U.S. patent application number 12/426739 was filed with the patent office on 2009-09-03 for treatment and prevention of abnormal cellular proliferation.
Invention is credited to Hari G. Garg, Charles A. Hales, Robert J. Linhardt, Lunyin Yu.
Application Number | 20090220606 12/426739 |
Document ID | / |
Family ID | 34994294 |
Filed Date | 2009-09-03 |
United States Patent
Application |
20090220606 |
Kind Code |
A1 |
Hales; Charles A. ; et
al. |
September 3, 2009 |
TREATMENT AND PREVENTION OF ABNORMAL CELLULAR PROLIFERATION
Abstract
This invention provides a method for inhibiting or preventing
the abnormal growth of cells, including transformed cells, by
administering an effective amount of O-acylated heparin derivative.
Abnormal growth of cells refers to cell growth independent of
normal regulatory mechanism (e.g. loss of contact inhibition). This
includes the abnormal growth of: (1) tumor cells (tumors); (2)
benign and malignant cells of other proliferative disease in which
aberrant cellular proliferation occurs; (3) aberrant smooth muscle
cell proliferation, such as might occur following treatment for
coronary atherosclerosis such as angioplasty or the insertion of a
stent into an occluded vessel.
Inventors: |
Hales; Charles A.; (Lincoln,
MA) ; Garg; Hari G.; (Belmont, MA) ; Yu;
Lunyin; (Allston, MA) ; Linhardt; Robert J.;
(Albany, NY) |
Correspondence
Address: |
DAVID S. RESNICK
NIXON PEABODY LLP, 100 SUMMER STREET
BOSTON
MA
02110-2131
US
|
Family ID: |
34994294 |
Appl. No.: |
12/426739 |
Filed: |
April 20, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11082213 |
Mar 16, 2005 |
7538096 |
|
|
12426739 |
|
|
|
|
60553800 |
Mar 16, 2004 |
|
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|
Current U.S.
Class: |
424/486 ; 514/56;
536/21 |
Current CPC
Class: |
A61K 31/727 20130101;
C08B 37/0075 20130101; A61P 9/00 20180101 |
Class at
Publication: |
424/486 ; 536/21;
514/56 |
International
Class: |
A61K 31/727 20060101
A61K031/727; C08B 37/10 20060101 C08B037/10; A61K 9/14 20060101
A61K009/14; A61P 9/00 20060101 A61P009/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made in part with U.S. Government support
under grant number 5 RO1 HL39150-15 awarded by the National
Institutes of Health. The U.S. Government has certain rights in
this application.
Claims
1. A composition comprising a hexanoyl derivative of low molecular
weight heparin with the chemical structure: ##STR00001## Wherein
m=8 and R.dbd.CH.sub.3(CH.sub.2).sub.4--.
2. A composition comprising a butanoyl derivative of low molecular
weight heparin with the chemical structure: ##STR00002## wherein
m=8 and R.dbd.CH.sub.3(CH.sub.2).sub.2--.
3. A pharmaceutical composition comprising: a) a hexanoyl
derivative of low molecular weight heparin with the chemical
structure: ##STR00003## wherein m=8 and
R.dbd.CH.sub.3(CH.sub.2).sub.4--; and b) a pharmaceutically
acceptable carrier.
4. A pharmaceutical composition comprising: a) a butanoyl
derivative of low molecular weight heparin with the chemical
structure: ##STR00004## wherein m=8 and
R.dbd.CH.sub.3(CH.sub.2).sub.2--; and b) a pharmaceutically
acceptable carrier.
5. The pharmaceutical composition of claim 3, which is formulated
for oral, rectal, topical or parenteral administration.
6. A composition for intravascular implant coating comprising a
hexanoyl derivative of low molecular weight heparin with the
chemical structure: ##STR00005## wherein m=8 and
R.dbd.CH.sub.3(CH.sub.2).sub.4--.
7. A composition for intravascular implant coating comprising a
butanoyl derivative of low molecular weight heparin with the
chemical structure: ##STR00006## wherein m=8 and
R.dbd.CH.sub.3(CH.sub.2).sub.2--.
8. A polymer matrix which has incorporated therein, a hexanoyl
derivative of low molecular weight heparin with the chemical
structure: ##STR00007## wherein m=8 and
R.dbd.CH.sub.3(CH.sub.2).sub.4--.
9. A polymer matrix which has incorporated therein, a butanoyl
derivative of low molecular weight heparin with the chemical
structure: ##STR00008## wherein m=8 and
R.dbd.CH.sub.3(CH.sub.2).sub.2--.
10. The pharmaceutical composition of claim 4, which is formulated
for oral, rectal, topical or parenteral administration.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims benefit under 35 U.S.C.
.sctn. 119(e) of U.S. Provisional Application Ser. No. 60/553,800
filed Mar. 16, 2004, the content of which is herein incorporated by
reference in its entirety.
FIELD OF INVENTION
[0003] The present invention is directed to a method of treating or
preventing abnormal cellular proliferation.
BACKGROUND OF THE INVENTION
[0004] Atherosclerosis, a common form of arteriosclerosis, results
from the deposition of fatty substances, primarily cholesterol, and
subsequent fibrosis in the inner layer (intima) of an artery,
resulting in plaque deposition on the inner surface of the arterial
wall and degenerative changes within it. The ubiquitous arterial
fatty plaque is the earliest lesion of atherosclerosis and is a
grossly flat, lipid-rich atheroma consisting of macrophages (white
blood cells) and smooth muscle fibers. The fibrous plaque of the
various forms of advanced atherosclerosis has increased intimal
smooth muscle cells surrounded by a connective tissue matrix and
variable amounts of intracellular and extracellular lipid. At the
luminal surface of the artery, a dense fibrous cap of smooth muscle
or connective tissue usually covers this plaque or lesion. Beneath
the fibrous cap, the lesions are highly cellular consisting of
macrophages, other leukocytes and smooth muscle cells. As the
lesions increase in size, they reduce the diameter of the arteries
and impede blood circulation resulting in coronary heart disease,
myocardial infarction (MI) and other serious complications.
[0005] Many therapies have been considered for the treatment of
atherosclerosis, including surgery and medical treatment. One
potential therapy is percutaneous transluminal angioplasty (balloon
angioplasty). More than 400,000 such procedures are performed each
year in the United States. In balloon angioplasty, a catheter
equipped with an inflatable balloon is threaded intravascularly to
the site of the atherosclerotic narrowing of the vessel. Inflation
of the balloon compresses the plaque enlarging the vessel.
[0006] While such angioplasty has gained wider acceptance, it
suffers from two major problems, i.e., abrupt closure and
restenosis. Abrupt closure refers to the acute occlusion of a
vessel immediately after or within the initial hours following a
dilation procedure. Abrupt closure occurs in approximately one in
twenty cases and frequently results in myocardial infarction and
death if blood flow is not restored in a timely manner.
[0007] As many as 50% of the patients who are treated by balloon
angioplasty require a repeat procedure within six months to correct
a re-narrowing of the artery. Restenosis refers to such
re-narrowing of an artery after an initially successful
angioplasty. Restenosis of the blood vessel is thought to be due to
injury to the endothelial cells of the blood vessel during
angioplasty, or during inflation of the balloon catheter. During
healing of the blood vessel after surgery, smooth muscle cells
proliferate faster than endothelial cells resulting in a narrowing
of the lumen of the blood vessel and starting the atherosclerotic
process anew. In recent years, smooth muscle cell proliferation has
been recognized as a major clinical problem limiting the long-term
efficacy of coronary angioplasty.
[0008] In an effort to prevent restenosis of the treated blood
vessel, the search for agents that can reduce or prevent excessive
proliferation of smooth muscle cells have been the object of much
research. (The occurrence and effects of smooth muscle cell
proliferation after these types of surgery have been reviewed, for
example, in Ip, et al., (June 1990) J. Am. College of Cardiology
15:1667-1687, and Faxon, et al. (1987) Am. J. of Cardiology 60:
5B-9B.). Such compounds have found little if any practical success.
There therefore exists a need to identify and successfully
administer compounds that inhibit smooth muscle cell
proliferation.
[0009] An alternative to angioplasty is the placement of
endovascular stents in the occluded blood vessel. Placement of a
stent at such a site, should mechanically block abrupt closure and
delay restenosis (Harrison's Principles of Internal Medicine,
14.sup.th Edition, 1998). Of the various procedures used to
overcome restenosis, stents have proven to be the most effective.
Stents are metal scaffolds that are positioned in the diseased
vessel segment to create a normal vessel lumen. Placement of the
stent in the affected arterial segment prevents recoil and
subsequent closing of the artery. By maintaining a larger lumen
than that created using balloon angioplasty alone, stents reduce
restenosis by as much as 30%. Despite their success, stents have
not eliminated restenosis entirely. (Suryapranata et al. 1998.
Randomized comparison of coronary stenting with balloon angioplasty
in selected patients with acute myocardial infarction. Circulation
97:2502-2502).
[0010] Unfortunately, the use of such stents are limited by direct
(subacute thrombosis) or indirect (bleeding, peripheral vascular
complications) complications. After stent implantation the patients
are threatened with stent thrombosis until the struts of the stent
are covered by endothelium. Thus, an aggressive therapy using
anticoagulation and/or antiplatelet agents is necessary during this
period of time. While these therapies are able to decrease the rate
of stent thrombosis, they are the main source of indirect
complications.
[0011] In addition to coronary artery occlusion, narrowing of the
arteries can occur in other vessels. Examples include the
aortoiliac, infrainguinal, distal profunda femoris, distal
popliteal, tibial, subclavian and mesenteric arteries. The
prevalence of peripheral artery atherosclerosis disease (PAD)
depends on the particular anatomic site affected as well as the
criteria used for diagnosis of the occlusion. Rates of PAD appear
to vary with age, with an increasing incidence of PAD in older
individuals. Data from the National Hospital Discharge Survey
estimate that every year, 55,000 men and 44,000 women had a
first-listed diagnosis of chronic PAD and 60,000 men and 50,000
women had a first-listed diagnosis of acute PAD. Ninety-one percent
of the acute PAD cases involved the lower extremity. The prevalence
of comorbid coronary artery disease (CAD) in patients with PAD can
exceed 50%. In addition, there is an increased prevalence of
cerebrovascular disease among patients with PAD.
[0012] PAD can be treated using percutaneous transluminal balloon
angioplasty (PTA). The use of stents in conjunction with PTA
decreases the incidence of restenosis. However, the post-operative
results obtained with medical devices such as stents do not match
the results obtained using standard operative revascularization
procedures, i.e., those using a venous or prosthetic bypass
material. (Principles of Surgery, Schwartz et al. eds., Chapter 20,
Arterial Disease, 7th Edition, McGraw-Hill Health Professions
Division, New York 1999).
[0013] Preferably, PAD is treated using bypass procedures where the
blocked section of the artery is bypassed using a graft.
(Principles of Surgery, Schwartz et al. eds., Chapter 20, Arterial
Disease, 7th Edition, McGraw-Hill Health Professions Division, New
York 1999). The graft can consist of an autologous venous segment
such as the saphenous vein or a synthetic graft such as one made of
polyester, polytetrafluoroethylene (PTFE), or expanded
polytetrafluoroethylene (ePTFE). Restenosis and thrombosis,
however, remain significant problems even with the use of bypass
grafts. For example, the patency of infrainguinal bypass procedures
at 3 years using an ePTFE bypass graft is 54% for a
femoral-popliteal bypass and only 12% for a femoral-tibial
bypass.
[0014] Consequently, there is a significant need to improve the
performance of both stents and synthetic bypass grafts in order to
further reduce the morbidity and mortality of CAD and PAD.
[0015] With stents, the approach has been to coat the stents with
various anti-thrombotic or anti-restenotic agents in order to
reduce thrombosis and restenosis. For example, impregnating stents
with radioactive material appears to inhibit restenosis by
inhibiting migration and proliferation of myofibroblasts. (U.S.
Pat. Nos. 5,059,166, 5,199,939 and 5,302,168). Irradiation of the
treated vessel can pose safety problems for the physician and the
patient. In addition, irradiation does not permit uniform treatment
of the affected vessel.
[0016] Numerous attempts to develop stents with a local
drug-distribution function have been made, most of which are
variances of the so called stent graft, a metal stent covered with
polymer envelope, containing a medicament. It would be of benefit
to coat a stent with a compound capable of diminishing or
eliminating restenosis.
[0017] Unlike the unwanted smooth muscle cell proliferation seen in
restenosis, cellular proliferation is a normal ongoing process in
all living organisms and is one that involves numerous factors and
signals that are delicately balanced to maintain regular cellular
cycles.
[0018] When normal cellular proliferation is disturbed or somehow
disrupted, the results can be inconsequential or they can be the
manifestation of an array of biological disorders. Disruption of
proliferation could be due to a myriad of factors such as the
absence or overabundance of various signaling chemicals or presence
of altered environments. Some disorders characterized by abnormal
cellular proliferation include cancer, abnormal development of
embryos, improper formation of the corpus luteum, difficulty in
wound healing as well as malfunctioning of inflammatory and immune
responses.
[0019] Cancer is characterized by abnormal cellular proliferation.
Cancer cells exhibit a number of properties that make them
dangerous to the host, often including an ability to invade other
tissues and to induce capillary ingrowth, which assures that the
proliferating cancer cells have an adequate supply of blood. One of
the defining features of cancer cells is that they respond
abnormally to control mechanisms that regulate the division of
normal cells and continue to divide in a relatively uncontrolled
fashion until they kill the host.
[0020] It is clear that aberrant cellular proliferation plays a
major role in the formation and progression of a cancer. If this
abnormal or undesirable proliferative activity could be repressed,
inhibited, or eliminated, then the tumor, although present, would
not grow. In the disease state, prevention of abnormal or
undesirable cellular proliferation could slow or abate the
progression of cancer. Additionally, compounds that could induce
apoptosis of abnormally proliferating cells would be especially
beneficial for complete removal or elimination of malignant cells,
helping to reduce relapses. Therapies directed at control of the
cellular proliferative processes could lead to the abrogation or
mitigation of such malignancies.
[0021] Pulmonary hypertension is caused largely by an increase in
pulmonary vascular resistance and is classified clinically as
either primary or secondary. Secondary pulmonary hypertension, the
more common form, is generally a result of (1) chronic obstructive
or interstitial lung disease; (2) recurrent pulmonary emboli; (3)
liver disease; or (4) antecedent heart disease. Primary pulmonary
hypertension is diagnosed only after all known causes of increased
pulmonary pressure are excluded.
[0022] At the moment there is no successful cure for pulmonary
hypertension. Administration of vasodilatating drugs has not proved
to be useful in patients suffering from pulmonary hypertension. The
prognosis is poor, with a median survival time of about 3
years.
[0023] Pulmonary fibrosis can occur in response to known stresses
such as asbestos or silica but most is idiopathic. There is a
spectrum of idiopathic fibrosis but most kinds are fatal in 3-5
years. At present, there is no effective therapy for most
cases.
[0024] What is needed therefore is a composition and method which
can inhibit abnormal or undesirable cellular proliferation,
especially the growth of smooth muscle cells after angioplasty,
stent placement, pulmonary hypertension, pulmonary fibrosis or the
proliferation of malignant cells. The composition should be able to
overcome the activity of endogenous growth factors in premetastatic
tumors and inhibit smooth muscle cell proliferation during
restenosis. Finally, the composition and method for inhibiting
cellular proliferation should preferably be non-toxic and produce
few side effects.
[0025] Heparin is a glycosaminoglycan that was first described by
McLean in 1916 and has been used clinically as an anticoagulant for
more than 50 years [McLean, Circulation 19, 75-78 (1959)]. Members
of the glycosaminoglycan family include hyaluronan, heparan
sulfate, dermatan sulfate, and chondroitin sulfate. Beyond its
well-recognized anticoagulant activity, heparin has other
activities. The antimetastatic activity of heparin has been known
for some time (see, for example, Drago, J. R. et al., Anticancer
Res., 4(3), 171-2, 1984).
[0026] Unfortunately, native and currently described modified
heparins are extremely anticoagulant. Their anticoagulant
properties are such that doses effective in the treatment of
malignancies and anti-proliferative disorders are not attainable.
It has therefore been suggested that altering the chemical
structure of heparin might decrease the anticoagulant properties of
heparin while maintaining its other important biological
activities, such as its antimetastatic activity (Barzu et al., J.
Med. Chem, 1993, 36, pg. 3546-3555).
[0027] Low molecular weight heparins have shown promise in reducing
anticoagulation while maintaining their antimetastatic activity.
For example, when compared to unmodified heparin, 2-O-desulfated
and 3-O-desulfated heparins had reduced anticoagulant activities,
but preserved their angiostatic, anti-tumor and anti-metastatic
properties (Masayuki et al., U.S. Pat. No. 5,795,875 (1997);
Lapierre et al., Glycobiology 6, 355-366 (1996)]. Nevertheless, the
use of currently available heparins and heparin derivitives for the
treatment of abnormal cellular proliferative disorders is not
practical due to their marked anticoagulant and antithrombotic
activities.
[0028] O-acylated heparins have been described. These molecules
have very low anticoagulative effects in vitro, yet retain activity
against HIV-1 and 2 induced cytopathicity (Barzu et al., J. Med.
Chem, 1993, 36, pg. 3546-3555).
[0029] A chemically modified heparin that can be used to treat
and/or prevent abnormal cellular proliferative disorders is needed.
Such a compound should have minimal anticoagulant properties while
maintaining antiproliferative properties. The anticoagulative
properties of the compound must not limit its use in the clinical
setting.
SUMMARY
[0030] This invention provides a method for inhibiting or
preventing the abnormal growth of cells, including transformed
cells, by administering an effective amount of O-acylated heparin
derivative. Abnormal growth of cells refers to cell growth
independent of normal regulatory mechanism (e.g. loss of contact
inhibition). This includes the abnormal growth of: (1) tumor cells
(tumors); (2) benign and malignant cells of other proliferative
disease in which aberrant cellular proliferation occurs; (3)
aberrant smooth muscle cell proliferation, such as might occur
following treatment for coronary atherosclerosis such as
angioplasty or the insertion of a stent into an occluded vessel.
The O-acylated heparin derivative is preferably an O-hexanoylated
heparin derivative or an O-butanoylated heparin derivative. In a
preferred embodiment, the o-acylated heparin is weakly
anticoagulant as compared to non-chemically modified heparins.
[0031] One embodiment of the present invention provides a method
for inhibiting or preventing tumor growth by administering an
effective amount of an o-acylated heparin, to a subject, e.g. a
mammal (and more particularly a human) in need of such treatment.
In particular, this invention provides a method for inhibiting the
growth of malignant cells by the administration of an effective
amount of an O-acylated heparin. In a preferred embodiment, the
methods are directed toward the treatment or prevention of lung and
colon cancer. Preferably, the O-acylated heparin derivative is an
O-hexanoylated or an O-butanoylated heparin derivative
[0032] In another embodiment, the invention provides a method of
preventing abnormal smooth muscle cell proliferation. The method
comprises presenting an O-acylated heparin near or into a site of
abnormal smooth muscle cell proliferation. In a preferred
embodiment, the methods are directed toward the prevention of
smooth muscle cell proliferation as occurs in restenosis. The
methods are used to prevent restenosis that occurs following
angioplasty or vascular stent placement. Alternatively, the methods
of the current invention are used to prevent restenosis following
coronary artery stent placement, peripheral artery stent placement,
or cerebral artery stent placement.
[0033] In yet another embodiment, the invention provides a medical
device coated with the heparin composition, e.g., a stent for
implantation in a blood vessel. The stent of the invention
comprises a coating containing an O-acylated heparin and
preferably, an O-hexanoylated or O-butanoylated heparin derivative.
In one embodiment, the stent is coated with an O-acylated heparin
and one or more compounds selected from the group consisting of a
polymer, fiber polymer, polyurethane, silicone rubber elastomer,
drug, hydrogel, or other acceptable compound or carrier known to
those of skill in the art. Other medical devices such as catheters
may also be coated with the O-acylated heparin.
[0034] Finally, the invention provides a method of treating
pulmonary hypertension and pulmonary fibrosis. The methods of the
present invention provide treating a subject with a therapeutic
amount of an o-acylated heparin, preferably a O-hexanoylated or
O-butanoylated heparin derivative. In one embodiment, the invention
provides for the treatment of primary pulmonary hypertension. In
another embodiment, the invention provides for the treatment of
secondary pulmonary hypertension. In a final embodiment, the
invention provides for the treatment of pulmonary fibrosis.
DESCRIPTION OF FIGURES
[0035] FIG. 1 shows the preparation of O-acylated heparin
derivatives.
[0036] FIG. 2 shows the results of .sup.1H NMR spectroscopy, where
approximately 10 mg of each sample was exchanged by lyophilization
three times from 0.5 ml portions of 99.9% 2H.sub.2O before being
redissolved in .sup.2H.sub.2O for NMR analysis. Chemical shifts are
reported relative to TMS at 0.00 ppm. The degree of substitution
(O-acylation) was determined from the ratio of the integrated area
of the peaks assigned to the aliphatic methyl protons of the
hexanoyl group (0.753 ppm) to the anomeric proton of IdoA2S (5.092
ppm).
[0037] FIG. 3 shows the structures of O-hexanoylated heparin and
O-butanoylated heparin.
[0038] FIG. 4 shows that hexanoylated heparin (HHP) significantly
inhibited pulmonary artery smooth muscle cell proliferation in
vivo.
[0039] FIG. 5 shows that hexanoylated heparin (HHP) significantly
inhibited the development of pulmonary hypertension induced by
hypoxia in the pig lung.
[0040] FIG. 6 shows a comparison of tumor growth in SCID mice
treated with various doses of native heparin (UHP) and butanoylated
heparin (BHP). Butanoylated heparin significantly inhibited A549
(non-small cell lung carcinoma) cell tumor growth in a dose
dependent manner.
[0041] FIG. 7 shows a comparison of tumor weights from SCID mice
treated with native heparin and butanoylated heparin. Butanoylated
heparin (BHP) significantly decreased A549 (non-small cell lung
carcinoma) cell tumor weight in a dose dependent manner.
[0042] FIG. 8 shows percent inhibition of tumor growth in SCID mice
treated with native heparin and butanoylated heparin. Butanoylated
heparin (BHP) significantly inhibited A549 (non-small cell lung
carcinoma) cell tumor growth in a dose dependent manner.
[0043] FIG. 9 shows tumor growth in SCID mice treated with native
heparin and butanoylated heparin. Butanoylated heparin (BHP)
significantly inhibited DMS79 (small cell lung carcinoma) cell
tumor growth in a dose dependent manner.
[0044] FIG. 10 shows tumor weight from SCID mice treated with
native heparin and butanoylated heparin. Butanoylated heparin (BHP)
significantly decreased DMS79 (small cell lung carcinoma) cell
tumor weight in a dose dependent manner.
[0045] FIG. 11 shows tumor growth in SCID mice treated with native
heparin and butanoylated heparin. Butanoylated heparin (BHP)
significantly inhibited HCT116 (colon cancer) cell tumor growth in
a dose dependent manner.
[0046] FIG. 12 shows tumor growth in SCID mice treated with native
heparin and butanoylated heparin. Butanoylated heparin (BHP)
significantly inhibited HCT116 (colon cancer) cell tumor growth in
a dose dependent manner.
[0047] FIG. 13 shows coagulation time for various heparin
compounds. Native heparin (UPJ HP) increased coagulation time in a
dose dependent manner. Butanoylated heparin had no significant
effect on coagulation time.
[0048] FIG. 14 shows histology of A549 cell tumor tissue grown in
SCID mice. Hemorrhage was detected in mice treated with 100 mg/kg
native, unfractionated heparin only.
[0049] FIG. 15 shows histology of heart from SCID mice bearing A549
cell tumor. No significant pathological change was observed in any
group.
[0050] FIG. 16 shows histology of kidney from SCID mice bearing
A549 cell tumor. No significant pathological change was observed in
any group.
[0051] FIG. 17 shows histology of liver from SCID mice bearing A549
cell tumor. No significant pathological change was observed in any
group.
[0052] FIG. 18 shows histology of lung from SCID mice bearing A549
cell tumor. No significant pathological change was observed in any
group.
[0053] FIG. 19 shows apoptosis index in different treatment groups.
Heparin significantly induces apoptosis in tumor grown in SCID
mice.
[0054] FIG. 20 shows expression of p27/KIP1 gene product in the
A549 cell tumor tissue grown in SCID mice treated with heparins.
BHP significantly inhibited p27/KIP1 in a dose dependent
manner.
[0055] FIG. 21 shows expression of Rb gene product in the A549 cell
tumor tissue grown in SCID mice treated with heparins. BHP
decreased Rb gene expression in a dose dependent manner although to
a lesser effect than on p27/KIP1.
[0056] FIG. 22 shows expression of E2F1 protein in A549 cell tumor
tissue grown in SCID mice treated with heparins. BHP significantly
inhibited E2F1 protein expression in a dose dependent manner.
[0057] FIGS. 23A and 23B show diagrams of a stent. FIG. 23A, a
stent; FIG. 23B, an end view of the stent of FIG. 23A
[0058] FIG. 24 shows a diagram of a stent having a second coating
formed on the outer surface.
DETAILED DESCRIPTION
[0059] The present invention is directed generally to compositions
and their use in the therapy and prevention of abnormal cellular
proliferative disorders, such as cancer, (i.e. lung and colon
cancer), restenosis (following angioplasy, vascular stent
placement, coronary artery stent placement, periphaeral artery
stent placement, or cerebral artery stent placement), pulmonary
hypertension (primary or secondary), and pulmonary fibrosis. The
administration of therapeutic levels of the O-acylated heparin
derivatives result in a decrease, cessation, or prevention of the
abnormal cellular proliferation.
O-Acylated Heparin
[0060] As described further below, compositions useful in the
present invention include, but are not restricted to, O-acylated
heparins, particularly O-hexanoylated heparin derivatives and
O-butanoylated heparin derivatives.
[0061] O-acylated heparins are prepared using any of a variety of
well known synthetic and/or recombinant techniques, an example of
which is further described below. Furthermore, O-acylated heparins,
useful in the present invention, have been described in Barzu et
al., J. Med. Chem, 1993, 36, 3546-3555 and U.S. Pat. No. 4,990,502
(Lormeau et al.). The structure of the O-acylated heparin
derivatives used in the present invention are shown in FIG. 3.
Preferably, the major disaccharide units (m) vary from about 4 to
about 14. Most preferably the major disaccharide units (m) vary
from about 7 to about 9. In the O-hexanoylated derivative,
R.dbd.CH.sub.3(CH.sub.2).sub.4- in the O-butanoylated derivative,
R.dbd.CH.sub.3(CH.sub.2).sub.2-.
[0062] Low-molecular weight heparins (LMWHs) are fragments of
conventional heparin. LMWHs were developed to provide more
selective inhibition of enzyme function and reduce adverse effects.
Heparin fragmentation produces products which maintain activity
against factor X.sub.a and release antithrombotic factors, but have
significantly less activity against factor II.sub.a. As a result,
treatment with LMWHs provides antithrombotic effects with less
anticoagulant effect, lessening the risk of hemorrhage. However, in
the generic sense, LMWHs have not proven beneficial in the
treatment of cancer due to their high anticoagulant activity.
Administration of Compounds
[0063] The heparins of the present invention can be administered
via any medically acceptable means which is suitable for the
compound to be administered, including oral, rectal, topical,
parenteral (including inhaled, subcutaneous, intramuscular and
intravenous) administration, or by coated stent, coated graft, or
coated catheter.
[0064] Effective doses for heparin-like substances are well known
to those of skill in the art. Generally, for heparin-like
substances, an effective dose is that which maintains the
anti-X.sub.a level between 0.5 and 1.0 units/ml. This range has
been shown to optimize antithrombotic activity while avoiding
adverse effects.
[0065] The total daily dose may be given as a single dose, multiple
doses, e.g., two to six times per day, or by intravenous infusion
for a selected duration. Dosages above or below the range cited
above are within the scope of the present invention and may be
administered to the individual patient if desired and necessary. If
discrete multiple doses are indicated, treatment might typically be
4-6,000 units of a compound given 4 times per day or if given
continuously, as is more often the case, then a loading dose of 80
units/kg followed by 18 units/kg/hr (Rascke R A, Reilly B M, Guidry
J R, et al. The weigh based heparin dosing nomogram compared with a
"standard care" nomogram: A randomized control trial. Ann Int Med
119:874-81, 1993).
[0066] Formulations
[0067] The compounds described above are preferably administered in
a formulation including an O-acylated heparin and/or an O-acylated
heparin together with an acceptable carrier for the mode of
administration. Any formulation or drug delivery system containing
the active ingredients, which is suitable for the intended use, as
are generally known to those of skill in the art, can be used.
Suitable pharmaceutically acceptable carriers for oral, rectal,
topical or parenteral (including inhaled, subcutaneous,
intraperitoneal, intramuscular and intravenous) administration are
known to those of skill in the art. The carrier must be
pharmaceutically acceptable in the sense of being compatible with
the other ingredients of the formulation and not deleterious to the
recipient thereof.
[0068] Formulations suitable for parenteral administration
conveniently include sterile aqueous preparation of the active
compound which is preferably isotonic with the blood of the
recipient. Thus, such formulations may conveniently contain
distilled water, 5% dextrose in distilled water or saline. Useful
formulations also include concentrated solutions or solids
containing the compound which upon dilution with an appropriate
solvent give a solution suitable for parental administration
above.
[0069] For enteral administration, a compound can be incorporated
into an inert carrier in discrete units such as capsules, cachets,
tablets or lozenges, each containing a predetermined amount of the
active compound; as a powder or granules; or a suspension or
solution in an aqueous liquid or non-aqueous liquid, e.g., a syrup,
an elixir, an emulsion or a draught. Suitable carriers may be
starches or sugars and include lubricants, flavorings, binders, and
other materials of the same nature.
[0070] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared by compressing in a suitable machine the active compound
in a free-flowing form, e.g., a powder or granules, optionally
mixed with accessory ingredients, e.g., binders, lubricants, inert
diluents, surface active or dispersing agents. Molded tablets may
be made by molding in a suitable machine, a mixture of the powdered
active compound with any suitable carrier.
[0071] A syrup or suspension may be made by adding the active
compound to a concentrated, aqueous solution of a sugar, e.g.,
sucrose, to which may also be added any accessory ingredients. Such
accessory ingredients may include flavoring, an agent to retard
crystallization of the sugar or an agent to increase the solubility
of any other ingredient, e.g., as a polyhydric alcohol, for
example, glycerol or sorbitol.
[0072] Formulations for rectal administration may be presented as a
suppository with a conventional carrier, e.g., cocoa butter or
Witepsol S55 (trademark of Dynamite Nobel Chemical, Germany), for a
suppository base.
[0073] Alternatively, the compound may be administered in liposomes
or microspheres (or microparticles). Methods for preparing
liposomes and microspheres for administration to a patient are well
known to those of skill in the art. U.S. Pat. No. 4,789,734, the
contents of which are hereby incorporated by reference, describes
methods for encapsulating biological materials in liposomes.
Essentially, the material is dissolved in an aqueous solution, the
appropriate phospholipids and lipids added, along with surfactants
if required, and the material dialyzed or sonicated, as necessary.
A review of known methods is provided by G. Gregoriadis, Chapter
14, "Liposomes," Drug Carriers in Biology and Medicine, pp. 287-341
(Academic Press, 1979).
[0074] Microspheres formed of polymers or proteins are well known
to those skilled in the art, and can be tailored for passage
through the gastrointestinal tract directly into the blood stream.
Alternatively, the compound can be incorporated and the
microspheres, or composite of microspheres, implanted for slow
release over a period of time ranging from days to months. See, for
example, U.S. Pat. Nos. 4,906,474, 4,925,673 and 3,625,214, and
Jein, TIPS19:155-157 (1998), the contents of which are hereby
incorporated by reference.
[0075] In one embodiment, the O-acylated heparin can be formulated
into a liposome or microparticle which is suitably sized to lodge
in capillary beds following intravenous administration. When the
liposome or microparticle is lodged in the capillary beds
surrounding ischemic tissue, the agents can be administered locally
to the site at which they can be most effective. Suitable liposomes
for targeting ischemic tissue are generally less than about 200
nanometers and are also typically unilamellar vesicles, as
disclosed, for example, in U.S. Pat. No. 5,593,688 to
Baldeschweiler, entitled "Liposomal targeting of ischemic tissue,"
the contents of which are hereby incorporated by reference.
[0076] Preferred microparticles are those prepared from
biodegradable polymers, such as polyglycolide, polylactide and
copolymers thereof. Those of skill in the art can readily determine
an appropriate carrier system depending on various factors,
including the desired rate of drug release and the desired
dosage.
[0077] In one embodiment, the formulations are administered via
catheter directly to the inside of blood vessels. The
administration can occur, for example, through holes in the
catheter. In those embodiments wherein the active compounds have a
relatively long half life (on the order of 1 day to a week or
more), the formulations can be included in biodegradable polymeric
hydrogels, such as those disclosed in U.S. Pat. No. 5,410,016 to
Hubbell et al. These polymeric hydrogels can be delivered to the
inside of a tissue lumen and the active compounds released over
time as the polymer degrades. If desirable, the polymeric hydrogels
can have microparticles or liposomes which include the active
compound dispersed therein, providing another mechanism for the
controlled release of the active compounds.
[0078] The formulations may conveniently be presented in unit
dosage form and may be prepared by any of the methods well known in
the art of pharmacy. All methods include the step of bringing the
active compound into association with a carrier which constitutes
one or more accessory ingredients. In general, the formulations are
prepared by uniformly and intimately bringing the active compound
into association with a liquid carrier or a finely divided solid
carrier and then, if necessary, shaping the product into desired
unit dosage form.
[0079] The formulations can optionally include additional
components, such as various biologically active substances such as
growth factors (including TGF.beta., basic fibroblast growth factor
(bFGF), epithelial growth factor (EGF), transforming growth factors
alpha and beta (TGF.alpha. and TGF.beta.), nerve growth factor
(NGF), platelet-derived growth factor (PDGF), and vascular
endothelial growth factor/vascular permeability factor (VEGF/VPF),
antivirals, antibacterials, antiinflammatories, immunosuppressants,
analgesics, vascularizing agents, cell adhesion molecules (CAM's),
and anticoagulants other than heparin or heparin-like
substances.
[0080] In addition to the aforementioned ingredients, the
formulations may further include one or more optional accessory
ingredient(s) utilized in the art of pharmaceutical formulations,
e.g., diluents, buffers, flavoring agents, binders, surface active
agents, thickeners, lubricants, suspending agents, preservatives
(including antioxidants) and the like.
[0081] Finally, compositions of the compound are presented for
administration to the respiratory tract as a snuff or an aerosol or
solution for a nebulizer, or as a microfine powder for
insufflation, alone or in combination with an inert carrier such as
lactose. In such a case the particles of active compound suitably
have diameters of less than 50 microns, preferably less than 10
microns, more preferably between 2 and 5 microns.
Stents, Grafts and Implants
[0082] The present invention further provides an intravascular
implant coating. The coating includes a therapeutically effective
amount of an O-acylated heparin. The coating can be used in any
type of implant. These include balloon catheters, stents, stent
graphs, drug delivery catheters, atherectomy devices, filters,
scaffolding devices, anastomothic clips, anastomotic bridges and
suture materials.
[0083] The coating can also include a polymer matrix, with the
polymer being a resorbable polymer selected from the group
consisting of poly-.alpha. hydroxyl acids, polyglycols,
polytyrosine carbonates, starch, gelatins, cellulose, and blends
and copolymers thereof. Examples of suitable poly-.alpha. hydroxyl
acids include polylactides, polyglycol acids, and blends and
co-polymers thereof.
[0084] According to the present invention, a coating for an
intravascular implant is provided. The coating can be applied
either alone, or within a polymeric matrix, which can be biostable
or bioabsorbable, to the surface of an intravascular device. The
coating can be applied directed to the implant or on top of a
polymeric substrate, i.e. a primer. If desired, a top coat can be
applied to the therapeutic coating.
[0085] It should be noted that the present invention relates to a
combinatorial therapy for delivery of more than one agent through a
coating on any intravascular implant. As used herein, implant means
any type of medical or surgical implement, whether temporary or
permanent. Delivery can be either during or after an interventional
procedure. Non-limiting examples of intravascular implants now
follow.
[0086] The outside surface of a balloon catheter may be coated with
the coating according to the present invention and could be
released immediately or in a time dependent fashion. When the
balloon expands and the wall of the vessel is in contact with the
balloon, the release of the o-acylated heparin can begin.
[0087] The surface of a stent may be coated with the combination of
agents and the stent is implanted inside the body. The stent struts
could be loaded with several layers of the agents or with just a
single layer. A transporter or a vehicle to load the agents on to
the surface can also be applied to the stent. The graft material of
the stent graft can also be coated (in addition to the stent or as
an alternative) so that the material is transported intravascularly
at the site of the location of the injury.
[0088] The drug delivery catheters that are used to inject drugs
and other agents intravascularly can also be used to deliver the
o-acylated heparins. Other intravascular devices through which the
transport can happen include atherectomy devices, filters,
scaffolding devices, anastomotic clips, anastomotic bridges, suture
materials etc.
[0089] The present invention envisions applying the coating
directly to the intravascular implant. However, the coating can be
applied to a primer, i.e. a layer or film of material upon which
another coating is applied. Furthermore, the o-acylated heparins
can be incorporated in a polymer matrix. Polymeric matrices
(resorbable and biostable) can be used for delivery of the
therapeutic agents. In some situations, when the agents are loaded
on to the implant, there is a risk of quick erosion of the
therapeutic agents either during the expansion process or during
the phase during which the blood flow is at high shear rates at the
time of implantation. In order to ensure that the therapeutic
window of the agents is prolonged over extended periods of time,
polymer matrices can be used.
[0090] These polymers could be any one of the following:
semitelechelic polymers for drug delivery, thermo responsive
polymeric micelles for targeted drug delivery, pH or temperature
sensitive polymers for drug delivery, peptide and protein based
drug delivery, water insoluble drug complex drug delivery matrices
polychelating amphiphilic polymers for drug delivery,
bioconjugation of biodegradable poly lactic/glycolic acid for
delivery, elastin mimetic protein networks for delivery,
generically engineered protein domains for drug delivery,
superporbus hydrogel composites for drug delivery, interpenetrating
polymeric networks for drug delivery, hyaluronic acid based
delivery of drugs, photocrosslinked polyanhydrides with controlled
hydrolytic delivery, cytokine-including macromolecular glycolipids
based delivery, cationic polysaccharides for topical delivery,
n-halamine polymer coatings for drug delivery, dextran based
coatings for drug delivery, fluorescent molecules for drug
delivery, self-etching polymerization initiating primes for drug
delivery, and bioactive composites based drug delivery.
[0091] One embodiment of the present invention discloses an
implant, e.g., a stent for implantation into a body, e.g., blood
vessel. The implant comprises a coating of O-acylated heparin or
o-acylated heparin in combination with one or more compounds
selected from the group consisting of (but not limited to) a
polymer, fiber polymer, polyurethane, silicone rubber elastomer,
drug, hydrogel, or other acceptable compound or carrier known to
those of skill in the art. Methods of coating an implant such as a
stent with heparin or heparin in combination with one or more of
the compounds listed above, are known to those of skill in the art
and are further described below and in the examples. Alternatively,
O-acylated heparins of the present invention may be coated alone or
in combination with the above polymer, fiber polymer, polyurethane,
silicone rubber elastomer, drug, hydrogel, or other acceptable
compound or carrier known to those of skill in the art onto a
bypass graft. The implant, e.g., graft or stent may be used in the
treatment of peripheral artery atherosclerosis disease (PAD).
[0092] Whereas the polymer of the coating may be any compatible
biostable material capable of being adhered to the stent material
as a thin layer, hydrophobic materials are preferred because it has
been found that the release of the biologically active species can
generally be more predictably controlled with such materials.
Preferred materials include silicone rubber elastomers and
biostable polyurethanes.
[0093] Heparin-loaded polymer can be applied by spraying or by
dipping the stent graft into a solution or melt, as disclosed, for
example, in U.S. Pat. Nos. 5,383,922, 5,824,048, 5,624,411 and
5,733,327. Additional methods for providing a drug-loaded polymer
are disclosed in U.S. Pat. Nos. 5,637,113 and 5,766,710, where a
pre-fabricated film is attached to the stent. Other methods, such
as deposition via photo polymerization, plasma polymerization and
the like, are also known in the art and are described in, e.g.,
U.S. Pat. Nos. 3,525,745, 5,609,629 and 5,824,049 and in the below
examples.
[0094] U.S. Pat. No. 5,549,663 discloses a stent graft having a
coating made of polyurethane fibers which are applied using
conventional wet spinning techniques. Prior to the covering
process, a medication is introduced into the polymer.
Alternatively, a metallic stent cam be coated with a polymeric
material and load the polymeric material with a drug.
[0095] The Figures have not been drawn to scale, and the dimensions
such as depth and thickness of the various regions and layers have
been over or under emphasized for illustrative purposes. Referring
to FIGS. 23A and 23B, a stent 10 is formed from a plurality of
struts 12. Struts 12 are separated by gaps 14 and may be
interconnected by connecting elements 16. Struts 12 can be
connected in any suitable configuration and pattern to form an a
tubular body. While a strut configuration is illustrated, any known
stent configuration may be used. Stent 10 is illustrated having an
outer surface or sidewall 18 (tissue-contacting surface) and an
inner surface 20 (blood-contacting surface). A hollow, central bore
22 extends longitudinally from a first open end 24 to a second end
26 of stent 10.
[0096] FIG. 24 illustrates stent 10 coated in accordance with the
present invention. The stent may have a first coating 28 containing
an O-acylated heparin on inner surface 20 and/or a second coating
32 containing an O-acylated heparin formed on outer surface 18 of
stent 10. The coatings can be of any suitable thickness. The
thickness of second coating 32 can be from about 0.1-15 microns,
more narrowly from about 3 microns to about 8 microns. By way of
example, second coating 32 can have a thickness of about 4
microns.
Cancer
[0097] In another embodiment of the present invention, methods are
disclosed for the treatment and or prevention of cancer.
Therapeutic amounts of O-acylated heparin, particularly
O-hexanoylated heparin derivatives and O-butanoylated heparin
derivatives are given to a patient alone or in combination with
other cancer therapies, known to those of skill in the art.
Compounds may be administered before, at the same time as, or after
the administration of other conventional cancer therapies.
O-acylated heparins of the present invention may be given prior to
the diagnosis of cancer, such as in the case of a patient having a
high-risk of developing cancer, or after the successful treatment
of cancer (ie. remission). The compounds of the present invention
may also be administered with the goal of reducing metastases.
[0098] Examples of tumors which may be inhibited, but are not
limited to, lung cancer (e.g. adenocarcinoma, small cell, and
including non-small cell lung cancer), pancreatic cancers (e.g.
pancreatic carcinoma such as, for example exocrine pancreatic
carcinoma), colon cancers (e.g. colorectal carcinomas, such as, for
example, colon adenocarcinoma and colon adenoma), prostate cancer
including the advanced disease, hematopoietic tumors of lymphoid
lineage (e.g. acute lymphocytic leukemia, B-cell lymphoma,
Burkitt's lymphoma), myeloid leukemias (for example, acute
myelogenous leukemia (AML)), thyroid follicular cancer,
myelodysplastic syndrome (MDS), tumors of mesenchymal origin (e.g.
fibrosarcomas and rhabdomyosarcomas), melanomas, teratocarcinomas,
neuroblastomas, gliomas, benign tumor of the skin (e.g.
keratoacanthomas), breast carcinoma (e.g. advanced breast cancer),
kidney carcinoma, ovary carcinoma, bladder carcinoma and epidermal
carcinoma.
[0099] For the treatment of the above conditions, the compound of
the invention may be advantageously employed in combination with
one or more other medicinal agents such as anti-cancer agents.
[0100] For example, O-acylated heparins of the invention may be
given in combination with one or more compounds selected from
platinum coordination compounds for example cisplatin or
carboplatin, taxane compounds for example paclitaxel or docetaxel,
camptothecin compounds for example irinotecan or topotecan,
anti-tumor vinca alkaloids for example vinblastine, vincristine or
vinorelbine, anti-tumor nucleoside derivatives for example
5-fluorouracil, gemcitabine or capecitabine, nitrogen mustard or
nitrosourea alkylating agents for example cyclophosphamide,
chlorambucil, carmustine or lomustine, anti-tumor anthracycline
derivatives for example daunorubicin, doxorubicin or idarubicin;
HER2 antibodies for example trastzumab; and antitumor
podophyllotoxin derivatives for example etoposide or teniposide;
and antiestrogen agents including estrogen receptor antagonists or
selective estrogen receptor modulators preferably tamoxifen, or
alternatively toremifene, droloxifene, faslodex and raloxifene, or
aromatase inhibitors such as exemestane, anastrozole, letrazole and
vorozole.
Aberrant Smooth Muscle Cell Proliferation
[0101] The methods of the present invention can be used to treat
disorders wherein smooth muscle cells abnormally proliferate. Such
conditions include, but are not limited to, restenosis (following
angioplasy, vascular stent placement, coronary artery stent
placement, peripheral artery stent placement, or cerebral artery
stent placement), pulmonary hypertension, and pulmonary fibrosis.
We have shown that heparin can inhibit fibroblast proliferation
(Dahlberg et al. Am Rev. Respir. Dis. 143:A357, 1993) and can
inhibit pulmonary fibrosis in the rat in response to bleomycin. We
also have unpublished data showing hexanoylated and butanoylated
heparins, which have virtually no anticoagulant property, can also
inhibit fibroblast proliferation and thus may offer a potent
therapeutic agent for human pulmonary fibrosis.
[0102] The methods of the invention provide for the treatment
(reduction or cessation) or prevention of disorders wherein smooth
muscle cells are abnormally proliferating. These methods include
the administration of O-acylated heparin compounds, preferably
O-hexanoylated or O-butanoylated heparin derivatives.
[0103] Administration of the compounds of the invention to treat
and/or prevent aberrant smooth muscle cell proliferation are known
to those skilled in the art and are presented above. Preferably,
O-acylated heparin is coated on an implantable stent, wherein the
delivery of the heparin is controlled and sufficient to reduce or
ablate aberrant smooth muscle cell proliferation.
Pulmonary Hypertension and Pulmonary Fibrosis
[0104] In yet another embodiment, the present invention is directed
to the treatment and/or prevention of pulmonary hypertension and
pulmonary fibrosis. Preferably, O-acylated heparins of the
invention are presented for administration to the respiratory tract
as a snuff or an aerosol or solution for a nebulizer, or as a
microfine powder for insufflation, alone or in combination with an
inert carrier such as lactose. In such a case the particles of the
active compound suitably have diameters of less than 50 microns,
preferably less than 10 microns, more preferably between 2 and 5
microns. The methods of the present invention are directed to the
treatment of both primary and secondary pulmonary hypertension and
pulmonary fibrosis.
Examples
Preparation of O-Acylated Heparins
[0105] In this example, we describe the synthesis of Low Molecular
Weight (LMW) heparin by periodate oxidation, its characterization,
and its O-acylation.
[0106] Heparin was fragmented by periodate oxidation based on a
modification of an earlier procedure (described in U.S. Pat. No.
4,990,502), wherein heparin sodium salt (20 g, 1.43 mmol) was
dissolved in 175 mL distilled water. The pH was adjusted to 5.0
using 1 N HCl. NaIO.sub.4 (15 g, 0.070 mol), dissolved in 500 mL
water, was added in a single portion with stirring. The pH was
readjusted to 5.0 using 1 N HCl and left for 24 hours at 4.degree.
C. in the dark. The solution was dialyzed against 4 volumes of
water (with one change of water) for 15 h at 4.degree. C. To the
approximately 1.5 L solution obtained after dialysis, 32 mL of 10 N
NaOH was added. The solution was stirred at room temperature for 3
h. To prevent the development of colored products, this step was
done in the dark.
[0107] NaBH.sub.4 (1 g, 0.026 mol) was added in one portion and the
approximately 1.5 L of solution was stirred for 4 hours. The pH was
then adjusted to 4.0 using 37% HCl and the solution was stirred for
an additional 15 min. The solution was neutralized to pH 7.0 using
1 N NaOH, NaCl (32.8 g, 0.56 mol) followed by 2.54 L of ethanol.
The solution was left for 3 h without stirring and the precipitate
was recovered by centrifugation at 15000 rpm (22,000.times.g) for
20 min. The precipitate was recovered by decantation and suspended
in 400 mL absolute ethanol. The solution was filtered using a
Buchner funnel and left to dry for 5 hours under vacuum affording
14.2 g of product.
[0108] The product was dissolved in 190 mL of water. NaCl (2.8 g,
0.05 mol) was added and the pH was adjusted to 3.5 using 1 N HCl.
The volume was adjusted to 280 mL using water. Absolute ethanol
(240 mL) was added with stirring. The solution was stirred 15 min
and then left without stirring for 10 hours at room temperature.
After decanting, the precipitate was recovered and dissolved in
water. The ethanol was removed by rotary evaporation under reduced
pressure and freeze-dried affording .about.10 g of LMW heparin
fragments (FIG. 1).
NMR Sample Preparation
[0109] For .sup.1H NMR spectroscopy, approximately 10 mg of each
sample was exchanged by lyophilization three times from 0.5 ml
portions of 99.9% .sup.2H.sub.2O before being redissolved in
.sup.2H.sub.2O for NMR analysis. Chemical shifts are reported
relative to TMS at 0.00 ppm. The degree of substitution
(O-acylation) was determined from the ratio of the integrated area
of the peaks assigned to the aliphatic methyl protons of the
hexanoyl group (0.753 ppm) to the anomeric proton of IdoA2S (5.092
ppm) (Table 1, FIG. 2).
Gradient PAGE Analysis
[0110] Gradient polyacrylamide gel electrophoresis (PAGE) was
performed on a 32 cm vertical slab gel unit PROTEAN II equipped
with Model 1000 power source from Bio-Rad IRichmond, Calif.).
Polyacrylamide linear gradient resolving gels (14.times.28 cm),
12-22%) total acrylamide) were prepared and run as previously
described (Edens et al., 1992, J. Pharm. Sci. 81, 823-827). The
molecular sizes of the oligosaccharide samples were determined by
comparing with a banding ladder of heparin oligosaccharide
standards prepared from bovine lung heparin. Oligosaccharides were
visualized by Alcian blue staining. The average MW of the product
was determined to be 6,000.
Anti-Factor Xa and Anti-Factor IIa Activities
[0111] LMW heparin and heparin standard were in diluted normal
human plasma. Chromogenic Xa substract S-2732
(Suc-IIc-Glu(gamma-piperidyl)-Gly-Arg-pNA) 2.9 MM in 50 mM Tris,
7.5 .mu.M EDTA, pH 8.4 buffer (200 .mu.L), was added to 25 .mu.L of
plasma containing sample and 200 .mu.L of bovine Factor Xa
(1.25/mL). After mixing, the reaction was incubated for 8 min. at
37 degrees Celsius and 200 .mu.L of 20% aqueous acetic was added.
Residual Factor Xa was then determined by measuring absorbance at
405 nm. Anti-factor IIa activity was determined by incubating 50 mL
of LMW heparin in NHP diluted 4-fold with water with 50 mL of human
thrombin (12 NIH units/mL) at 37.degree. C. for 30 s. then 50 mL of
(2.5 mmol/mL of Chromogenic TH (ethylmalonyl-Pro-Aeg-p-nitroanilide
hydrochloride) was added, and the amidolytic thrombin activity was
measured at 405 nm. Measurements were performed on an ACL 300 plus
from Instrumentation (Lexington, Mass.) and calculated in
comparison with USP Heparin Reference Standard (K-3) supplied by
U.S. Pharmacopeial Convention (Rockville, Md.). The product
exhibited no measurable anti-factor Xa or anti-factor IIa
activity.
O-Acylated LMW Heparin Derivatives
[0112] (1) O-Hexanoyl derivative of periodate-oxidized heparin
fragments. These were obtained by treating the tributylaminmonium
salt of periodate oxidized heparin fragments with hexanoic
anhydride as described previously (Gohda et al., 2001,
Biomacromolecules, 2(4):1178-83) (Lormeau U.S. Pat. No. 4,990,502).
Briefly, the tributylammonium salt (11.9 g) was dissolved in dry
DMF (114 mL), kept under Ar and cooled to 0 degrees Celsius.
4-Dimethylaminopyridine (0.695 g, 5.69 mmol), hexanoic anhydride
(26.2 mL, 0.113 mol), and tributylamine (227 mL, 0.113 mol) were
successively added in single portions, and the reaction was allowed
to proceed under argon at room temperature for 24 hours. After
cooling to 0.degree. C., 5% NaHCO3 in water (227 mL) was gradually
added and the solution was stirred at room temperature for 48 h.
Excess NaHCO3 was eliminated by slow, dropwise addition of 1 N HCL
(.about.200 mL) until pH4 was reached and then readjusted to pH 7
with 1 N NaOH (.about.150 mL). Cold denatured (95%) ethanol (5 L, 5
vol) was added with stirring. The sample was allowed to sit
overnight at 4 degrees Celsius to afford precipitate. The
precipitate was recovered by decanting and dissolved in 0.2 M NaCl
(114 mL), and the precipitation procedure was repeated by adding
absolute ethanol (570 mL). The precipitate was recovered by
centrifugation at 15000 rpm for 20 minutes, dissolved in water (114
mL), and passed through a column (300 mL) of Dowez 50WX8(H.sup.+)
cation-exchange resine and 600 ml was recovered. The acid was
neutralized to pH 7 with 10 N NaOH and the solution was filtered
through a 0.22 .mu.m Millipore filter. After lyophilization,
O-hexanoyl heparin oligosaccharides (7.12 g) was obtained as an
off-white powder (FIG. 3).
O-Butanoylated LMW Heparin
[0113] This derivative was prepared from the tributylaminmonium
salt of LMW heparin by treatment with butyric anhydride under the
same condition as described for hexanoyl derivative (see
above).
Application of Heparin to Stent
[0114] O-acylated heparin derivatives can be coated on stents using
the methods set forth in U.S. Pat. No. 6,620,194. The method is
generally as follows.
[0115] The application of the coating material to the stent is
quite similar for all of the materials and the same for the heparin
and one or more additional suspensions prepared as in the above
Examples. The suspension to be applied is transferred to an
application device, typically a paint jar attached to an air brush,
such as a Badger Model 150, supplied with a source of pressurized
air through a regulator (Norgren, 0-160 psi). Once the brush hose
is attached to the source of compressed air downstream of the
regulator, the air is applied. The pressure is adjusted to
approximately 15-25 psi and the nozzle condition checked by
depressing the trigger.
[0116] Any appropriate method can be used to secure the stent for
spraying and rotating fixtures. Both ends of the relaxed stent can
be fastened to the fixture by two resilient retainers, commonly
alligator clips, with the distance between the clips adjusted so
that the stent remains in a relaxed, unstretched condition. The
rotor is then energized and the spin speed adjusted to the desired
coating speed, nominally about 40 rpm.
[0117] With the stent rotating in a substantially horizontal plane,
the spray nozzle is adjusted so that the distance from the nozzle
to the stent is about 2-4 inches and the composition is sprayed
substantially horizontally with the brush being directed along the
stent from the distal end of the stent to the proximal end and then
from the proximal end to the distal end in a sweeping motion at a
speed such that one spray cycle occurs in about three stent
rotations. Typically a pause of less than one minute, normally
about one-half minute, elapses between layers. Of course, the
number of coating layers will vary with the particular application.
For example, for a coating level of 3-4 mg of heparin per cm.sup.2
of projected area, 20 cycles of coating application should be
required and about 30 ml of solution will be consumed for a 3.5 mm
diameter by 14.5 cm long stent.
[0118] The rotation speed of the motor, of course, can be adjusted
as can the viscosity of the composition and the flow rate of the
spray nozzle as desired to modify the layered structure. Generally,
with the above mixes, the best results will be obtained at
rotational speeds in the range of 30-50 rpm and with a spray nozzle
flow rate in the range of 4-10 ml of coating composition per
minute, depending on the stent size. It is contemplated that a more
sophisticated, computer-controlled coating apparatus will
successfully automate the process demonstrated as feasible in the
laboratory.
[0119] The coated stent can be thereafter subjected to a curing
step in which the pre-polymer and crosslinking agents cooperate to
produce a cured polymer matrix containing the biologically active
species. The curing process involves evaporation of the solvent
xylene, THF, etc. and the curing and crosslinking of the polymer.
Certain silicone materials can be cured at relatively low
temperatures, (i.e. RT-50.degree. C.) in what is known as a room
temperature vulcanization (RTV) process. More typically, however,
the curing process involves higher temperature curing materials and
the coated stents are put into an oven at approximately 90.degree.
C. or higher for approximately 16 hours. The temperature may be
raised to as high as 150.degree. C. for dexamethasone containing
coated stents. Of course, the time and temperature may vary with
particular silicones, crosslinkers, and biologically active
species.
[0120] Stents coated and cured in the manner described need to be
sterilized prior to packaging for future implantation. For
sterilization, gamma radiation is a preferred method particularly
for heparin containing coatings; however, it is possible that
stents coated and cured according to the process of the invention
subjected to gamma sterilization may be too slow to recover their
original posture when delivered to a vascular or other lumen site
using a catheter unless a pretreatment step as at 24 is first
applied to the coated, cured stent.
[0121] The pretreatment step can involve an argon plasma treatment
of the coated, cured stent in the unconstrained configuration. In
accordance with this procedure, the stents are placed in a chamber
of a plasma surface treatment system such as a Plasma Science 350
(Himont/Plasma Science, Foster City, Calif.). The system is
equipped with a reactor chamber and RF solid-state generator
operating at 13.56 mHz and from 0-500 watts power output and being
equipped with a microprocessor controlled system and a complete
vacuum pump package. The reaction chamber contains an unimpeded
work volume of 16.75 inches (42.55 cm) by 13.5 inches (34.3 cm) by
17.5 inches (44.45 cm) in depth.
[0122] In the plasma process, unconstrained coated stents are
placed in a reactor chamber and the system is purged with nitrogen
and a vacuum applied to 20-50 mTorr. Thereafter, inert gas (argon,
helium or mixture of them) is admitted to the reaction chamber for
the plasma treatment. A highly preferred method of operation
consists of using argon gas, operating at a power range from 200 to
400 watts, a flow rate of 150-650 standard ml per minute, which is
equivalent to 100-450 mTorr, and an exposure time from 30 seconds
to about 5 minutes. The stents can be removed immediately after the
plasma treatment or remain in the argon atmosphere for an
additional period of time, typically five minutes.
[0123] After this, the stents can be exposed to gamma sterilization
at 2.5-3.5 Mrad. The radiation may be carried out with the stent in
either the radially non-constrained status--or in the radially
constrained status.
[0124] With respect to the anticoagulant material heparin, the
percentage in the tie layer is nominally from about 20-50% and that
of the top layer from about 0-30% active material. The coating
thickness ratio of the top layer to the tie layer varies from about
1:10 to 1:2 and is preferably in the range of from about 1:6 to
1:3.
[0125] Suppressing the burst effect also enables a reduction in the
drug loading or in other words, allows a reduction in the coating
thickness, since the physician will give a bolus injection of
antiplatelet/anticoagulation drugs to the patient during the
stenting process. As a result, the drug imbedded in the stent can
be fully used without waste. Tailoring the first day release, but
maximizing second day and third day release at the thinnest
possible coating configuration will reduce the acute or subacute
thrombosis.
RESULTS
[0126] Effect on smooth muscle cell proliferation in vitro and in
vivo. Hexanoylated LMW heparin significantly inhibited pulmonary
artery smooth muscle cell proliferation in vivo (FIG. 4) and the
development of pulmonary hypertension induced by hypoxia in pig
lung (FIG. 5)
[0127] In comparison to non-acylated heparin fragments,
hexanoylated LMW heparin significantly enhanced the
antiproliferative effect of bovine pulmonary artery smooth muscle
cells in vitro.
[0128] Effect of O-acylation of heparin on tumor growth in vivo. As
seen in FIGS. 6-10, butanoylated heparin significantly inhibited
the growth of both A549 non-small cell lung carcinoma and DMS79
small cell lung carcinoma in SCID mice (FIG. 6-10). In addition,
FIGS. 11 and 12 demonstrate that butanoylated heparin significantly
inhibited the growth of HCT116 colonic carcinoma in SCID mice.
[0129] FIGS. 13 and 14 demonstrate that the above butanoylated
heparin compounds exhibit very low anticoagulant effects (compared
to non-acylated controls). Butanoylated heparin had no toxic effect
on heart, liver, kidney, and lung of the animals tested (FIG.
15-18). Furthermore, the anti-tumor effect of butanoylated heparin
is associated with the induction of apoptosis (FIG. 19). The
mechanism by which butanoylated heparin inhibits tumor growth of
lung cancer and colon cancer may involve p27- and p21-RB-E2F
pathway (FIG. 20-22). Similar antiproliferative effects were seen
with O-hexanoylated LMW heparin on anti-tumor cell growth in
vitro.
TABLE-US-00001 TABLE 1 Assignment of selected signals in the
.sup.1H NMR spectrum of the O-hexanoyl heparin derivative. Chemical
(ppm) shift Residue H-1 H-2 H-3 H-4 H-5 GlcNS6S 5.302 3.093 3.539
3.629 3.892 IdoA2S 5.092 4.218 4.008 3.920 4.709
* * * * *