U.S. patent application number 11/296153 was filed with the patent office on 2007-07-19 for method of inhibiting restenosis using bisphosphonates.
Invention is credited to Haim Danenberg, Gershon Golomb.
Application Number | 20070166385 11/296153 |
Document ID | / |
Family ID | 29254006 |
Filed Date | 2007-07-19 |
United States Patent
Application |
20070166385 |
Kind Code |
A1 |
Golomb; Gershon ; et
al. |
July 19, 2007 |
Method of inhibiting restenosis using bisphosphonates
Abstract
A method of inhibiting the activity or production of cytokines
or growth factors associated with vascular restenosis, by
administering to an individual an effective amount of an active
ingredient comprising a bisphosphonate particle or a bisphosphonate
particulate. The bisphosphonate may be encapsulated, embedded or
adsorbed within the particle, dispersed uniformly in the polymer
matrix, adsorbed on the particle surface, or in combination of any
of these forms. The particles include liposomes or inert polymeric
particles, such as microcapsules, nanocapsules, nanoparticles,
nanospheres, or microparticles. The particulates include any
suspended or dispersed form of the bisphosphonate which is not
encapsulated, entrapped, or adsorbed within a polymeric particle.
The particulates include suspended or dispersed colloids,
aggregates, flocculates, insoluble salts and insoluble complexes of
the active ingredient. The cytokines and growth factors include,
but are not limited to interleukin 1-.beta., matrix
metalloproteinase-2, and platelet-derived growth factor .beta.
(PDGF.beta.).
Inventors: |
Golomb; Gershon; (Efrat,
IL) ; Danenberg; Haim; (Brookline, MA) |
Correspondence
Address: |
MORGAN & FINNEGAN, L.L.P.
3 World Financial Center
New York
NY
10281-2101
US
|
Family ID: |
29254006 |
Appl. No.: |
11/296153 |
Filed: |
December 6, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10160207 |
May 30, 2002 |
7008645 |
|
|
11296153 |
Dec 6, 2005 |
|
|
|
10126248 |
Apr 19, 2002 |
6984400 |
|
|
10160207 |
May 30, 2002 |
|
|
|
09743705 |
Mar 22, 2001 |
6719998 |
|
|
PCT/IL99/00387 |
Jul 14, 1999 |
|
|
|
10126248 |
Apr 19, 2002 |
|
|
|
Current U.S.
Class: |
424/489 ;
514/102; 514/89 |
Current CPC
Class: |
A61K 33/24 20130101;
A61K 31/66 20130101; A61K 9/5153 20130101; A61K 31/663 20130101;
A61K 31/675 20130101; A61P 9/10 20180101; Y10S 514/951 20130101;
A61K 9/127 20130101; Y10S 514/824 20130101; A61K 31/00
20130101 |
Class at
Publication: |
424/489 ;
514/089; 514/102 |
International
Class: |
A61K 31/675 20060101
A61K031/675; A61K 31/66 20060101 A61K031/66; A61K 9/14 20060101
A61K009/14 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 14, 1998 |
IL |
125336 |
Claims
1. A pharmaceutical composition comprising bisphosphonate that has
been formulated into an insoluble particulate having a size of
0.01-1.0 microns, together with a pharmaceutically acceptable
carrier.
2. The pharmaceutical composition according to claim 1, wherein the
bisphosphonate particulate is selected from the group consisting of
aggregates, flocculates, colloids, polymer chains, insoluble salts
and insoluble complexes.
3. The pharmaceutical composition according to claim 1, further
comprising a diluent.
4. The pharmaceutical composition according to claim 1 further
comprising a stabilizer.
5. The pharmaceutical composition according to claim 1, wherein the
bisphosphonate has the following formula (I): ##STR7## wherein
R.sub.1 is H, OH or a halogen atom; and R.sub.2 is a 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.
6. The pharmaceutical composition according to claim 1, wherein
said bisphosphonate is clodronate, etidronate, tiludronate,
pamidronate, alendronate, or ISA.
7. The pharmaceutical composition according to claim 1, for
intravenous (IV), intrarterial (IA), intramuscular (IM),
subcutaneous (SC), parenteral, or intraperitoneal (IP)
administration.
8. A pharmaceutical composition comprising an active ingredient
encapsulated in, embedded in, absorbed onto, or linked to an
insoluble particle wherein the particle is non-liposomal and is
selected from the group consisting of a bisphosphonate
microparticle, a bisphosphonate nanoparticle, a bisphosphonate
nanosphere, a bisphosphonate microcapsule, a bisphosphonate
nanocapsule, and a bisphosphonate polymeric particle together with
a pharmaceutically acceptable carrier, the particle having a size
of 0.01-1.0 microns in diameter.
9. The pharmaceutical composition according to claim 8, wherein the
size of the particle is 100-300 nm in diameter.
10. The pharmaceutical composition according to claim 8, further
comprising a diluent.
11. The pharmaceutical composition according to claim 8 further
comprising a stabilizer.
12. The pharmaceutical composition according to claim 8, wherein
the bisphosphonate has the following formula (I): ##STR8## wherein
R.sub.1 is H, OH or a halogen atom; and R.sub.2 is a 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.
13. The pharmaceutical composition according to claim 8, wherein
said bisphosphonate is clodronate, etidronate, tiludronate,
pamidronate, alendronate, or ISA.
14. The pharmaceutical composition according to claim 8, for
intravenous (IV), intrarterial (IA), intramuscular (IM),
subcutaneous (SC), or intraperitoneal (IP) administration.
15. A pharmaceutical composition comprising an active ingredient
encapsulated in, embedded in, absorbed onto, or linked to an
insoluble particle wherein the particle is a liposome having a size
of 0.01-1.0 microns in diameter.
16. The pharmaceutical composition according to claim 15, wherein
the size of the liposome is 100-300 nm in diameter.
17. The pharmaceutical composition according to claim 15, wherein
the liposome comprisies distearoyl-phosphatidylcholine (DSPC) and
cholesterol.
18. The pharmaceutical composition according to claim 17, wherein
the liposome comprisies DSPC and cholesterol in a weight ratio of
3:1.
19. The pharmaceutical composition according to claim 17, wherein
the liposome further comprisies distearoylphosphatidylglycerol
(DSPG).
20. The pharmaceutical composition according to claim 19, wherein
the liposome comprises DSPG, DSPC, and cholesterol in a weight
ratio of 1:3:1.
21. The pharmaceutical composition according to claim 15, further
comprising a diluent.
22. The pharmaceutical composition according to claim 15 further
comprising a stabilizer.
23. The pharmaceutical composition according to claim 15, wherein
the bisphosphonate has the following formula (I): ##STR9## wherein
R.sub.1 is H, OH or a halogen atom; and R.sub.2 is a 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.
24. The pharmaceutical composition according to claim 15, wherein
said bisphospho nate is clodronate, etidronate, tiludronate,
pamidronate, alendronate, or ISA.
25. The pharmaceutical composition according to claim 15, for
intravenous (IV), intrarterial (IA), intramuscular (IM),
subcutaneous (SC), or intraperitoneal (IP) administration.
Description
[0001] This application is a continuation of application Ser. No.
10/160,207 filed on May 30, 2002, which is a continuation-in-part
of application Ser. No. 10/126,248 filed on Apr. 19, 2002, which is
a continuation-in-part of application Ser. No. 09/743,705 filed on
Mar. 22, 2001, which is a 35 U.S.C .sctn.371 filing of PCT
application No. PCT/IL99/00387 filed on Jul. 14, 1999, which is a
continuation-in-part of Israeli application no. 125336 filed on
Jul. 14, 1998.
FIELD OF THE INVENTION
[0002] The present invention is concerned with compositions capable
of preventing, inhibiting or reducing restenosis (sometimes
referred to in the art as "accelerated arteriosclerosis" and
"post-angioplasty narrowing"). Specifically, the invention relates
to the use of bisphosphonates ("BP") to inhibit and/or prevent
restenosis.
BACKGROUND OF THE INVENTION
[0003] Over the past decade, mechanical means of achieving
revascularization of obstructive atherosclerotic vessels have been
greatly improved. Percutaneous transluminal coronary angioplasty
(PTCA) procedures include, but are not limited to, balloon
dilatation, excisional atherectomy, endoluminal stenting,
rotablation and laser ablation. However, revascularization induces
thrombosis, and neointimal hyperplasia, which in turn cause
restenosis in a substantial proportion of coronary arteries after
successful balloon angioplasty and in aortacoronary saphenous vein
bypass graft and other coronary grafts. Furthermore, intimal
hyperplasia causes restenosis in many superficial femoral
angioplasties, carotid endarterectomies, and femoro-distal vein
bypasses. Restenosis is the formation of new blockages at the site
of the angioplasty or stent placement or the anastomosis of the
bypass. As a result, the patient is placed at risk of a variety of
complications, including heart attack or other ischemic disease,
pulmonary embolism, and stroke. Thus, such procedures can entail
the risk of precisely the problems that its use was intended to
ameliorate. The introduction of endovascular stents has reduced the
incidence of restenosis, but this problem still remains
significant, since restenosis or "over exuberant" tissue healing
may occur at the site of stent placement. (Waller, B. F. et al.,
1997, Clin-Cardiol., 20(2):153-60; Anderson, W. D et al., 1996,
Curr-Opin-Cardiol., 11(6):583-90; Moorman, D. L. et al., 1996,
Aviat-Space-Environ-Med., 67(10):990-6; Laurent, S. et al., 1996,
Fundam. Clin. Pharmacol., 10(3):243-57; Walsh, K. et al., 1996,
Semin-Interv-Cardiol., 1(3):173-9; Schwartz, R. S., 1997, Semin
Interv Cardiol., 2(2):83-8; Allaire, E. et al., 1997, Ann. Thorac.
Surg., 63:582-591; Hamon, M. et al., 1995, Eur. Heart J.,
16:33s-48s; Gottsauner-Wolf, M., et al., 1996, Clin. Cardiol.,
19:347-356).
[0004] Despite extensive research on the incidence, timing,
mechanisms and pharmacological interventions in humans and animal
models to date, no therapy exists which consistently prevents
coronary restenosis (Herrnan, J. P. R. et al., 1993, Drugs,
46:18-52; Leclerc, G. et al., 1995, Elsevier Science, 722-724,
Topol, E., 1997, The NY Academy of Sciences, 225-277). Compositions
and methods for the reduction or prevention of restenosis are still
greatly desired. Accordingly, it would be desirable to develo p
novel compositions and methods that are effective in treating
restenosis and preventing its reoccurrence.
[0005] Bisphosphonates ("BPs") (formerly called diphosphonates) are
compounds characterized by two C--P bonds. If the two bonds are
located on the same carbon atom (P--C--P) they are termed geminal
bisphosphonates. The BPs are analogs of the endogenous inorganic
pyrophosphate which is involved in the regulation of bone formation
and resorption. The term bisphosphonates is generally used for
geminal and non-geminal bisphosphonates. The BPs may at times form
polymeric chains. BPs act on bone because of their affinity for
bone mineral and also because they are potent inhibitors of bone
resorption and ectopic calcification. BPs have been clinically used
mainly as (a) antiosteolytic agents in patients with increased bone
destruction, especially Paget's disease, tumor bone disease and
osteoporosis; (b) skeletal markers for diagnostic purposes (linked
to .sup.99mTc); (c) inhibitors of calcification in patients with
ectopic calcification and ossification, and (d) antitartar agents
added to toothpaste (Fleisch, H., 1997, in: Bisphosphonates in bone
disease. Parthenon Publishing Group Inc., 184-186). Furthermore,
being highly hydrophilic and negatively charged, BPs in their free
form are almost incapable of crossing cellular membranes.
SUMMARY OF THE INVENTION
[0006] In one embodiment, the present invention relates to a method
of treating or preventing restenosis by administering to an
individual an effective amount of an active ingredient comprising a
bisphosphonate, a bisphosphonate salt, a bisphosphonate ester, or a
bisphosphonate complex, wherein the active ingredient is in a
particle dosage form. The particles include, but are not limited
to, inert polymeric particles, such as microcapsules, nanocapsules,
nanospheres, microspheres, nanoparticles, microparticles, or
liposomes.
[0007] In a further embodiment, the present invention relates to a
method of treating or preventing restenosis by administering to an
individual an effective amount of an active ingredient comprising a
bisphosphonate, an insoluble bisphosphonate salt, an insoluble
bisphosphonate ester, or an insoluble bisphosphonate complex,
wherein the active ingredient is in a free particulate dosage
form.
[0008] Effective phagocytosis of both the bisphosphonate particles
and the bisphosphonate free particulates by the
monocytes/macrophages can affect the activity of such phagocytic
cells. The active ingredient affects restenosis by inhibiting
phagocytic cells involved in the restenotic cascade, such as
macrophages/monocytes and fibroblasts. The delivery system affects
smooth-muscle cells (SMC) and extracellular matrix production
indirectly by inhibiting the cells that trigger their migration
and/or proliferation. Nevertheless, a direct effect on SMC may also
occur. The active ingredient may be administered by any route which
effectively transports the active compound to the desirable site of
action. In a preferred embodiment, the mode of administration
includes intra-arterial, intravenous or subcutaneous
administration.
[0009] In a further embodiment, the present invention includes a
method of treating or preventing restenosis by administering to an
individual, an effective amount of any compound or composite known
to inactivate or inhibit blood monocytes and tissue macrophages,
thereby treating or preventing restenosis.
[0010] In a further embodiment, the present invention includes a
pharmaceutical composition comprising an active ingredient selected
from the group consisting of a bisphosphonate particle, a
bisphosphonate particulate, or a salt, ester, or complex of
bisphosphonate, together with a pharmaceutically acceptable
carrier, stabilizer or diluent for the prevention or treatment of
vascular restenosis.
[0011] In yet a further embodiment, the present invention includes
a method of inhibiting the activity and/or production of cytokines
and growth factors associated with vascular restenosis, by
administering an effective amount of an active ingredient
comprising a bisphosphonate, a bisphosphonate salt, a
bisphosphonate ester, or a bisphosphonate complex, wherein the
active ingredient is in a particle dosage form.
[0012] In still yet a further embodiment, the present invention
includes a method of inhibiting the activity and/or production of
cytokines and growth factors associated with vascular restenosis,
by administering to an individual an effective amount of an active
ingredient comprising a bisphosphonate, an insoluble bisphosphonate
salt, an insoluble bisphosphonate ester, or an insoluble
bisphosphonate complex, wherein the active ingredient is in a free
particulate dosage form.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIGS. 1-3 are bar graphs of results demonstrating the effect
of clodronate encapsulated in liposomes on the reduction of
restenosis in an experimental rat carotid catheter injury model as
compared to the effect of control liposomes which did not contain
clodronate on the same rats. In these figures:
[0014] FIG. 1 shows the mean neointimal area to the area of the
media in rats treated with clodronate containing liposomes as
compared to rats treated with control liposomes. The medial area is
the difference between the total arterial area and the original
lumen area.
[0015] FIG. 2 shows the % stenosis in rats treated with clodronate
containing liposomes as compared to the % stenosis in rats treated
with control liposomes.
[0016] FIG. 3 shows the extent of medial area as an indirect index
of smooth muscle cell viability and determined as the difference
between the total arterial area and the original lumen area
(External elastic lamina bound area-Internal elastic lamina bound
area) in rats treated with clodronate containing liposomes as
compared to rats treated with control liposomes only.
[0017] FIG. 4 illustrates the antirestenotic effects of liposomal
clodronate in the balloon-injured rat and atherosclerotic rabbit
carotid arterial models.
[0018] FIGS. 5a, 5b, 5c and 5d illustrate the effect of liposomal
clodronate treatment on interleukin 1-.beta. (IL-1.beta.)
concentration and matrix metalloproteinase-2 (MMP-2) activity in
the arteries of rats and rabbits following balloon injury.
Specifically, FIGS. 5a and 5b illustrate the effect of liposomal
clodronate treatment on IL-1.beta. concentration in the rat and
rabbit models, respectively, and FIGS. 5c and 5d illustrate the
effect of liposomal clodronate treatment on MMP-2 activity in the
rat and rabbit models, respectively.
[0019] FIG. 6 illustrates the effect of liposomal clodronate on
IL-1.beta. transcription in rabbits' arteries following balloon
injury.
[0020] FIGS. 7a, 7b, and 7c illustrate the effect of liposomal
clodronate treatment on the platelet-derived growth factor (PDGF)
system in the arterial walls of rats following balloon injury.
Specifically, FIG. 7a illustrates the effect on platelet-derived
growth factor .beta. receptor (PDGF.beta.R) activation (i.e.,
tyrosine phosphorylation), FIG. 7b illustrates the effect on the
PDGF.beta.R protein, and FIG. 7c illustrates the effect on the
PDGF-B protein.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention relates to compositions and methods
for reducing, delaying or eliminating restenosis. Reducing
restenosis includes decreasing the thickening of the inner blood
vessel lining that results from stimulation and proliferation of
smooth muscle cell and other cell migration and proliferation, and
from extracellular matrix accumulation, following various
angioplasty procedures. Delaying restenosis includes delaying the
time until angiographic re-narrowing of the vessel appears or until
the onset of clinical symptoms which are attributed to stenosis of
this vessel. Eliminating restenosis following angioplasty includes
reducing hyperplasia to an extent which is less than 50% of the
vascular lumen, with lack of clinical symptoms of restenosis.
Methods of intervening include re-establishing a suitable blood
flow through the vessel by methods such as, for example, repeat
angioplasty and/or stent placement, or coronary artery bypass graft
(CABG).
[0022] The present invention includes a method of treating or
preventing restenosis by administering to an individual, 'an
effective amount of any compound or composite known to inactivate
or inhibit blood monocytes and tissue macrophages.
[0023] One example of a group of drugs useful in the present
invention to inhibit restenosis, are bisphosphonates ("BPs"). BPs
inhibit smooth muscle cell migration and proliferation by
transiently depleting and/or inactivating cells that are important
triggers in the restenosis cascade, namely macrophages and/or
monocytes. Bisphosphonates, when encapsulated in liposomes or
nanoparticles in a "particle" dosage form, or when in a "free
particulate" dosage form, such as, for example, in aggregates of a
specific size, are taken-up, by way of phagocytosis, very
efficiently by the macrophages and monocytes, and to some extent by
other cells with phagocytic activity such as fibroblasts. Once
inside the macrophages, the liposomal structure of the cell is
disrupted and the bisphosphonates are released, thereby inhibiting
the activity and/or killing the macrophages. Since macrophages, in
their normal state, are recruited to the areas traumatized by
angioplasty or other intrusive intervention and initiate the
proliferation of smooth-muscle cells (SMC), inhibiting the
macrophages' activity inhibits the migration and proliferation of
SMC. After being taken-up by the macrophages, the bisphosphonates
have a sustained inhibitory activity on the macrophages. Thus,
prolonged release of the bisphosphonates is not required in order
to sustain inhibition. Accordingly, the method of inhibiting or
reducing restenosis by administering a bisphosphonate in a particle
or free particulate form is preferably a systemic therapy, in that
the bisphosphonate particles and particulates target the
circulating monocytes and macrophages.
[0024] It should be noted, however, that some bisphosphonate
particles and particulates may have a direct effect on SMC
activity. Additionally, some of the bisphosphonate particles and
particulates may also inactivate other phagocytic cells and cells
of the white-blood cell lineage in the body, such as liver and
spleen macrophages and macrophages in the arterial walls.
[0025] Furthermore, the delivery system of the present invention
not only retains the BP for a sufficient time so that the free BP
is not released in the body fluids, but also efficiently discharges
the drug within the target cell. The free BP drug, as opposed to BP
particles, is ineffective since it is not taken-up by phagocytic
cells.
[0026] An additional example of a group of drugs useful in the
present invention to inhibit restenosis are inactivators of
monocytes/macrophages, such as gallium or gold.
[0027] In accordance with the present invention, a bisphosphonate
or a compound or composite which inactivates monocytes/macrophages
(collectively herein: "active ingredient") is used for treatment or
prevention of vascular restenosis. The tenn bisphosphonate as used
herein, denotes both geminal and non-geminal bisphosphonates. The
term "active ingredient" encompasses in its scope, not only BP and
compounds which inactivate monocytes/macrophage, but also polymeric
chains of the BPs and the monocyte/macrophage inactivators,
particularly such chains consisting of up to 40 BP monomers.
Preferred active ingredients are compounds of the following formula
(I) ##STR1## wherein R.sub.1 is H, OH or a halogen atom; and [0028]
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.
[0029] The present invention thus provides the use of said active
ingredient, a complex of said active ingredient or a
pharmaceutically acceptable salt or ester thereof for the
preparation of a composition for the prevention or treatment of
vascular restenosis. In one embodiment, the composition comprises a
"particle" dosage form, wherein the active ingredient is
encapsulated, embedded, and/or adsorbed within a particle,
dispersed in the particle matrix, adsorbed or linked on the
particle surface, or in combination of any of these forms. The
particle includes any of the liposomes, microparticles,
nanoparticles, nanospheres, microspheres, microcapsules, or
nanocapsules known in the art (M. Donbrow in: Microencapsulation
and Nanoparticles in Medicine and Pharmacy, CRC Press, Boca Raton,
Fla., 347). The term particle includes both polymeric and
non-polymeric preparations of the active ingredient. In a further
embodiment, the composition comprises a "free particulate" dosage
form of the active ingredient, such as an insoluble salt, insoluble
ester, or insoluble complex of the active ingredient. Typically,
"insoluble" refers to a solubility of one (1) part of a compound in
more than ten-thousand (10,000) parts of a solvent. A "free
particulate" dosage form includes any insoluble suspended or
dispersed particulate form of the active ingredient which is not
encapsulated, entrapped or adsorbed within a polymeric particle.
Free particulates include, but are not limited to, suspended or
dispersed colloids, aggregates, flocculates, insoluble salts and
insoluble complexes. Additionally, in both the particle and free
particulate dosage forms, suspending agents and stabilizers may be
used. In yet a further embodiment, the composition comprises
polymeric chains of the active ingredient.
[0030] The present invention also provides a method of treatment of
restenosis, comprising administering to an individual in need an
effective amount of said active ingredient, a complex thereof or a
pharmaceutically acceptable salt or ester thereof.
[0031] The present invention still further provides a
pharmaceutical composition for the prevention or treatment of
restenosis comprising, an effective amount of the active
ingredient, a complex or a salt thereof, optionally together with a
pharmaceutically acceptable carrier or diluent. Carriers include,
but are not limited to, liposomes, particles, and lipid
particles.
[0032] The present invention also provides a method of inhibiting
the activity, production, and/or transcription of certain cytokines
and growth factors that are associated with restenosis or with any
of the cell types involved in the restenotic cascade, by
administering a bisphosphonate particle or particulate. The select
cytokines and growth factors that are associated with restenosis or
with the cell types involved in the restenotic cascade include, but
are not limited to, interleukin-1 (IL-1), matrix metalloproteinases
(MMPs), and platelet-derived growth factor .beta. (PDGF.beta.). For
example, IL-1.beta. and MMP-2 are major products of activated
macrophages, that are secreted following arterial injury, and
contribute to the process of neointimal proliferation.
Additionally, PDGF-BB is a strong chemoattractant for vascular
smooth muscle cells and is involved in neointima formation
secondary to vascular injury. (Fishbein, I., et al., 2000,
Arterioscler. Thromb. Vasc. Biol., 20:667-676; Jawien, A., et al.,
1992, J. Clin. Invest., 507-511; Ross, R., 1993, Nature,
362:801-809; Panek, R. L., et al., 1997, Arterioscler. Thromb.
Vasc. Biol., 17:1283-1288; Waltenberger, J, 1997, Circulation,
96:4083-4094; Deguchi, J., et al., 1999, Gene Ther.,
6:956-965.)
[0033] The term "effective amount" denotes an amount of the active
ingredient, which is effective in achieving the desired therapeutic
result, namely prevention, reduction, or elimination of vascular
restenosis. The effective amount may depend on a number of factors
including: weight and gender of the treated individual; the type of
medical procedure, e.g. whether the vascular restenosis to be
inhibited is following balloon angioplasty, balloon angioplasty
followed by deployment of a stent; the mode of administration of
the active ingredient (namely whether it is administered
systemically or directly to the site); the type of carrier being
used (e.g. whether it is a carrier that rapidly releases the active
ingredient or a carrier that releases it over a period of time);
the therapeutic regime (e.g. whether the active ingredient is
administered once daily, several times a day, once every few days,
or in a single dose); clinical factors influencing the rate of
development of restenosis such as diabetes, smoking,
hypercholesterolemia, renal diseases; anatomical factors such as
whether there is severe preangioplasty stenosis, total occlusion,
left anterior descending coronary artery location, saphenous vein
graft lesion, long lesions, multivessel or multilesion PTCA; and on
the dosage form of the composition. Moreover, procedural variables
may also have bearing on the dosage, such as greater residual
stenosis following PTCA, severe dissection, intimal tear,
appropriate size of balloon, and the presence of thrombus. The
artisan, by routine type experimentation should have no substantial
difficulties in determining the effective amount in each case.
[0034] The invention is applicable for the prevention, reduction or
treatment of vascular restenosis and mainly, but not limited to,
coronary restenosis after angioplasty. Vascular restenosis
primarily results from various angioplasty procedures including
balloon angioplasty, intravascular stent deployment or other
methods of percutaneous angioplasty (including angioplasty of
coronary arteries, carotid arteries, and other vessels amenable for
angioplasty) as well as for restenosis resulting from vascular
graft stenosis (e.g. following by-pass surgery) (Braunwald, E.,
1997, Heart Disease in: A textbook of cardiovascular medicine; 5th
Ed., W.B. Saunders Company: Philadelphia).
[0035] In addition, the invention is also applicable for use in
prevention, reduction or treatment of vascular restenosis in
peripheral arteries and veins.
[0036] One exemplary application of the invention is to prevent and
treat in-stent restenosis. It is a widely acceptable medical
procedure to deploy a stent within a blood vessel within the
framework of an angioplastic procedure, to support the walls of the
blood vessel. However, very often restenosis occurs notwithstanding
the presence of the stent within the blood vessel. In accordance
with the invention, the above noted active ingredient may be
administered, either systemically or directly to the site, in order
to prevent or inhibit such restenosis. The active ingredient may be
formulated in a manner allowing its incorporation onto the stent
which, in fact, yields administration of said active ingredient
directly at the site. The active ingredient may be formulated in
that manner, for example, by including it within a coating of the
stent. Examples of coatings are polymer coatings, (e.g., made of
polyurethane), gels, fibrin gels, hydrogels, carbohydrates,
gelatin, or any other biocompatible gel.
[0037] The active ingredient used in accordance with the invention
may be formulated into pharmaceutical compositions by any of the
conventional techniques known in the art (see for example, Alfonso,
G. et al., 1995, in: The Science and Practice of Pharmacy, Mack
Publishing, Easton Pa., 19th ed.). The compositions may be prepared
in various forms suitable for injection, instillation or
implantation in body such as suspensions of the nanoparticles, as
in a coating of a medical device such as a stent (see above). In
addition, the pharmaceutical compositions of the invention may be
formulated with appropriate pharmaceutical additives for parental
dosage forms. The preferred administration form in each case
depends on the desired delivery mode, which is usually that which
is the most physiologically compatible with the patient's condition
and with the other therapeutic treatments which the patient
currently receives.
[0038] In a preferred embodiment of the invention, the active
ingredient is selected from the group of bisphosphonates. One
preferred active ingredient for this group is the compound
clodronate, (dichloromethylene) diphosphonic acid, (Fleisch, H.,
1997, in: Bisphosphonates in bone disease. Parthenon Publishing
Group Inc., 184-186) having the following formula (II):
##STR2##
[0039] Clodronate was previously described for use in the treatment
of hypercalcemia resulting from malignancy in the treatment of
tumor associated osteolysis (Fleisch, H., 1997, in: Bisphosphonates
in bone disease. Parthenon Publishing Group Inc., 184-186).
Clodronate 5 was also found to inhibit macrophages in vitro and to
suppress macrophage activity in the spleen and liver tissues of
mice. (Monkkonen, J. et al, 1994, J. Drug Target, 2:299-308;
Monkkonen, J. et al., 1993, Calcif. Tissue Int., 53:139-145).
[0040] Other preferred active ingredients of this group are
etidronate and tiludronate having the following formulae (III) and
(IV) respectively: ##STR3##
[0041] Additional BPs having activities similar to that of
clodronate are also preferred in accordance with the invention.
Such BPs may be selected on the basis of their ability to mimic the
biological activity of clodronate. This includes, for example: in
vitro activity in inhibiting phagocytic activity of phagocytic
cells, e.g. macrophages and fibroblasts; inhibition of secretion of
IL-1 and/or IL-6 and/or TNF-.alpha. from macrophages; reduction of
MMP activity, for example, MMP-2 activity; inhibition of
PDGF.beta.R activation and/or reduction of PDGF-B protein levels;
and, in vivo activity, e.g. the ability of the tested BP to prevent
or reduce restenosis in an experimental animal model such as, for
example, the rat or rabbit carotid catheter injury model described
in Example 1 below, or porcine model of restenosis.
[0042] The most preferred group of active ingredients in accordance
with the invention are the amino-BPs and any other
nitrogen-containing BPs having the following general formula (V):
##STR4## wherein X represents C.sub.1-C.sub.10 alkylamino or
C.sub.3-C.sub.8 cycloalkylamino, where the amino may be primary,
secondary or tertiary; or X represents NHY where Y is hydrogen,
C.sub.3-C.sub.8 cycloalkyl, aryl or heteroaryl.
[0043] The BPs belonging to this group are believed not to be
metabolized and have been shown at relatively low concentrations to
induce secretion of the interleukin, IL-1, and cause, at relatively
high concentrations, apoptosis in macrophages (Monkkonen, J. et
al., 1993, Calcif. Tissue Int., 53:139-145). Preferred BPs
belonging to this group are for example, pamidronate and
alendronate having the following formulae (VI) and (VII),
respectively. ##STR5## ##STR6##
[0044] Although the geminal BPs are preferred BPs in accordance
with the invention, non-geminal BPs, monophosphonates of BPs,
termed generally as phosphonates may also be used as active
ingredients in accordance with the invention.
[0045] Additional bisphosphonates include, but are not limited to,
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.
[0046] Thus, suitable bisphosphonates for use in the present
invention include the acid compounds presented above, any
acceptable salts thereof, and crystalline and amorphous BPs. 20
Additionally, preferred bisphosphonates are the
amino-bisphosphonates such as alendronate, zolendronate, and
risendronate.
[0047] The composition of the invention may comprise said active
ingredient either encapsulated within a particle, adsorbed on the
particle surface, complexed with metal cations such as calcium,
magnesium or organic bases, formed into non-soluble salts or
complexes, or 25 polymerized to yield polymers of up to 40
monomers. The salts may be sodium, potassium, ammonium, gallium or
calcium salts or salts formed with any other suitable cation (e.g.
organic amino compounds). The salts or polymers may be in a
micronized particulate form having a diameter within the range of
about 0.01-1.0 .mu.m, preferably within a range of about 0.1-0.5
.mu.m. The active ingredients in their salt form may be with or
without water of crystallization (hydrous and anhydrous).
Additionally, additives such as polyvinyl alcohol (PVA), pluronics,
and other surface active agents, may be used to stabilize the salt
and or complex to establish a colloidal or nano-size suspension. In
one embodiment for example, the composition may comprise a Ca--BP
salt and or complex.
[0048] In one embodiment of the invention, the active ingredient is
encapsulated in liposomes. The liposomes may be prepared by any of
the methods known in the art (regarding liposome preparation
methods see Monkkonen, J. et al, 1994, J. Drug Target, 2:299-308,
and Monkkonen, J. et al., 1993, Calcif. Tissue Int., 53:139-145).
The liposomes may be positively charged, neutral or negatively
charged (negatively charged liposomes being currently preferred),
and may be single or multilamellar. Suitable lip osomes in
accordance with the invention are preferably non toxic liposomes
such as, for example, those prepared from phosphatidyl-choline
phosphoglycerol, and cholesterol, e.g. as described below. In many
cases, use of liposomal delivery results in enhanced uptake of the
active ingredient by cells not only via endocytosis but also via
other pathways such as fusion (such uptake may play a role in the
therapeutic effect). The diameter of the preferred liposomes may
range from 0.15 to 300 nm. However, this is merely a non-limiting
example, and liposomes of other size ranges may also be used.
[0049] In a further preferred embodiment, the active ingredient or
bisphosphonate may be encapsulated or embedded in inert particles.
In yet a further embodiment, the active ingredient may be adsorbed
onto the surface of, or adsorbed within, a blank particle, wherein
a blank particle is a particle which has no drug encapsulated or
embedded therein. Alternatively, the active ingredient may form a
particulate, which includes a colloid, aggregate, flocculate or
other such structure known in the art for the preparation of
particulates of drugs. Furthermore, such particulates may be
aggregates of the polymerized active ingredient.
[0050] Particulates of the active ingredient may be obtained by
using an insoluble salt or complex that can be obtained in-situ,
i.e., starting with the soluble drug and "salting-out" the drug by
adding for example, Ca at the appropriate concentration and pH. The
dispersed or free particulates are formed and then stabilized by
the aid of surface active agents, suspending agents, deflocculating
agents or by thickening agents, such those used in gels. The active
ingredient may be further precipitated by adding a trivalent
cation, for example, gallium, thereby forming a precipitate of
gallium-BP salt/complex.
[0051] The active ingredient may be encapsulated within or adsorbed
onto particles, e.g., nanoparticles by utilizing, for example, a
modified nano-precipitation method. In this embodiment of the
invention, the polymeric nanoparticle containing the active
ingredient is formed by mixing water and organic solutions of the
drug and polymer (PLGA or other polymers), respectively. Thus, the
nanoparticle containing drug formed is suspended in water and can
be lyophilized. Additionally, the active ingredient may be
entrapped or adsorbed into blank polymeric nanoparticles, and/or
adsorbed on the surface of the blank polymeric nanoparticles.
(Blank nanoparticles are particles which have no drug encapsulated,
embedded, and/or adsorbed therein).
[0052] One advantage of particulate dosage forms of the active
ingredient itself, or of polymeric particle dosage forms (e.g.
nanoparticles), is the possibility of lyophilization and of
sterilization methods other than filter-sterilization. Thus, these
forms of the active ingredient have an extended shelf-life and ease
of handling.
[0053] In a further embodiment, the bisphosphonates may be
encapsulated in nanoparticles ("NP"). Nanoparticles are 30-1000 nm
diameter, spherical or non-spherical polymeric particles. The drug
can be encapsulated in the nanoparticle, dispersed uniformly or
non-uniformly in the polymer matrix (monolithic), adsorbed on the
surface, or in combination of any of these forms. It is the
submicron nature of this compositional form, which makes it more
efficient in therapeutic applications. The submicron size
facilitates uptake by phagocytic cells such as monocytes and
macrophages, and avoids uptake in the lungs. In a preferred
embodiment, the polymer used for fabricating nanoparticles is the
biocompatible and biodegradable, poly(DL-lactide-co-glycolide)
polymer (PLGA). However, any polymer which is biocompatible and
biodegradable may be used. Therefore, additional polymers which may
be used to fabricate the NP include, but are not limited to,
polyanhydrides, polyalkyl-cyanoacrylates (such as
polyisobutylcyanoacrylate), polyetheyleneglycols,
polyethyleneoxides and their derivatives, chitosan, albumin,
gelatin and the like. The size of the nanoparticle used to
encapsulate the active ingredient or bisphosphonate depends on the
method of preparation and the mode of administration (e.g. IV, IA,
etc.) Preferably, the nanoparticles range in size from 70-500 nm.
However, depending on preparation and sterilization techniques, the
more preferred ranges include, but are not limited to, 100-300 nm
and 100-220 nm.
[0054] The pharmaceutical carrier or diluent used in the
composition of the invention may be any one of the conventional
solid or liquid or semisolid carriers known in the art. A solid
carrier, for example, may be lactose, sucrose, gelatins, and other
carbohydrates. A liquid carrier, for example, may be a
biocompatible oil suitable for injection such as peanut oil, water
or mixtures of biocompatible liquids, or a biocompatible viscous
carrier such as a polyethylene or gelatin gel.
[0055] The composition of the active ingredient used for injection
may be selected from emulsions, suspensions, colloidal solutions
containing suitable additives, and additional suitable compositions
known to the skilled artisan.
[0056] The compositions of the invention may be administered by any
route which effectively transports the active compound to the
appropriate or desirable site of action. By a preferred embodiment
of the invention, the modes of administration are intravenous (IV)
and intra-arterial (IA) (particularly suitable for on-line
administration). Other suitable modes of administration include
intramuscular (IM), subcutaneous (SC), or intraperitonal (IP). Such
administration may be bolus injections or infusions. The
compositions may also be administered locally to the diseased site
of the artery, for example, by means of a medical device which is
coated with the active ingredient. Another mode of administration
may be by perivascular delivery. Combinations of any of the above
routes of administration may also be used in accordance with the
invention.
[0057] The dosage of the active ingredient to be used also depends
on the specific activity of the active ingredient selected, on the
mode of administration (e.g. systemic administration or local
delivery), the form of the active ingredient (e.g. polymer,
encapsulated in a particle such as a liposome, nanoparticle etc.),
the size of the particle, the type of bisphosphonate, the
administration route, the number of injections, the timing of
injections, the biology/pathology of the patient in need, and other
factors as known per se.
[0058] In one embodiment, the dosage for clodronate-containing
liposomes (liposomal clodronate, ("LC")) in humans preferably
ranges from 0.015 mg/kg (per kg of body weight) to 150 mg/kg; more
preferably, however, the dosage ranges from 0.15 to 15 mg/kg.
Dosages outside these preferred ranges may also be used, as can be
readily determined by the skilled artisan. When IV/IA injections or
local delivery methods are used, i.e. via a balloon catheter, the
dosage is at the lower end of the range. However, when IM or SC
administration modes are used the dosage is approximately 10 times
that used for IV administration.
[0059] In accordance with a preferred embodiment of the invention,
treatment of an individual with the active ingredient may be for
the purpose of preventing restenosis before its occurrence. For
prevention, the active ingredient may be administered to the
individual before angioplasty procedure, during the procedure or
after the procedure as well as combination of before, during and
after procedural administration. Furthermore, the active ingredient
may be administered via IV, IA, IM, SC, IP or any other suitable
type of administration. For example, the active ingredient may be
administered via IA the day of the angioplasty procedure (day 0),
via IV the day before the procedure (-1) and/or on day 0, or both
via IV the day before the procedure (-1) and also after the
procedural administration, for example, on day 6.
[0060] In accordance with a further embodiment of the invention,
the active ingredient is administered to an individual suffering
from restenosis for the purpose of reducing or treating restenosis.
In such a case, the active ingredient may also be administered to
the individual at different periods of time after restenosis is
discovered, either alone or in combination with other kinds of
treatments.
[0061] In addition, the active ingredient may be administered
before any other conditions which may yield accelerated
arteriosclerosis, as well as acutely after the process has begun to
inhibit further development of the condition.
EXAMPLES
[0062] The invention will now be demonstrated by way of
non-limiting examples with reference to the accompanying drawings.
The animal models used in the examples below include the
balloon-injured rat carotid arterial model and the balloon-injured
hypercholesterolemic rabbit carotid arterial model. The rat is an
acceptable model in evaluating the antirestenotic effects of drugs
and composites; however, the rabbit is more preferred since it,
unlike the rat, is both atherosclerotic and contains a significant
number of macrophages in the arterial wall.
Example 1
Liposomes of Clodronate
[0063] Stock solutions of clodronate were prepared by dissolving
the drug in deionized water at a concentration of 0.11 M, pH=7.
Liposome Preparation
[0064] 38.9 mg of distearoylphosphatidylglycerol (DSPG), 118.5 mg
of distearoyl-phosphatidylcholine (DSPC) and 38.7 mg of cholesterol
were accurately weighed and dissolved in 20 ml of chloroform:
methanol (9:1) in a round bottom vial. The vial was gently warmed,
and the solvent was then evaporated in rotavapor. 20 mls of
hydrated diisopropylether were then added and the vial was put into
a water bath until the contents were dissolved. 8 mls of the
clodronate solution prepared as described above were then added,
and the solution was sonicated at 55.degree. C. for a period of 45
minutes. The organic phase was then evaporated in rotavapor
(55.degree. C., 100 rpm). Similarly, other drug-containing
liposomes can be prepared.
Purification of Prepared Liposomes
[0065] A Sephadex gel was prepared by dissolving 2.6 grams of
Sephadex G-50 in 40 mls of water and stabilizing overnight. The
column was rinsed with 100 mls of buffer (50 mM Mes+50 mM HEPES+75
mM NaCl, pH 7.2). The liposomes were applied to the column and the
column was rinsed with the buffer. The liposome was seen as a band
which can be followed in the column by its color. About 20 drops
were collected from the column into each tube.
Animals
[0066] Animals were obtained and housed in the animal facilities of
the Faculty of Medicine, The Hebrew University of Jerusalem,
conforming to the standards for care and use of laboratory animals
of the Hebrew University of Jerusalem. Male rats of Sabra strain
weighing 350-420 g were used. The animals were fed standard
laboratory chow and tap water ad libitum. All in vivo experiments
were conducted under general anaesthesia achieved with 80 mg/kg
ketamine and 5 mg/kg xylazine administered IP.
Rat Carotid Catheter Injury Model
[0067] The distal left common and external carotid arteries were
exposed through a midline incision in the neck. The left common
carotid artery was denuded of endothelium by the intraluminal
passage of a 2 F balloon catheter introduced through the external
carotid artery. The catheter was passed three times with the
balloon distended sufficiently with saline to generate a slight
resistance. The catheter was then removed and the external carotid
artery was ligated, and the wound was closed with surgical
staples.
[0068] Seven rats served as the control group, and 6 rats as the
treated group (randomly chosen). Liposomal clodronate was injected
IV to the "treated group" one day prior to the arterial injury (6
mg of clodronate per rat) and repeated on day 6. In the control
group similar injections were administered but with "empty" or
blank liposomes (no clodronate).
[0069] All animals were sacrificed 14 days after injury by an
overdose of pentobarbital. Arteries were perfusion-fixed with 150
ml of 4% formaldehyde solution pH 7.4 at 100 mm Hg. The right
atrium was dissected and an 18 G catheter connected to the
perfusion system was inserted in the left ventricle. The arterial
segments were dissected, cut, gently separated from the polymer,
and postfixed for at least 48 hours in the same fixative solution.
The arterial segments were embedded in paraffin and cut at 8-10
sites 600 .mu.m apart. Sections of 6 .mu.m were then mounted and
stained with Verhoeffs elastin stain for histologic
examination.
Morphometric Analysis
[0070] The slides were examined microscopically by an investigator
blinded to the type of the experimental group. Six to eight
sections in each slide were evaluated by computerized morphometric
analysis and the averaged section data were further used as a
representative of a whole slide for comparisons between groups. The
residual lumen, the area bounded by the internal elastic lamina
(original lumen), and the area circumscribed by the external
elastic lamina ("total arterial area") were measured directly. The
degree of neointimal thickening was expressed as the ratio between
the area of the neointimal and the original lumen (% stenosis), and
as the ratio between the neointimal area to the area of the media
(N/M). The medial area, an indirect index of SMC viability, was
determined as the difference between the total arterial area and
the original lumen area.
[0071] The surgical procedure and treatment did not cause mortality
or apparent morbidity of the animals.
[0072] As seen in FIG. 1 the ratio between the neointimal area to
the area of the media (N/M) was significantly reduced following
treatment with clodronate-encapsulated in liposomes. The N/M ratio
in clodronate treated rats was 0.28.+-.0.23 as compared to
1.42.+-.0.26 in the control group (mean.+-.SD, p<0.01).
Similarly as seen in FIG. 2, significant inhibition of % stenosis
was achieved in the treated group: 9.8.+-.7.76 vs. 41.53.+-.7.9,
treated and control groups, respectively (mean.+-.SD, p<0.01).
There were no apparent systemic side effects nor any effects on
somatic growth as illustrated in FIG. 3.
[0073] Thus, the results of the experiments indicate that treatment
of rats with clodronate-containing liposomes significantly reduces
restenosis observed as neointimal formation following
balloon-injury of the carotid artery.
Example 2
[0074] The antirestenotic effects of liposomal clodronate
injections were studied in the balloon-injured rat and
hypercholesterolemic rabbit carotid arterial models. The rats were
treated by clodronate-containing liposomes, empty liposomes
(control), and clodronate in solution (additional control). The
dose of clodronate injected was 1.5 and 15 mg/kg administered one
day before procedure (-1) and/or on day 6 (+6) post injury. The
rabbits (following 30 days of atherosclerotic diet) were treated
one day prior to balloon angioplasty by liposomal clodronate (10
mg/kg). The lumen, neointimal, medial and vessel areas and volumes
were measured in the treated and control animal groups by digital
planimetry of histological sections, at 14 and 30 days post injury
in the rat and rabbit models, respectively.
[0075] The results of the antirestenotic effects of liposomal
clodronate are shown in FIG. 4. As illustrated, no significant
differences were found between treatments with empty liposomes, and
free clodronate in solution, which both exhibited marked neointimal
formation. The extent of mean neointimal formation, mean neointimal
to media ratio (N/M), and % stenosis following treatment with
clodronate-laden liposomes was significantly reduced. However, the
medial area was not affected by the various treatments indicating
no deleterious effects on quiescent cells. Moreover, there were
neither apparent systemic side effects nor any effects on bone and
somatic growth. Significantly, more potent treatments were
evaluated, specifically, 1.times.15 mg/kg (-1) and 2.times.15 mg/kg
(-1, and +6) injections, with no significant difference between
them. Similar findings of no adverse effects were also observed in
the rabbits' study. Liposomal clodronate was significantly
effective in reducing neointimal formation and % stenosis.
[0076] Furthermore, injection of silica particles also reduces
intimal formation (FIG. 4). This observation can be attributed to
the known inhibiting effect of silica on macrophages.
[0077] The results of the experiment indicated that treatment by
clodronate-containing liposomes significantly reduces neointimal
formation following balloon-injury both in rat and rabbit models.
There were neither apparent systemic and local side effects nor any
effects on somatic growth. It should be noted that although BPs are
known as affecting bone, no effects on the bone or on calcium and
phosphorus levels in bone and blood were observed following
treatment with liposomal preparation of clodronate.
Example 3
Effect of Liposomal Clodronate on IL-1.beta. Production and
Transcription and MMP-2 Activity
[0078] The effects of liposomal clodronate on interleukin 1-.beta.
(IL-1.beta.) production and transcription and matrix
metalloproteinase-2 (MMP-2) activity were studied in the
balloon-injured rat and the hypercholesterolemic rabbit carotid
arterial models. A group of male Sabra Rats was prepared according
to the rat carotid catheter injury model described supra, in
Example 1. The hypercholesterolemic rabbit model consisted of New
Zealand White rabbits weighing 2.5-3.5 kg. The rabbits were fed an
atherogenic diet of 2% cholesterol and 6% peanut oil starting 30
days before angioplasty and hypercholesterolemia was established
(plasma cholesterol>1,200 mg/dL). The rabbits were then
anesthesized by xylazine (7 mg/kg) and ketamine (40 mg/kg). Heparin
(200 units/kg), atropine (0.05 mg) and norfloxacin nicotinate (70
mg) were also administered to the rabbits. Thereafter, balloon
injury was performed on the left common carotid artery with a 3 mm
angioplasty balloon catheter (Cordis, Miami, Fla., USA, 2.times.1
min inflation at 8 atm). In both the rabbit and rat models,
liposomal clodronate (LC) was injected IV to the "treated group"
both one day prior to the arterial injury (-1d) and six days after
(+6) at a dosage of 15 mg/kg. The control animals were treated with
empty liposomes, i.e., liposomes with no bisphosphonates
encapsulated therein.
IL-.beta. Production and Transcription
[0079] Arteries and livers were homogenized in collagenase buffer
(5 mM CaCl.sub.2, 50 mM Tris, 0.02% Brij 35, 0.2 M NaCl, pH 7.6).
IL-1.beta. was measured using commercial ELISA kits (R&D
Systems, Minneapolis, Minn., USA). For RT-PCR analysis, RNA from
the carotid arteries was extracted using a RNA isolation kit. (Life
Technologies Inc., USA). Quality, size and quantity of RNA were
examined by conventional 1.0% agarose gel electrophoresis (Sigma)
and spectrophotometry. Total RNA (2 .mu.g) was used for the
synthesis of first strand cDNA using Superscript reverse
transcriptase and a mixture (1:1) of oligo (dT) and random
hexanucleotides in 20 ml reaction volume. First strand cDNA was
amplified by PCR. To ensure the quality of the RNA preparation and
to normalize the RT-PCR protocol, .beta.-actin RT-PCR products were
also produced for all samples. cDNA (2 .mu.l) was added to a 50 ml
reaction mixture containing 5 .mu.l 10*PCR reaction buffer, 2.0 mM
MgCl.sub.2, 20 MM of each dATP, dCTP, dGTP, and dTTP, 200 nM of
each oligonucleotide primer, and 1.0 unit Taq DNA polymerase.
Oligonucleotide primers for rabbit IL-1.beta., and .beta.-actin
were synthesized based on the following nucleotide sequences:
IL-1.beta. sense primer 5'-TAC AAC AAGAGC TTC CGG CA (SEQ. ID. NO.
1); IL-1beta antisense primer 5'-GGC CAC AGG TAT CTT GTC GT (SEQ.
ID. NO. 2); .beta.-actin sense primer 5'-ACG TTC AAC ACG CCG GCC AT
(SEQ. ID. NO. 3); .beta.-actin antisense primer 5'-GGA TGT CCA CGT
CGC ACT TC (SEQ. ID. NO. 4). Amplification was performed using a
DNA thermal cycler for 37 cycles, where a cycle profile consisted
of 1 minute at 94.degree. C. for denaturation, 1 minute at
55.degree. C. annealing, and 1 minute at 72.degree. C. for
extension. The size of amplified fragments was 354 and 493 bp for
IL-1.beta. and .beta.-actin, respectively. Electrophoresis of 10
.mu.l of the reaction mixture on a 1.5% agarose gel containing
ethidium bromide was performed to evaluate amplification and size
of generated fragments. PCR marker (Promega, USA) was used as a
standard size marker. The bands' intensity was quantified by
densitometry, and values of the bands were normalized to
.beta.-actin mRNA expression.
MMP-2 Activity
[0080] The supernatant of arteries homogenate in collagenase buffer
(see above) was analyzed for collagenase activity. Samples of
arteries were separated on gelatin-impregnated (1 mg/ml: Difco,
Detroit, Mich., USA) SDS 8% polyacrylamide gels under non-reducing
conditions, followed by 30 minutes of shaking in 2.5% Triton X-100
(BDH, Poole, UK). The gels were incubated for 16 hours at
37.degree. C. in a collagenase buffer, and stained with 0.5%
Coomassie G-250 (BioRad, Richmond, Calif.) in methanol/acetic
acid/H.sub.2O (30:10:60). Band intensity was determined by
cornputerized densitometry (Molecular Dynamics type 300A).
[0081] As illustrated in FIG. 5A, analysis of IL-1.beta. levels in
rat arterial tissue following balloon injury (control animals)
revealed a bell shape pattern peaking at 6 days following injury
(37.3.+-.9.6 pg/mg protein) and returning to basal levels after 30
days. However, a significant decrease of IL-1.beta. levels was
observed on days 3 and 6 post-injury, following LC-treatment. As
illustrated in FIG. 5B, a similar response was observed in the
rabbit artery, with a significant decrease of IL-1.beta. levels on
days 2, 4 and 6 post-injury, following LC-treatment.
[0082] The reduction in arterial IL-1.beta. levels following
LC-treatment was associated with a marked decrease in IL-1.beta.
transcription. IL-1.beta. mRNA transcription in rabbits' arteries
was analyzed following LC-treatment, which was administered the day
before balloon injury (-1). The gel electrophoresis of the
resultant reaction mixture following RT-PCR analysis is illustrated
in FIG. 6. The RT-PCR analysis illustrates that in control animals
(no treatment with LC), IL-1.beta. MRNA transcription was stronger
three days after the injury (+3) than one day after the injury
(+1). However, IL-1.beta. transcription on both day one (+1) and
day three (+3) after the injury, was significantly reduced by LC
treatment. In FIG. 6, Lane 1 represents PCR markers (50, 150, 300,
500, 750, 1000 bp); lanes 2 and 3 represent LC-treated and
untreated (control), on day +1, respectively; and lanes 4 and 5
represent LC-treated and untreated (control), on day +3,
respectively. Note the strong signal (at 354 bp) of IL-1.beta. mRNA
expression in untreated (control) animals (lanes 2 and 4) that was
suppressed by LC treatment (lanes 3 and 5). Expression of
.beta.-actin mRNA expression (493 bp) was used as loading control
in the same samples (lower panel). IL-1.beta. mRNA levels
(densitometry analysis relative to .beta.-actin mRNA) were found to
be 0.45.+-.0.24 and 0.37.+-.0.44 on day +1, 0.59.+-.0.2 and
0.12.+-.0.1 on day +3, LC-treated and untreated animals,
respectively (3 independent RT-PCR reactions).
[0083] Additionally, IL-1.beta. levels in the liver were also
examined. A significant reduction was noted after a single
injection of LC on day -1 inclining to basal levels at 30 days
(data not shown).
[0084] As illustrated in FIG. 5c, MMP-2 activity in rats' arterial
tissue increased following injury, exhibiting a bell shape pattern
peaking at 14 days (252.+-.12 and 402.+-.44, at 6 and 14 days,
respectively), and returning to basal levels at 30 days. However,
treatment with LC resulted in a significant reduction of MMP-2
activity at 6 and 14 days (152.+-.23 and 284.+-.17, respectively).
Similarly, in the rabbit's artery, the surge of MMP-2 activity was
less than that of the rat's artery, but the effect of LC-treatment
was more pronounced (See, FIG. 5d). As illustrated in FIG. 5d,
MMP-2 activity at 6 days was 248.+-.42 and 52.+-.5, in control and
LC-treated rabbits, respectively, returning to the baseline
approximately 14 days after injury.
Example 4
Effect of Liposomal Clodronate on PDGF-BB, PDGF.beta.R and
PDGF.beta.R Tyrosine Phosphorylation
[0085] The effect of liposomal clodronate on PDGF-BB,
platelet-derived growth factor .beta. receptor (PDGF.beta.R) and
PDGF.beta.R tyrosine phosphorylation was studied in the
balloon-injured rat arterial model. A group of male Sabra Rats was
prepared according to the rat carotid catheter injury model
described supra, in Example 1. Carotid arteries were rapidly
retrieved before injury and at day 14, rinsed in cold PBS and
immediately deep-frozen (-70.degree. C.) until further processing.
Frozen segments were mechanically minced on dry ice. There was a
total of 12 animals in each group, with four arteries being pooled
for each run. Liposomal clodronate (LC) was injected IV to the
"treated group" one day prior to the arterial injury (-1d) and six
days later (+6) at a dosage of 15 mg/kg. Proteins were extracted
using lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1% Triton X-100,
10 mM EDTA, 1 mM PMSF, 100 .mu.M sodium orthovanadate and 1%
aprotinin) and pooled for 4 animals in each group. Protein content
was determined using a modified Lowry protocol, and samples of 100
.mu.g were subjected to SDS-PAGE (7.5% or 12%) and blotted onto a
nitrocellulose membrane (Hybond C extra, Amersham) of PVDF membrane
(Roth) for the analysis of PDGF.beta.R or PDGF-B chain,
respectively. PDGF.beta.R protein was detected using a polyclonal
antibody (SC-431, Santa Cruz, USA) and a polyclonal alkaline
phosphatase-conjugated goat anti rabbit antiserum (Tropix, USA),
and tyrosine phosphorylated proteins were detected using a mixture
of monoclonal antibodies PY20 (Transduction Laboratories, USA) and
4G10 (UBI, USA) followed by the application of a
chemoluminescence-based detection system including a polyclonal
alkaline phosphatase-conjugated anti-mouse antiserum (CDP Star,
TROPIX). PDGF-BB protein was detected using the monoclonal antibody
PGF007 (Mochida), a horseradish-conjugated rabbit anti-mouse
antiserum (DAKO) and the detection system Super Signal Ultra
(Pierce, Germany). Quantification of the data was made by means of
LAS-1000 Imager (Fuji, Japan).
[0086] As illustrated in FIG. 7a, the activation of PDGF.beta.R
(i.e., tyrosine phosphorylation) markedly increased to 135% of the
baseline levels in the balloon-injured artery of untreated rats,
while it was barely detectable in LC treated rats, i.e. below
baseline activity. Note the band representing activated PDGF.beta.R
at 190 kDa. Additionally, as depicted in FIG. 7b, vascular injury
resulted in the upregulation of PDGF.beta.R protein both in the
untreated group (121%) and the LC-treated group (233%). Injury
resulted in a strong accumulation of PDGF-B protein within the
vessel wall on days 1 and 3 after injury (46 kDa), reaching 333%
and 219% of the baseline level, respectively (FIG. 7c). Further
illustrated in FIG. 9c is that in LC-treated rats, this
accumulation of PDGF-B was strongly reduced (181% and 168%, on days
1 and 3, respectively), in correlation with the reduced activation
of PDGF.beta.R at these time points.
[0087] The results of the experiments described supra, clearly
indicate that treatment of rats and rabbits with liposomal
clodronate significantly reduces the transcription and production
of interleukin 1-.beta., the activity of matrix
metalloproteinase-2, the activation of platelet-derived growth
factor .beta. receptor (PDGF.beta.R), and the levels of PDGF-B
protein.
Sequence CWU 1
1
4 1 20 DNA Rabbit IL-1beta primer 1 tacaacaaga gcttccggca 20 2 20
DNA Rabbit IL-1beta primer 2 ggccacaggt atcttgtcgt 20 3 20 DNA
Rabbit beta-actin primer 3 acgttcaaca cgccggccat 20 4 20 DNA Rabbit
beta-actin primer 4 ggatgtccac gtcgcacttc 20
* * * * *