fed normal chow and water ad libitum. The rabbits were randomly
administered saline (control) or liposomal alendronate (3 mg/kg,
i.v.) as a single infusion simultaneous with coronary artery
occlusion. The rabbits were anesthetized by Ketamine/Xylazine (35
mg/kg; 5 mg/kg) and Isoflurane. The experiment was performed with
respiratory support given by intubation and mechanical ventilation
with isoflurane in balance oxygen, and continuous echocardiogram
(ECG) and arterial blood pressure (catheter in ear artery)
monitoring. Thoracotomy was performed through the left 4.sup.th
intercostal space, followed by pericardiotomy and creation of a
pericardial cradle. The left main coronary artery was identified
and a large branch was encircled by a 5-0 silk suture and a snare.
Thereafter, the snare was tightened for 30 minutes. Ischemia was
verified by ECG changes (ST-T segment elevation), changes of
segment coloration and hypokinesia. After thirty minutes, the snare
was released and resumption of blood flow was confirmed. The suture
was left in place, released, and the chest cavity was closed in
layers. Buprenex was administered to the rabbits for analgesia for
2-3 additional days. Following euthanasia with Penthotal, the
rabbits were sacrificed after 7 days and the hearts were harvested.
The coronary arteries were perfused through the ascending aorta
with saline, followed by tightening of the suture on the previously
occluded coronary artery and perfusion of the coronary arteries
with 0.5% Evans blue solution (Sigma) to stain areas of
re-endothelialization (presence of blood). The left ventricular
area unstained by Evans blue was defined as the area at risk. The
hearts were then frozen at -20.degree. C. for 24 hours and cut into
transverse sections 2 mm apart. Slices of the hearts were incubated
for 30 minutes in the vital stain tritetrazolium chloride (TTC, 1%,
Sigma), fixed in 10% natural buffered formalin to stain cells that
had been alive previous to tissue processing. The left ventricular
area not stained by TTC (white) was defined as the area of infarct.
The stained sections were then photographed and processed by
digital planimetry (Photoshop).
[0082] Rabbits treated with liposomal alendronate had a zone of
infarct that was 29.5.+-.6% of the area at risk. This was
contrasted with the control rabbits (untreated with liposomal
alendronate) which showed an infarct zone that was 42.+-.5.5% of
the area at risk (FIG. 1). Accordingly, liposomal alendronate was
effective in reducing the zone of infarct. No adverse effects were
observed in the treatment group.
6.2 Effect of Liposomal Alendronate On Myocardial Morphology
[0083] Rabbits as treated in Section 6.1 showed variation in
myocardial morphology as exhibited by Hemotoxylin and Eosin
staining. The control rabbits have a distorted myocardial
morphology (FIG. 2A) while the rabbits treated with liposomal
alendronate exhibit a more normal morphology (FIG. 2B).
6.3 Effect of Liposomal Alendronate on Macrophage Infiltration
[0084] Rabbits as treated in Section 6.1 showed a reduction in
macrophage infiltration in rabbits treated with liposomal
alendronate. Representative sections of the rabbits' hearts were
subjected to immunostaining for RAM11+macrophages. Sections from
rabbits treated with liposomal alendronate (FIG. 3B) showed less
staining and therefore had less RAM11+macrophages accumulation than
sections from control rabbits (FIG. 3A).
[0085] Liposomal alendronate was also shown to reduce the number of
circulating monocytes systemically. Rabbits were administered
saline (control) or liposomal alendronate (3 mg/kg, i.v.) Monocyte
levels in circulating blood were determined using FACS analysis for
CD-14. At 48 hours after injection with liposomal alendronate, the
blood monocyte population was reduced by 75-95% as compared to the
control group.
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