U.S. patent application number 11/644411 was filed with the patent office on 2007-05-31 for method for treating atherosclerosis or restenosis using microtubule stabilizing agent.
This patent application is currently assigned to The Government of the U.S.A. as represented by the Secretary of the Dept. of Health & Human Services. Invention is credited to James L. Kinsella, Steven J. Sollott.
Application Number | 20070123583 11/644411 |
Document ID | / |
Family ID | 22272458 |
Filed Date | 2007-05-31 |
United States Patent
Application |
20070123583 |
Kind Code |
A1 |
Kinsella; James L. ; et
al. |
May 31, 2007 |
Method for treating atherosclerosis or restenosis using microtubule
stabilizing agent
Abstract
The present invention is a method of preventing or reducing
atherosclerosis or restenosis, and a pharmaceutical preparation
used therefor. In particular, it is a method of preventing or
reducing atherosclerosis or resenosis after arterial injury by
treatment with a low dose of a microtubule stabilizing agent such
as taxol or a water soluble taxol derivative. The low dose used in
the present invention prevents artery blockage while minimizing any
negative side effects associated with the drug.
Inventors: |
Kinsella; James L.;
(Baltimore, MD) ; Sollott; Steven J.; (Baltimore,
MD) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 S.W. SALMON STREET
SUITE #1600
PORTLAND
OR
97204-2988
US
|
Assignee: |
The Government of the U.S.A. as
represented by the Secretary of the Dept. of Health & Human
Services
|
Family ID: |
22272458 |
Appl. No.: |
11/644411 |
Filed: |
December 21, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11304362 |
Dec 14, 2005 |
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11644411 |
Dec 21, 2006 |
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10272496 |
Oct 15, 2002 |
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11304362 |
Dec 14, 2005 |
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10121500 |
Apr 11, 2002 |
6500859 |
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10272496 |
Oct 15, 2002 |
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08821906 |
Mar 21, 1997 |
6429232 |
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10121500 |
Apr 11, 2002 |
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08633185 |
Apr 18, 1996 |
5616608 |
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08821906 |
Mar 21, 1997 |
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08099067 |
Jul 29, 1993 |
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08633185 |
Apr 18, 1996 |
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Current U.S.
Class: |
514/449 |
Current CPC
Class: |
A61L 31/16 20130101;
A61L 2300/602 20130101; A61P 9/12 20180101; A61P 9/00 20180101;
A61K 31/335 20130101; A61K 31/337 20130101; A61L 2300/416 20130101;
Y10S 514/824 20130101; A61K 9/0024 20130101; A61P 3/10 20180101;
A61P 9/10 20180101 |
Class at
Publication: |
514/449 |
International
Class: |
A61K 31/337 20060101
A61K031/337 |
Claims
1-13. (canceled)
14. A method of inhibiting or reducing restenosis or
atherosclerosis in a patient comprising: infusing a patient with a
pharmaceutical preparation comprising a therapeutically effective
amount of taxol or a taxol derivative, wherein the pharmaceutical
preparation is in-line filtered during infusion.
15. The method of claim 14, wherein the taxol or taxol derivative
is administered at a dose lower than a dose used to treat human
cancers.
16. A method of inhibiting or reducing restenosis or
atherosclerosis in a patient comprising: administering to the
patient about 0.5 to about 2 mg/kg of taxol or a taxol derivative
over about a 24 hour time period prior to vascular surgery;
administering to the patient about 0.25 to about 2 mg/kg of taxol
or a taxol derivative over about a 24 hour time period after the
vascular surgery; and then administering to the patient about 0.25
to about 2 mg/kg of taxol or a taxol derivative over about a 24
hour time period every 21 days for 1 to 6 cycles.
17. The method of claim 14, wherein each administration of taxol or
a taxol derivative is via continuous intravenous infusion.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method of treating
patients at risk of developing atherosclerosis or restenosis.
[0002] More particularly, the invention relates to treatment of
these patients with a low dose taxol solution to prevent or reduce
the development of atherosclerosis or restenosis.
BACKGROUND OF THE INVENTION
[0003] Vascular diseese is the leading cause of death and
disability in the developed world, particularly afflicting the
elderly. In the United States alone, despite recent encouraging
declines, cardiovascular disease is still responsible for almost
one million fatalities each year and more than one half of all
deaths; almost 5 million persons afflicted with cardiovascular
disease are hospitalized each year. The cost of this disease in
terms of human suffering and of material resources is almost
incalculable.
[0004] Atherosclerosis is the most common form of vascular disease
and leads to insufficient blood supply to critical body organs,
resulting in heart attack, stroke, and kidney failure.
Additionally, atherosclerosis causes major complications in those
suffering from hypertension and diabetes, as well as tobacco
smokers. Atherosclerosis is a form of chronic vascular injury in
which some of the normal vascular smooth muscle cells ("VSMC") in
the artery wall, which ordinarily control vascular tone regulating
blood flow, change their nature and develop "cancer-like" behavior.
These VSMC become abnormally proliferative, secreting substances
(growth factors, tissue-degradation enzymes and other proteins)
which enable them to invade and spread into the inner vessel
lining, blocking blood flow and making that vessel abnormally
susceptible to being completely blocked by local blood clotting,
resulting in the death of the tissue served by that artery.
[0005] Restenosis, the recurrence of stenosis or artery stricture
after corrective surgery, is an accelerated form of
atherosclerosis. Recent evidence has supported a unifying
hypothesis of vascular injury in which coronary artery restenosis
along with coronary vein graft and cardiac allograft
atherosclerosis can be considered to represent a much accelerated
form of the same pathogenic process that results in spontaneous
atherosclerosis (Ip, J. H., et al., (1990) J Am Coll Cardiol,
15:1667-1687; Muller, D. W. M., et al., (1992) J Am Coll Cardiol,
19:418-432). Restnosis is due to a complex series of
fibroproliferative responses to vascular injury involving potent
growth-regulatory molecules, including platelet-derived growth
factor (PDGF) and basic fibroblast growth factor (bFGF), also
common to the later sg in atherosclerotic lesions, resulting in
vascular smooth muscle cell prolifeation, migration and neointimal
accumulation.
[0006] Restenosis occurs after coronary artery bypass surgery
(CAB), endarterectomy, and heart transplantation, and particularly
after heart balloon angioplasty, atherectomy, laser ablation or
endovascular stenting (in each of which one-third of patients
redevelop artery-blockage (restenosis) by 6 months), and is
responsible for recurrence of symptoms (or death), often requiring
repeat revascularization surgery. Despite over a decade of research
and significant improvements in the primary success rate of the
various medical and surgical treatments of atherosclerotic disease,
including angioplasty, bypass grafting and endarterectomy,
secondary failure due to late restenosis continues to occur in
30-50% of patients (Ross, R. (1993) Nature, 362:801-809).
[0007] As a result, a need exists for a successful chemotherapeutic
therapy to reduce or prevent artery-blockage. The most effective
way to prevent this disease is at the cellular level, as opposed to
repeated revascularization surgery which can carry a significant
risk of complications or death, consumes time and money, and is
inconvenient to the patient.
[0008] Microtubules, cellular organeles present in all eukaryotic
cells, are required for healthy, normal cellular activities. They
are an essential component of the mitotic spindle needed for cell
division, and are required for maintaining cell shape and other
cellular activities such as motility, anchorage, transport between
cellular organelles, extracellular secretary processes (Dustin, P.
(1980) Sci. Am., 243: 66-76), as well as modulating the
interactions of growth factors with cell surface receptors, and
intracellular signal transduction. Furthermore, microtubules play a
critical regulatory role in cell replication as both the c-mos
oncogene and CDC-2-kinase, which regulate entry into mitosis, bind
to and phosphorylate tubulin (Verde, F. et al. (1990) Nature,
343:233-238), and both the product of the tumor suppressor gene,
p53, and the T-antigen of SV-40 bind tubulin in a ternary complex
(Maxwell, S. A. et al. (1991) Cell Growth Differen., 2:115-127).
Microtubules are not static, but are in dynamic equilibrium with
their soluble protein subunits, the .alpha.- and .beta.-tubulin
heterodimers. Assembly under physiologic conditions equires
guanosine trphosphate (GTP) and certain microtubule associated and
organizing proteins as cofactors; on the other hand, high calcium
and cold temperature cause depolymerization.
[0009] Interference with this normal equilibrium between the
microtubule and its subunits would therefore be expected to disrupt
cell division and motility, as well as other activities dependent
on microtubules. This strategy has been used with
significant-success in the treatment of certain malignancies.
Indeed, antimicrotubule agents such as colchicine and the vinca
alkaloids are among the most important anticancer drugs. These
antimicrotubule agents, which promote microtubule disassembly, play
principal roles in the chemotherapy of most curable neoplasms,
including acute lymphocytic leukemia, Hodgkin's and non-Hodgkin's
Lymphomas, and genn cell tumors, as well as in the palliative
treatment of many other cancers.
[0010] The newest and most promising antimicrotubule agent under
research is taxol. Taxol is an antimicrotubule agent isolated from
the stem bark of Taxus brevifolia, the western (Pacific) yew tree.
Unlike other antimicrotubules such as colchicine and the vinca
alkaloids which promote microtubule disassembly, taxol acts by
promoting the formation of unusually stable microtubules,
inhibiting the normal dynamic reorganization of the microtubule
network required for mitosis and cell proliferation (Schiff, P. B.,
et al. (1979) Nature 277: 665; Schiff, P. B., et al. (1981)
Biochemistry 20: 3247).
[0011] In the presence of taxol, the concentration of tubulin
required for polymerization is significantly lowered; microtubule
assembly occurs without GTP and at low temperatures, and the
microtubules formed are more stable to depolymerization by
dilution, calcium, cold, and inhibitory drugs. Taxol will
reversibly bind to polymerized tubulin, and other tubulin-binding
drugs will still bind to tubulin even in the presence of taxol.
[0012] Taxol has one of the broadest spectrum of an antineoplastic
activity, renewing serious interest in chemotherapeutic strategies
directed against microtubules (Rowinsky, E. K., et al. (1990) Jrnl.
of the Nat'l. Cancer Inst., 82:1247-1259). In recent studies, taxol
has shown significant activity in advanced and refractory ovarian
cancer (Einzig, A. I., et al. (1992) J. Clin. Oncol., 10:1748),
malignant melanoma (Einzig, A. I. (1991) Invest. New Drugs,
9:59-64), as well as in cancers of the breast (Holmes, F. A., et
al. (1991) JNCI, 83:1797-1805), head and neck, and lung.
[0013] Taxol has been studied for its effect in combating tumor
growth in several clinical trials using a variety of administration
schedules. Severe alligic reactions have been observed following
administration of taxol. However, it is has been demonstrated that
the incidence and severity of allergic reactions is affected by the
dosage and rate of taxol infusion (Weiss, R. B., et al. (1990) J.
Clin. Oncol. 8: 1263).
[0014] Cardiac arrhythmias are associated with taxol
administration, and like allergic reactions, their incidence is
affected by the dosage and rate of taxol administration. Sinus
bradycardia and Mobitz II arrhythmia will develop in approximately
40% and 5% of patients, respectively, beginning 4-6 hours after the
start of a taxol infusion, and continuing for 48 hours after its
completion. In most patients, the abnormal rhythm is transient,
asymptomatic, hemodynamically stable, and does not require cardiac
medications or electrical pacing. Additionally, it has been
observed that the incidence of severe cardiac events is low in
patients receiving taxol alone. Thus, infusion times up to 24 hours
have been used in treatment with taxol to decrease the incidence of
toxicity and allergic reaction to the drug.
[0015] During angioplasty, intraarterial balloon catheter inflation
results in deendothelialization, disruption of the internal elastic
lamina, and injury to medial smooth muscle cells. While restenosis
likely results from the interdependent actions of the ensuing
inflammation, thrombosis, and smooth muscle cell accumulation
(Ferrell, M., et al. (1992) Circ., 85:1630-1631), the final common
pathway evolves as a result of medial VSMC dedifferentiation from a
contractile to a secretory phenotype. This involves, principally,
VSMC secretion of matrix metalloproteinases degrading the
surrounding basement membrane, proliferation and chemotactic
migration into the intima, and secretion of a large extracellular
matrix, forming the neointimal fibropoliferative lesion. Much of
the VSMC phenotypic dedifferentiation after arterial injury mimics
that of neoplastic cells (i.e., abnormal proliferation,
growth-regulatory molecule and protease secretion, migration and
basement invasion).
[0016] Although others have investigated the use of the
antimicrotubule agent colchicine in preventing restenosis, opposite
conclusions have been reported (See Currier, et al., "Colchicine
Inhibits Restenosis After Iliac Angioplasty In The Atherosclerotic
Rabbit" (1989) Circ., 80:II-66; O'Keefe, et al., "Ineffectiveness
Of Colchicine For The Prevention Of Restenosis After Coronary
Angioplasty" (1992) J. Am. Coll. Cardiol., 19:1597-1600). The art
fails to suggest the use of a microtubule stabilizing agent such as
taxol in preventing or reducing this disease. Thus, the method of
the present invention is to prevent or reduce the development of
atherosclerosis or restenosis using a microtubule stabilizing agent
such as taxol or a water soluble taxol derivative. This microtubule
stabilizing mechanism of atherosclerosis or restenosis prevention
is supported by the analogous results in experiments on cellular
proliferation and migration using taxol and .sup.3H.sub.2O
(deuterium oxide), which exert comparable microtubule effects via
different underlying mechanisms.
[0017] Accordingly, an object of this invention is to provide a
method to reduce or prevent the development of atherosclerosis or
restenosis using treatment with a drug which promotes highly
stabilized tubule formation.
[0018] An additional object of this invention is to provide a
method of preventing or reducing atherosclerosis or restenosis
using a pharmaceutical preparation containing a low dosage of taxol
or water soluble taxol derivative.
[0019] All references cited are herein incorporated by
reference.
SUMMARY OF THE INVENTION
[0020] In accordance with these and other objects of the present
invention, a method of preventing or reducing atherosclerosis or
restenosis is provided, which comprises treatment with a
therapeutically effective amount of a microtubule stabilizing agent
such as taxol or a water soluble taxol derivative. A
therapeutically effective amount of agent is an amount sufficient
to prevent or reduce the development of atherosclerosis or
restenosis.
[0021] This method provides an effective way of preventing or
reducing the development of atherosclerosis or restenosis in those
patients susceptible to such disease. Additionally, because of the
low dose of chemotherapeutic agent used, the chance of a patient
developing adverse reactions is potentially reduced.
BRIEF DESCRIPTION OF FIGURES
[0022] FIG. 1 depicts the taxol induced impairment of the ability
of VSMC to invade filters coated with basement membrane proteins,
and taxol inhibition of cultured VSMC [.sup.3H]-thymidine
incorporation.
[0023] FIG. 2 shows taxol inhibition of smooth muscle cell
neointimal accumulation after balloon catheter injury of the rat
carotid artery.
[0024] FIG. 3 depicts deuterium oxide dose-dependent inhibition of
VSMC chemoinvasion, and deuterium oxide inhibition of cultured VSMC
bromodeoxyuridine (BrDU) incorporation.
[0025] FIG. 4 shows concentrations of taxol caused dose-dependent
microtubule bundling in VSMC's cultured on plastic.
[0026] FIG. 5 shows deuterium oxide induced microtubule bundling in
cultured VSCM's.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The practice of an embodiment in the present invention may
be accomplished via several alternative drug delivery routes, such
as intraperitoneal or subcutaneous injection, continuous
intravenous infusion, oral ingestion or local (direct) delivery, or
a combination of two or more. When formulating a solution for
injection or continuous infusion, one must first prepare a taxol
solution. Taxol is supplied through CTEP, DCT, NCI (IND#22850) as a
concentrated solution, 6 mg/ml, in 5 ml vials (30 mg/vial) in a
polyoxyethylated castor oil (Cremophor EL.RTM.) 50% and dehydrated
alcohol, USP (50%) vehicle. The intact vials should be stored under
refrigeration and diluted prior to use. When diluted in either 5%
Dextrose Injection or 0.9% Sodium Chloride, taxol concentrations of
0.3-1.2 mg/ml are physically and chemically stable for at least 12
hours at room temperature. (NCI Investigation Drugs; Pharmaceutical
Data (1990)). It has also been demonstrated that taxol
concentrations of 0.6 mg/ml diluted in either D5W or NS and 1.2
mg/ml diluted in NS prepared in polyolefin containers are stable
for at least 25 hours at ambient temperatures (20-23.degree. C.).
(Waugh, et al. (1990) Am. J. Hosp. Pharm. 48, 1520). Although these
concentrations have exhibited stability for the above periods of
time, they are not meant to limit the practice of the present
invention wherein any concentration of taxol may be utilized.
[0028] All solutions of taxol exhibit a slight haziness directly
proportional to the concentrations of drug and time elapsed after
preparation. Formulation of a small number of fibers in the
solution (within acceptable limits established by the USP
Particulate Matter Test for LVP's) has been observed after
preparation of taxol infusion solutions. While particulate
formation does not indicate loss of drug potency, solutions
exhibiting excessive particulate matter formation should not be
used. Therefore, when administering via continuous infusion,
in-line filtration may be necessary and can be accomplished by
incorporating a hydrophilic, microporous filter with a pore size no
greater than 0.22 microns (WEX-HP In Line Filter Set-SL, 15'',
Abbott model # 4525 or equivalent) into the fluid pathway distal to
an infusion pump.
[0029] Taxol must be prepared in non-plasticized solution
containers (e.g., glass, polyolefin, or polypropylene) due to
leaching of diethylhexylphthlalate (DEHP) plasticizer from
polyvinyl chloride (PVC) bags and intravenous tubing. Taxol must
not be administered through PVC intravenous or injection sets.
Therefore, polyolefin- or polyethylene-line sets, such as IV
nitroglycerin sets (or equivalent) should be used to connect the
bottle or bag (containing the taxol solution for a continuous
infusion) to the IV pump, a 0.22 micron filter is then attached to
the IV set, and then may be directly attached to the patient's
central access device. If necessary, a polyolefin-line extension
set (Polyfin.TM. Extension Set, MiniMed Technologies, Model #126)
can be used to provide additional distance between the IV pump and
the patient's central access device.
[0030] One category of taxol use would encompass the prevention of
recurrent stenosis (restenosis) post therapeutic coronary- or
peripheral-attery angioplasty or atherectomy, after coronary bypass
graft or stent surgery, or after peripheral vascular surgery (e.g.,
carotid or other peripheral vessel endarterectomy, vascular bypass,
stent or prosthetic graft procedure). A human dosing schedule can
consist of (but not be limited to) 24-hour continuous IV
pretreatment with up to about 0.5-2 mg/kg (20-80 mg/m.sup.2) prior
to the vascular procedure, about 0.25-2 mg/kg (10-80 mg/m.sup.2)
continuous IV infusion over the 24 hours post-procedure, then about
0.25-2 mg/kg (10-80 mg/m.sup.2) continuous IV infusion over 24
hours every 21 days for 1 to 6 cycles. Such a dosage is
significantly lower than that used to treat human cancers
(approximately 4-6 mg/kg).
[0031] Another category of taxol use would encompass the primary
prevention, or the attenuation, of vascular disease
(atherosclerosis) development. Certain of these applications
(examples of which include the prevention of cardiac allograft
(transplant) atherosclerosis, the multi-organ system failure
resulting from the vascular complications of diabetes mellitus or
accelerated, medically-refractory atherosclerosis in patients who
are poor surgical candidates) may require the later treatment
cycles to be continuous low-dose (1-5 mg/m.sup.2/day) IV infusions
over 5-7 days. Each of the taxol treatments will generally require
retreatment with dexamethasone 20 mg orally 14 and 7 hours prior to
taxol, diphenhydramine 50 mg IV and cimetidine 300 mg IV 30 min
prior to taxol, to minimize potential episodes of allergic
reaction. Additional applications that may not be associated with a
surgical procedure include treatment of vascular fibromuscular
dysplasia, polyarteritis nodosa, and Takayasu's arteritis. Each of
the aforementioned applications may also be amenable to selective,
localized application of sustained-release preparations of taxol
(or other microtubule-stabilizing agent) which would enable high
dosage local drug delivery with little systemic toxicity.
[0032] Additionally, water soluble derivatives of taxol can also be
used in the present invention. The water soluble derivatives of
taxol, as described in U.S. Pat. No. 5,157,049 to Haugwitz, et al.
(incorporated herein by reference) include, but are not limited to,
2'-succinyl-taxol; 2'-succinyl-taxol triethanolamine;
2'-glutaryl-taxol; 2'-glutaryl-taxol triethanolamine salt;
2'-O-ester with N-(dimethylaminoethyl) glutamide; 2'-O-ester with
N-(dimethylaminoethyl) glutamide hydrochloride salt. These water
soluble taxol derivatives can be administered in a dosage schedule
analogous to that given above for taxol with the appropriate
modifications pending clarification of the pharmacokinetics of
these agents.
[0033] A pharmaceutical composition comprising an effective amount
of water soluble derivative of taxol as an active ingredient is
easily prepared by standard procedures well known in the art, with
pharmaceutically acceptable non-toxic sterile carriers, if
necessary. Such preparations could be administered orally or in
injectable form, or directly to an affected area, to a patient at
risk of developing or suffering from atherosclerosis to prevent or
reduce the development of the disease.
[0034] The following examples illustrate the effectiveness of taxol
(or other microtubule-stabilizing agents including, but not limited
to, water soluble derivatives of taxol) in inhibiting the
proliferation and migration of vascular smooth muscle cells, and
should not be used to limit the scope of the present invention.
EXAMPLE 1
[0035] The in vitro ability of cultured VSMCs, pretreated with
different taxol concentrations, to invade filters coated with
reconstituted basement membrane proteins was tested to evaluate how
taxol-induced microtubule bundling would impair cell processes
necessary for in vivo neointimal formation.
[0036] Vascular Smooth Muscle Cells (VSMCs) were isolated by
collagenase/elasase enzymatic digestion of the medial layers of the
rat aorta obtained from 6 month old Wistar rats. The cells were
maintained in culture with 10% fetal calf serum, high glucose DMEM,
and amino acid supplement. Cell cultures were maintabed at
37.degree. C. in 5% CO.sub.2.
[0037] After 18 -hour taxol pre-treatment in culture, cells were
fixed in 3.7% formalin, permeabilized with 1% Triton X-100, and
polymerized tubulin was labelled with mouse anti-.beta.-tubulin
antibody (SMI 62 monoclonal antibody to polymerized .beta.-tubulin,
Paragon Biotec, Inc., Baltimore, Md.). Secondary labelling was
achieved with silver-enhanced, 1 nm gold-conjugated rabbit
anti-mouse antibody (Goldmark Biologicals, Phillipsburg, N.J.).
Representative light photomicrographs from (A) control, (B) 0.1 nM
taxol, (C) 1 nM taxol, and (D) 10 nM taxol treated VSMCs are shown
in FIG. 4.
[0038] Chemonivasion (Boyden chamber) assays were performed using
modified Boyden chamber (Albini, et al. (1987) Cancer Res.,
47:3239-3245), comprising an upper chamber separated from a lower
chamber by a porous PVPF filter. PVPF filters (8 .mu.m pore
diameter, Nucleopore Filters, Pleasonton, Calif.) were coated and
air dried consecutively with solutions containing 100 .mu.g/ml type
I collagen, 5 .mu.g/ml fibronectin, and 5 .mu.g reconstituted
basement membrane (produced from the Englebreth-Holm-Swarm tumor
(Kleinman, et al. (1986) Biochemistry, 25:312-318), producing a
continuous 10 .mu.m thick coating of matrix material. Boyden
chambers were assembled by adding 10 ng/ml PDGF BB in DMEM to the
lower (chemoattractant) chamber. Cells (approximately 200,000)
suspended in DMEM containg 0.1% BSA were then added to the upper
chamber. Some of the cells used in this assays were pretreated 18
hours with taxol (concentration 30 pM to 100 nM) in culture. In the
taxol-treated groups, taxol was added to the upper and lower
chambers at the same concentration as that used for pretreatment.
The chambers were then incubated for 4 hours at 37.degree. C. in a
5% CO.sub.2 atmosphere. At the end of the incubation period, the
cells were fixed and stained with hematoxylin and eosin. The cells
on the upper surface (non-invaders) were mechanically removed and
the cells on the underside of the filter (invaders) were counted
under 400.times. magnification (four random fields were counted per
filter and all experiments were run in triplicate, and each
triplicate assay was repeated at least three times on separate
occasions using different VSMC preparations). Chemotaxis was
assayed in analogous fashion in the Boyden chambers described
above, except that the reconstituted basement membrane was omitted.
This chemoinvasion assay is accepted by those skilled in the art as
exhibiting high correlation between invasiveness in vitro and
cellular behavior as it occurs in vivo (Iwamoto, Y., et al. (1992)
Advances In Experimental Medicine & Biology, 324:141-9).
[0039] Using the PDGF-BB as an attractant, taxol inhibited VSMC
invasion with half-maximal inhibitory concentration of 0.5 nM.
Taxol caused essentially complete inhibition at 100 nM, and
significant inhibition was still resolvable at 30 pM (the lowest
dose used) (FIG. 1). A chemotaxis assay filter coated only with
fibronectin and collagen I, without basement membrane proteins
occluding the filter pores) with PDGF-BB as the attractant was
performed in analogous fashion, yielding the identical outcome.
These results demonstrate that taxol, at least at nanomolar drug
levels, inhibits VSMC invasion primarily via inhibition of
locomotion and/or shape changes, rather than by inhibiting cellular
secretion of collagenases and metalloproteinases, which are known
to be necessary for VSMC to penetrate basement membrane proteins in
this assay.
[0040] Gelatinase zymography was performed on the supernatants
removed after the 4 hour conclusion of the Boyden assays described
above. Gelatin-degrading proteinases secreted into the media by
VSMCs were analyzed by non-reducing sodium dodecyl
sulfate-polyacryramide gel electrophoresis in 10% polyacrylamide
gels containing 0.1% (w/v) gelatin. Following electrophoresis, the
gelatinases were renatured by incubating the gel for 30 min. at
23.degree. C. in 2.5% (v/v) Triton X-100 followed by 18 hour
incubation at 37.degree. C. in 0.2 M NaCl, 5 mM CaCl.sub.2 0.02%
Brij 35 (w/v), 50 mM Tris-HCl (pH 7.6). The gels were stained for
90 minutes with 0.5% Coomassie Brilliant Blue G-250 and destained
with 10% acetic acid, 40% methanol. Gelatinolytic activity was
indicated by a clear band against the background of blue-stained
gelatin.
[0041] These gelatinase zymography assays from the Boyden chamber
invasion experiments confirm that the level of VSMC collagenase
secetion did not vary significantly over the taxol range 30 pM to
100 nM, compared to control (FIG. 2, inset).
EXAMPLE 2
[0042] To confirm the fact that microtubule stabilization and
hyperpolymerization is the critical and sufficient factor involved
in taxol-inhibition of VSMC invasiveness, the chemoinvasion (Boyden
chamber) assay was run with deuterium oxide (.sup.2H.sub.2O, heavy
water). Deuterium oxide enhances microtubulel/tubulin
polymerization via a mechanism distinct from that of taxol. A
combination of the isotope and solvent effects of deuterium oxide
reversibly increases microtubule polymerization both by reducing
the critical concentration for polymerization for
.alpha..beta.-tubulin heterodimers via enhanced tubulin hydrophobic
interactions (Itoh, T. J., et al. (1984) Biochim. Biophys. Acta.,
800:21-27), and by converting a population of unpolymerizable
tubulin to the polymerizable form (Takahashi, T. C., et al. (1984)
Cell Struct. Funct., 9:45-52).
[0043] VSMC's were isolated by collagense/elastase enzymatic
digestion of the medial layers of the rat aorta obtained from 6
month old Wistar rats. The cells were maintained in culture with
10% fetal calf serum, high glucose DMEM, and amino acid supplement.
Cell cultures were maintained at 37.degree. C. in 5% CO.sub.2.
[0044] In deuterium oxide-treated cells, .sup.2H.sub.2O (v/v) was
substituted for water (H.sub.2O) in the preparation of the DMBM
from concentrated stock. After 18-hour deuterium oxide
pre-treatment in culture, cells were fixed in 3.7% formalin,
permeabilized with 1% Triton X-100, and polymerized tubulin was
labelled with mouse anti-.beta.-tubulin antibody (SMI 62 monoclonal
antibody to polymenze .beta.-tubulin, Paragon Biotec, Inc.,
Baltimore, Md.). Secondary labelling was achieved with
silver-enhanced, 1 nm gold-conjugated rabbit anti-mouse antibody
(Goldmark Biologicals, Phillipsburg, N.J.). Reresentative light
photomicrographs from (A) control, and (B) 75% deuterium oxide
treated VSMCs arm shown in FIG. 5.
[0045] Chemoinvasion assays were performed using a modified Boyden
chamber, consisting of an upper chamber separated from a lower
chamber by a porous PVPF filter. PVPF filters (8 .mu.m pore
diameter, Nucleoport Filters, Pleasonton, Calif.) were coated and
air dried consecutively with solutions containing 100 .mu.g/ml type
I collagen, 5 .mu.g/ml fibronectin, and 5 .mu.g reconstituted
basement membrane (produced from the Englebreth-Holm-Swarm tumor),
producing a continuous 10 .mu.m thick coating of matrix material.
Boyden chambers were assembled with PDGF-BB 10 ng/ml in DMEM in the
lower (chemoattractant) chamber, then cells (approximately 200,000)
suspended in DMEM containing 0.1% BSA were added to the upper
chamber. Some of the cells used in these assays were pretreated 18
hours with deuterium oxide (25%, 50%, or 75% v/v substitution for
H.sub.2O) in culture. In the deuterium oxide-treated groups,
.sup.2H.sub.2O substituted DMEM (v/v) was added to the upper and
lower chambers at the same concentration as that used for
pretreatment. The chambers were then incubated for 4 hours at
37.degree. C. in a humidified 5% CO.sub.2 atmosphere. At the
conclusion of the experiment, the filters were removed and the
cells were fixed and stained with hematoxylin and eosin. After the
cells on the upper surface of the filter (non-invaders) were
mechanically removed, the cells on the underside (invaders) were
counted under 400.times. magnification (four random fields were
counted per filter and all experiments were run in triplicate).
[0046] Pretreating cultured VSMCs for 18 hours with 25%, 50% or 75%
deuterium oxide caused dose-dependent microtubule
hyperpolymerization similar to that observed with taxol. This
treatment likewise inhibited PDGF-mediated. VSMC Boyden chamber
invasion in a dose-dependent fashion, achieving half-maximal
inhibition at 25% .sup.2H.sub.2O, and nearly complete inhibition at
75% .sup.2H.sub.2O
EXAMPLE 3
[0047] In addition to cell recruitment and migration, the various
growth regulatory molecules elaborated after arterial injury, such
as PDGF and bFGF, are also implicated in mitogenesis and cellular
proliferation. To measure the effect of taxol on VSMC DNA
synthesis, [.sup.3H]thymidine incorporation was measured. VSMCs
were plated at 4.5.times.10.sup.4 on 24-well plates. Following 5
hr. incubation in 10% FCS+DMEM, 0.5 mCi [.sup.3H]thymidine was
added and the incubation continued for an additional 16 hrs. Cells
were washed twice with phosphate-buffered saline, extracted with
10% TCA for 2 hrs. on ice, then centrifuged at 2,000 g for 10 mins.
Supernatants were decanted and pellets were solubilized in 0.5 ml
of 1 N NaOH. After neutralizing with 0.5 ml of 1 N HCl,
[.sup.3H]thymidine uptake was determined by a Beckman liquid
scintillation counter. VSMCs were treated with the various
concentrations of taxol for both the 18 hr. prior to the addition
of the thymidine and during thymidine incorporation. Each condition
of these experiments was performed in triplicate.
[0048] Taxol inhibited cultured VSMC [.sup.3H]thymidine
incorporation, an index of cell division, in a dose-dependent
fashion, with a half-maximal inhibitory concentration of 5 nM.
Taxol caused essentially complete inhibition at 100 nM, and
significant inhibition was resolvable at 1 nM (FIG. 1). That this
inhibitory profile differs somewhat from that of invasion and
chemotaxis, demonstrating one log-concentration-unit lower
sensitivity but with steeper concentration-dependence, likely
arises because of the considerably different roles played by
mircotubules between these processes. Taxol also inhibited
PDGF-BB-stimulated c-fos mRNA expression in this cultured VSMC
model, in a dose-dependent fashion, with a half-maximal inhibitory
concentration of 1 nM, with essentially complete inhibition above
20 nM. Thus, inhibition of immediate early gene induction is
another important mechanism by which taxol blocks growth. factor
stimulation in VSMCs, and may underlie, at least in part, the
thymidine incorporation results.
[0049] Thus, taxol significantly inhibits cultured VSMC in vitro
invasion and proliferation through interference with micratubule
function, disrupting locomotion and the ability to alter shape, as
well as growth-factor stimulated early gene expression and cell
proliferation, at concentrations one hundred- to one thousand-fold
lower than used to treat human cancer.
EXAMPLE 4
[0050] Incorporation of the thymidine analog, bromodeoxyuridine
(BrDU) was measured to determine the effect of deuterium oxide on
VSMC DNA synthesis. VSMCs were plated at 4.5.times.10.sup.4 on
24-well plates. Following 20 hr incubation in 10% FCS+DME at
various .sup.2H.sub.2O concentrations, 10 .mu.M BrDU was added and
the incubation continued for an additional 4 hr. Cells were washed
twice with phosphate-buffered saline (PBS) and fixed with 100%
methanol (-20.degree. C.) for 10 minutes. The cells were incubated
for 2 hr with 1N HCl to denature the DNA, and subsequently washed 4
times in PBS. Mouse monoclonal BrDU antibody (Boehringer Mannheim)
in 2% BSA-PBS was incubated with cells for 1 hr. After PBS wash,
goat anti-mouse antibody conjugated with alkaline phosphatase was
added. Cell nuclei containing BrDU substituted for thymidine
stained red with alkaline phosphatase substrate, while all other
nuclei stained blue. The fraction of BrDU-positive nuclei was
compared between control (defined as 100%) and that of the
deuterium oxide-pretreated groups.
[0051] The results indicated that deuterium oxide, similar to
taxol, inhibited cultured VSMC proliferation and DNA synthesis in a
dose-dependent fashion, consistent with the critical balance of
microtubule-tubulin dynamics in VSMC proliferation.
[0052] While taxol and deuterium oxide potentially have multiple
intracellular effects, the coincidence of their parallel effects on
microtubules (despite different mechanisms of action) and on VSMC
functionality at multiple levels, indicates that the common
microtubule stabilizing mechanism of action is responsible for the
observed functional changes. Thus, based on the results of
experiments with both taxol and deuterium oxide, it is evident that
micrtubules are involved in the control of the most critical and
sensitive intracellular mechanisms necessary for VSMCs to undergo
the multiple transformations involved in the development of
atherosclerosis and restenosis after arterial injury, making
microtubules particularly strategic targets to influence the
outcome.
EXAMPLE 5
[0053] Under a protocol approved by the National Institute on Aging
Aninal Care and use Committee, 6 month Wistar rats from the GRC
colony were anesthetized with 20 mg/kg body weight pentobarbital, 2
mg/kg body weight ketamine, and 4 mg/kg body weight xylazine
intraperitoneally. The left external carotid artery was cannulated
with 2-French Fogarty embolectomy catheter, inflated with saline
and passed three times up and down the common carotid artery to
produce a distending, deendothelializing injury. The animals were
treated with 2 mg/kg body weight taxol solution or the control
animals with vehicle alone (13.4 ml/kg body weight per day of
1:2:2:165 DMSO:Cremophor. EL:Dehydrated ethanol:phosphate buffered
saline) by intraperitoneal injection beginning 2 hours after
injury. The taxol solution or vehicle alone was administered once
daily, as an intraperitoneal injection, for the next 4 days. After
11 days the animals (8 taxol-treated and 10 vehicle-treated) were
anesthetized as above and the carotid artery was isolated and fixed
in 10% buffered formalin and embedded in paraffin. Cross sections
of the carotids were mounted on microscope slides and stained with
hematoxylin and eosin stain. The image of the carotid artery was
projected onto a digitizing board and the cross sectional areas of
the intima and the media were measured. The results are shown in
FIG. 2. As indicated in the prior art (Ferns, G. A. A. et al.
(1991) Science, 253:1129-1132) the rat carotid artery injury model
of restenosis can be useful, in the study of human restenosis, and
indicate potential therapeutic action in humans.
[0054] Quantitative analysis of injured carotid segments showed
that taxol treatment reduced the neointimal area by 70% compared to
vehicle treated animals (Table I) (*P<0.001; .sup..dagger.P=NS;
.sup..dagger-dbl.P<0.001). Several of the taxol-treated animals
showed virtually no discemable neointima (in the presence of
denuded endothelium, proving injury), while all vehicle treated
animals demonstrated at least modest neointimal thickening.
[0055] While the in vivo systemic taxol dose used in these
epperiMCeMs (2 mg/kg) is significantly lower than that ordinarily
used to treat human cancers (approximately 3-6 mg/kg), dramaicaly
lower systemic dosing with sustained or even improved efficacy
could be possible combining a pretreatment regimen with the optimal
treatment duration. Furthermore, since the goal of therapy is to
keep the "activated" VSMCs in check, or preferably to prevent
activation in the first place, until the stimulus for growth and
migration has resolved (rather than causing cytotoxicity resulting
in cell death), the goal of short-term therapy with limited
toxicity may be possible in humans. Ultimately, local
sustained-release delivery systems may offer the best solution to
prevent restenosis post-angioplasty, enabling high local
concentrations of drug delivery and essentialy eliminating problems
of systemic toxicity. Drug delivery systems that can be valuable
include drug-impregnated polymer-coated metallic stents,
biodegradable drug-eluting polymer stents, and genetically primed
endothelial celis to coat metallic stents or be delivered directly
as a local endothelial cell covering. (Muller, D. W. M. et al.
(1991) JACC 17:126b-131b). These systems allow safe use of a
chemotherapeutic agent without systemic side effects.
Alternatively, treatment may involve a period of pretreatment
(i.e., before vascular surgery) via continuous intravenous infusion
for a period of time, followed by a different therapy during
(local, direct delivery) or after (oral, injection) surgery.
[0056] The above examples teach taxol's (or other
microtubule-stabilizing agent including, but not limited to, water
soluble derivatives of taxol) potential beneficial uses to prevent
artery blockage and thereby reduce the possibility of, or prevent,
heart attacks, strokes, kidney failure and renal dialysis,
blindness, limb amputations, nerve loss, need for corrective
vascular surgery/angioplasty or organ transplantation, and
premature and permanent disability requiring chronic
hospitalization. The invention has been described in detail, but it
will be understood that the invention is capable of other different
embodiments. As is readily apparent to those skilled in the art,
variations and modifications can be affected within the spirit and
scope of the invention. Accordingly, the foregoing disclosure and
description are for illustrative purposes only, and do not in any
way limit the invention, which is defined only by the claims.
TABLE-US-00001 TABLE 1 Group Intima (mm.sup.2) Media (mm.sup.2) I/M
Vehicle 0.09 .+-. 0.01 0.14 .+-. 0.01 0.66 .+-. .08 Taxol 0.03 .+-.
0.01* 0.16 .+-. 0.02.sup..dagger. 0.18 .+-.
.04.sup..dagger-dbl.
* * * * *