U.S. patent application number 16/297006 was filed with the patent office on 2019-07-04 for apparatus and methods for preventing or treating failure of hemodialysis vascular access and other vascular grafts.
The applicant listed for this patent is Vascular Therapies, Inc.. Invention is credited to Sriram S. Iyer, Nicholas N. Kipshidze, Victor V. Nikolaychik.
Application Number | 20190201383 16/297006 |
Document ID | / |
Family ID | 22996277 |
Filed Date | 2019-07-04 |
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United States Patent
Application |
20190201383 |
Kind Code |
A1 |
Iyer; Sriram S. ; et
al. |
July 4, 2019 |
Apparatus and Methods for Preventing or Treating Failure of
Hemodialysis Vascular Access and Other Vascular Grafts
Abstract
This invention is a prosthetic device generally placed on the
outside surface of the vessel or graft which then elutes
antiproliferative drugs or agents from a drug-eluting matrix
material. Methods of perivascular antiproliferative drug
administration also are disclosed.
Inventors: |
Iyer; Sriram S.; (New York,
NY) ; Kipshidze; Nicholas N.; (New York, NY) ;
Nikolaychik; Victor V.; (Mequon, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vascular Therapies, Inc. |
Cresskill |
NJ |
US |
|
|
Family ID: |
22996277 |
Appl. No.: |
16/297006 |
Filed: |
March 8, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14509279 |
Oct 8, 2014 |
10272073 |
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16297006 |
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11931143 |
Oct 31, 2007 |
8858982 |
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14509279 |
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10832048 |
Apr 26, 2004 |
7807191 |
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11931143 |
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10051708 |
Jan 16, 2002 |
6726923 |
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10832048 |
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60262132 |
Jan 16, 2001 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 7/02 20180101; A61M
39/0247 20130101; A61L 2300/416 20130101; A61K 31/337 20130101;
A61K 31/727 20130101; A61L 27/44 20130101; A61M 2202/0468 20130101;
A61L 27/225 20130101; A61K 31/122 20130101; A61P 9/00 20180101;
A61L 27/20 20130101; A61P 37/06 20180101; A61K 31/573 20130101;
A61M 1/16 20130101; A61M 2202/0478 20130101; A61K 31/395 20130101;
A61L 2300/252 20130101; A61P 9/10 20180101; A61M 2039/0261
20130101; A61K 9/0024 20130101; A61M 2039/0258 20130101; A61P 31/00
20180101; A61K 31/436 20130101; A61L 2300/204 20130101; A61P 41/00
20180101; A61P 7/08 20180101; A61M 1/3655 20130101; A61P 43/00
20180101; A61L 27/54 20130101; A61L 31/16 20130101 |
International
Class: |
A61K 31/436 20060101
A61K031/436; A61M 39/02 20060101 A61M039/02; A61K 31/122 20060101
A61K031/122; A61K 31/337 20060101 A61K031/337; A61K 31/395 20060101
A61K031/395; A61K 31/573 20060101 A61K031/573; A61K 31/727 20060101
A61K031/727; A61L 31/16 20060101 A61L031/16; A61M 1/36 20060101
A61M001/36; A61L 27/20 20060101 A61L027/20; A61L 27/22 20060101
A61L027/22; A61L 27/44 20060101 A61L027/44; A61L 27/54 20060101
A61L027/54; A61K 9/00 20060101 A61K009/00 |
Claims
1. A method of preventing or treating vasculoproliferative disease
in vascular structures, which comprises the step of: administering
extravascularly and locally an antiproliferative effective amount
of an antiproliferative agent to the vascular structure.
2. A method according to claim 1 wherein the agent comprises
rapamycin.
3. A method according to claim 1 wherein the antiproliferative
agent is administered perivascularly.
4. A method according to claim 1 wherein extravascular, local
administration is accomplished by means of an implantable,
antiproliferative agent eluting, perivascular vascular sleeve, the
sleeve comprising a matrix material imbibed with the agent.
5. A method according to claim 4 wherein the sleeve is
substantially circumvascular.
6. A method according to claim 4 wherein the matrix material
comprises fibrin.
7. A method according to claim 4 wherein the agent comprises
rapamycin and heparin
8. A method according to claim 4 wherein the matrix material
comprises collagen.
9. A method according to claim 4 wherein the matrix material
comprises chitosan.
10. An implantable, antiproliferative agent-administering
perivascular sleeve adapted to be placed in contact with the
exterior of a vascular structure comprising: a) A flexible,
bioabsorbing, agent-eluting matrix material, the material having a
vascular-sized lumen passing substantially through said matrix
material, the matrix material having dispersed therein: b) an
antiproliferative agent.
Description
CROSS-REFERENCED TO RELATED APPLICATIONS
[0001] This application is a continuation patent application of
U.S. patent application Ser. No. 14/509,279 filed Oct. 8, 2014,
which is a divisional patent application of U.S. patent application
Ser. No. 11/931,143 filed Oct. 31, 2007, now U.S. Pat. No.
8,858,982, which is a continuation patent application of U.S.
patent application Ser. No. 10/832,048 filed Apr. 26, 2004, now
U.S. Pat. No.7,807,191, which is a continuation patent application
of U.S. patent application Ser. No. 10/051,708, filed Jan. 16,
2002, now U.S. Pat. No. 6,726,923, which claims priority to U.S.
Provisional Patent Application Ser. No. 60/262,132, filed Jan. 16,
2001, each of which are incorporated herein by reference in their
entireties.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not Applicable
BACKGROUND OF THE INVENTION
[0003] Failure of hemodialysis vascular access and other vascular
grafts becomes evident as compromise of the lumen of the native
vessel (vein or artery) or of the prosthetic conduit at o or away
from the anastamotic site. Compromise of the lumen manifests as
either stenosis or occlusion and is a result of either intraluminal
thrombus and/or a vasculoproliferative response. The etiology of
graft failures may be related to a variety of physical (e.g., shear
stress causing hemodynamic disturbance), chemical and/or biological
stimuli as well as infection and foreign body rejection which may
explain why fistulae which do not involve a foreign body (in this
case, for example, polytetrafluroethylene, PTFE) remain patent for
a longer time compared to vascular access grafts that involve
interposition of a PTFE graft.
[0004] The present invention relates generally to therapeutic
implant, apparatus and methods useful for preventing, suppressing
(inhibiting) or treating failure of hemodialysis vascular access
and other vascular grafts.
[0005] Vascular access grafts, specifically, hemodialysis access
grafts are well known to the art. Approximately 100,000 vascular
access procedures are performed yearly in the United States.
Hemodialysis vascular access can be constructed in one of several
ways: as an arterio-venous fistula (e.g.; Brecisa-Cimino), or as a
graft, interposing either prosthetic (e.g., PTFE) or biologic
tissue (e.g., vein) between the artery and the vein. Such grafts
are usually constructed using a tubular or cylindrical segment of
suitably bio-compatible, substantially inert material such as
polytetrafluoroethylene (PTFE). In fact, PTFE is the most common
material used for prosthetic dialysis access. In one approach, a
segment of PTFE is surgically interposed between an artery and a
vein in the arm, forearm or thigh. The graft is then available for
repeated vascular access for performing hemodialysis.
[0006] Subsequent to placement of the access graft the sutured
sites in the artery and the vein undergo healing. Sixty percent of
these grafts fail each year, usually because of narrowing
(stenosis) at the venous end. Similar lesions develop in PTFE
grafts placed in the arterial circulation, where there is a similar
tendency for the distal end of the graft to be affected.
Dysfunction or failure of veing grafts and/or other graft conduits
used in coronary artery bypass graft surgery or in peripheral
vascular surgery (e.g., aorta-iliac, femoral-femoral,
femoral-popliteal, femoral tibial, etc.) are well known.
Development of arterial access graft stenosis is not as rapid as
development of access graft stenosis at the venous end.
Proliferation and migration of smooth muscle cells resulting in
intimal hyperplasia in the vein and the adjacent graft orifice has
been described in human dialysis access stenosis. As the stenosis
in the graft becomes progressively more severe, the graft becomes
dysfunctional and hemodialysis is suboptimal. If the stenosis in
the graft is not treated, it eventually leads to occlusion and to
graft failure.
[0007] The reasons why the venous ends of access graft have such a
marked propensity for narrowing are multifactorial. Features unique
to this location include exposure to arterial pressures and
arterial flow rates, dissipation of acoustic (vibratory) energy in
the vessel wall and surrounding tissue, repeated puncture of the
graft, and infusion of processed blood. In addition, the venous end
of the graft may be bathed in mitogens released during passage of
the blood through the dialysis tubing or during activation of
platelets at the site of needle puncture.
[0008] Tissue samples collected from the graft-vein anastomosis
site of stenotic PTFE grafts during surgical revision showed
significant narrowing of the lumen and were characterized by the
(i) presence of smooth muscle cells, (ii) accumulation of
extra-cellular matrix, (iii) angiogenesis within the neointima and
adventitia, and (iv) presence of an active macrophage cell layer
lining the PTFE graft material. A large variety of cytokines and
cell growth stimulating factors like platelet-derived growth factor
(PDGF), basic fibroblast growth factor (bFGF), and vascular
endothelial growth factor (VEGF) were expressed by smooth muscle
cells/myofibroblasts within the venous neointima, by macrophages
lining both sides of the PTFE graft, and by vessels within the
neointima and adventitia. It has been suggested that macrophages,
specific cytokines (bFGF, PDGF, and VEGF), and angiogenesis within
the neointima and adventitia are likely to contribute to the
pathogenesis of venous neointimal hyperplasia (VNH) a manifestation
of the vasculoproliferative response in PTFE dialysis grafts.
[0009] Survival of patients with chronic renal failure depends on
optimal regular performance of dialysis. If this is not possible
(for example as a result of vascular access dysfunction or
failure), it leads to rapid clinical deterioration and unless the
situation is remedied, these patients will die. Vascular access
dysfunction is the most important cause of morbidity and
hospitalization in the hemodialysis population in the United States
at an estimated cost of approximately one billion US dollars per
annum. Venous neointimal hyperplasia characterized by stenosis and
subsequent thrombosis accounts for the overwhelming majority of
pathology resulting in PTFE dialysis graft failure. Despite the
magnitude of the problem and the enormity of the cost, there are
currently no effective therapies for the prevention or treatment of
venous neointimal hyperplasia in PTFE dialysis grafts.
Consequently, interventions aimed at the specific mediators and
processes may be successful in reducing the very significant human
and economic costs of vascular access dysfunction.
[0010] Once the stenosis has occurred, one of the current methods
of treatment involves reduction or obliteration of the narrowing
and restoration of blood flow through the graft (permitting the
performance of adequate hemodialysis) by means of non-surgical,
percutaneous catheter based treatments such as balloon angioplasty.
Balloon angioplasty, in one aspect, involves deployment of a
balloon catheter at the site of the blockage and inflating the
balloon to increase the minimum luminal diameter (MLD) of the
vessel by compressing the material causing the restriction against
the interior of the vessel wall, thereby dilating the vessel.
Depending upon the length and severity of the restriction, the
procedure may be repeated several times (by inflating and deflating
the balloon). When completed, the balloon catheter is withdrawn
from the system.
[0011] Although balloon angioplasty can be used as a "stand alone"
procedure, it is frequently accompanied by deployment of what is
called a stent. A stent is an expandable scaffolding or support
device which is placed within the vasculature to prevent mechanical
recoil and reduce the chance of renarrowing (restenosis) at the
site of the original restriction. Stents are either
"balloon-expandable" or "self-expanding" and when deployed
endovascularly, abut against the inner vessel wall. Whether or not
a stent is placed, this form of treatment has a high risk of
failure i.e., the risk of renarrowing (restenosis) at the treatment
site is very high. Unless stenosis within the access graft can be
effectively and permanently treated, graft failure tends to follow.
In the event of graft failure, the patient has to undergo an
endovascular procedure i.e., a non-surgical, catheter-based
percutaneous procedure, repeat vascular surgery e.g., thrombectomy
to "declot" the graft or to place another vascular access graft or
a shunt (as it is sometimes referred to) at a different site,
unless the patient receives a kidney transplant. Given the obvious
problems of repeat surgery(ies) and the limited availability of
transplants, there is a need for a treatment that is both effective
and long lasting (durable) in the prevention and treatment of
dialysis graft stenosis.
[0012] The vast majority of current approaches for reducing or
preventing the vasculoproliferative response (believed to be the
pathophysiological basis of restenosis), are based on treatment
options that originate from within the vascular or graft lumen. One
current, novel approach utilizes drug coated or drug impregnated
stents which are then deployed within the lumen of the blood
vessel. Examples of drugs used to coat stents include Rapamycin
commercially available from the Wyeth Ayerst company
(Sirolimus.RTM.), and Paclitaxel commercially available from the
Bristol-Myers Squibb Company (Taxol.RTM.). In this stent-based
approach, Rapamycin or Paclitaxel is gradually eluted from the
stent and diffuses into the vessel wall from the intima (the
innermost layer of the vessel wall) to the adventitia (the
outermost layer of the vessel wall). Studies have shown that
Rapamycin and Paclitaxel tend to inhibit smooth muscle cell
proliferation.
[0013] Delivery from the perivascular or extravascular space
through the arterial or vascular wall utilizing a synthetic matrix
material (ethylene-vinyl acetate copolymer, EVA) together with an
anticoagulant that also has antiproliferative properties e.g.,
heparin, has been suggested. There are two disadvantages of this
approach: heparin is a soluble substance and rapidly disappears
from the vascular wall and, ethylene-vinyl acetate copolymer is not
biodegradable potentially raising concerns about long term effects,
in vivo.
[0014] If a therapeutic agent is delivered locally using a matrix
material-based system, the matrix material should preferably have
the following characteristics:
[0015] 1. The matrix material has to permit the loading of adequate
quantity of the therapeutic agent.
[0016] 2. The matrix material must elute the therapeutic agent at
an appropriate, well defined rate.
[0017] 3. The matrix material should preferably be implantable and
biodegradable. Thus, physical removal of the matrix material from
recipient's tissue following drug delivery would not be necessary
and obviates concerns about the long term effects of the residual
matrix.
[0018] 4. Neither matrix material nor its biodegradation products
should provoke a significant inflammatory or proliferative tissue
response, nor should they alter or interfere with the recipient's
natural defense systems or healing.
[0019] 5. The device (comprising the matrix material and the drug)
should be flexible enough to mould to the contours of the
vasculature and
[0020] 6. The device should be amenable to be fixed in place
preventing its migration to an unintended location.
[0021] Polymer matrix materials used for drug delivery within the
context of implantable devices can be either natural or synthetic.
Examples include but are not limited to polymers composed of
chemical substances like polyglycolic acid or polyhydroxybutyrate,
EVA or natural polymers like collagen, fibrin or polysaccharides
like chitosan. However, not all of these matrix materials are
ideal; inappropriate features include poor mechanical
characteristics, potential immunogenicity, and cost. In addition,
some may produce toxic degradation products and induce inflammatory
reactions or a proliferative response.
[0022] A well known biocompatible, biodegradable, resorbable matrix
material for drug delivery is collagen. The use of collagen as a
material for fabrication of biodegradable medical devices is and
has undergone serious scrutiny. U.S. Pat. Nos. 6,323,184,
6,206,931; 4,164,559; 4,409,332; 6,162,247. One current focus
involves delivery of pharmaceutical agents including antibiotics
and physiologically active proteins and peptides such as growth
factors.
[0023] Under scanning electron microscopy, the collagen matrix has
a morphology of condensed laminated film with a textured surface
and a range of pore sizes. It can be produced in a wide range of
effective pore sizes from 0.001 microns to 100 microns or even
larger. This internal pore network (porous material) creates a high
surface area and serves as a microreservoir for storage and
delivery of the therapeutic agent. Several features make collagen
an excellent and ideal matrix material for drug delivery. Collagen
exhibits a high degree of flexibility and mechanical durability, as
well as intrinsic water wettability, semipermeability and
consistent flow characteristics. More importantly, collagen, a
naturally occurring substance is biodegradable and non-toxic. In
addition, collagen has favorable biodegradation characteristics and
time to complete degradation or resorption i.e., durability of the
collagen matrix for drug delivery can be modified.
[0024] A second protein matrix suitable for drug delivery is
fibrin. A fibrin matrix is comprised of cross-linked fibrin units
that are a reticular network of thrombin-modified fibrinogen
molecules. This matrix is similar to a natural blood clot. In
contrast to natural clot, the size of pores in a fibrin matrix can
be controlled and varies from 0.001 millimicrons to 0.004
millimicrons, so-called micropores. The differences in pore sizes
between collagen and fibrin matrices permit the binding of
therapeutic agents with distinct rates of drug release. The ability
to control bleeding, to remain firmly fixed in place, and to be
naturally biodegradable have all made fibrin a good matrix material
for drug delivery and confers fibrin some advantages over synthetic
matrices. Most of the early applications of fibrin as a matrix were
for delivery of antibiotics and other biologics.
[0025] The fibrin matrices are prepared in a dry granular form.
(cf., PCT/EP99/08128). This formulation, manufactured by HyQ
Solvelopment, Buhlmhle, Germany, contains D-mannitol, D-Sorbit,
fibrinogen-aqueous solution, and a thrombin-organic suspension. The
formulation is manufactured by fluid bed granulation. The
applications for dry fibrin are manifold: wound closure, promotion
of healing, and homeostasis. However, application for drug delivery
is limited since such a formulation does not allow for a
target-oriented shaping of solid particles around the vessel wall
and delivery of exact dosages is difficult. Porosity and capacity
of dry fibrin particles are low, physical stability is poor.
[0026] Another group of potentially useful resorbable, natural
polymer matrix material is chitosan. Chitosan has proven to be a
useful biocompatible aminopolysaccharide and a matrix for
controlled release of the agent for local delivery. Chitosan
implants cause no systemic and local side effects or immunologic
responses, and are suitably biodegradable. Chitosan can be prepared
from the degradation of slow chitin (molecular weight
1.times.10.sup.6) using high temperature sodium hydroxide
hydrolysis to a molecular weight of 5.times.10.sup.5. The inability
to control porosity is a disadvantage of this matrix material.
BRIEF DESCRIPTION OF THE PRESENT INVENTION
[0027] The present invention is unique in at least two respects: 1)
Whereas the majority of current methods of preventing suppressing
or treating the vasculoproliferative response (smooth muscle cell
hyperplasia, restenosis, vascular occlusion) do so from inside the
vascular (i.e., vein and/or artery) or graft lumen, the present
invention is a method of doing so extravascularly or perivascularly
i.e., from outside the vascular or graft lumen and through the
vascular wall. 2) All current treatment approaches are relevant
only after the narrowing or stenosis has actually taken place. The
current invention is, in one aspect, a method of preventing or
suppressing vasculoproliferative disease, in contradistinction to
curing it.
[0028] In a further embodiment, the present invention is an
implantable prosthetic device placed on the outer surface of the
vessel or graft which then elutes anti-vasculoproliferative drugs
or agents such as Rapamycin, Paclitaxel, Tacrolimus, and other cell
cycle inhibitor or similarly-functioning agents. In addition to a
resorbable matrix material, e.g., protein, and an antiproliferative
agent, this implantable device contains optionally, agents that
inhibit collagen accumulation in the tunica media and adventitia of
the vascular wall and pharmaceuticals that help reduce
calcification of the vascular wall. This invention provides a
method of preventing or treating neo intimal hyperplasia (an
expression of the vasculoproliferative response) and calcification
by extravascular delivery of an effective amount of an
antiproliferative agent with low water solubility alone or in
combination with adjuvants, and other antiproliferative agents.
Rapamycin is a particularly preferred drug with antiproliferative
properties for use with the present invention. A mixture of
suitable drugs may be used. The Rapamycin diffuses from the outside
and through the vessel and/or graft wall to the interior of the
vein and/or artery and/or graft. Elution of Rapamycin (and other
drugs with antiproliferative effect), into and through the vascular
wall from the outside starts soon after the device is implanted and
the drug will inhibit smooth muscle cell proliferation within the
hemodialysis and other vascular grafts and/or at their anastamotic
sites. Thus, in one aspect, the present invention is a method of
inhibiting smooth muscle cell proliferation of a vascular access
graft or shunt by the gradual elution or timed release of a drug
from outside the vascular access site vessel wall to the vessel
interior i.e., by extravascular or perivascular delivery.
[0029] In another aspect the present invention is a prosthetic
device comprising a cylindrical, antiproliferative-imbibed, protein
interior layer and, optionally, an exterior support or skeletal
structure or layer. In one embodiment, the imbibed protein layer is
collagen and the exterior skeletal support structure is a sheet of
PTFE. The antiproliferative drug, in this embodiment, is preferably
Rapamycin. Paclitaxel (or Taxol) is another antiproliferative drug
or agent well-suited to the embodiment of the invention.
[0030] A third embodiment of the present invention is a method of
inhibiting stenosis of hemodialysis access graft comprising the
method of placing a prosthetic device (described above) over a
graft or vascular structure and/or at the site of anastomosis and
anchoring the prosthetic device at the desired site (e.g., by
suturing).
[0031] A device of this invention may employ a biocompatible matrix
material such as collagen, fibrin or chitosan. An important factor
in the selection of a particular matrix material is the porosity of
the material and a controllable rate of biodegradation. Use of a
matrix material is important because it creates a delivery
reservoir and controls the agent delivery kinetics.
[0032] A preferred device of this invention comprises a collagen
matrix material imbibed with Rapamycin, which will be placed in
position so as to extravascularly deliver the agent.
[0033] In a preferred embodiment, about 120 micrograms/cm.sup.2 of
Rapamycin (Range: 50 micrograms to 10 mg/cm.sup.2) is combined with
a collagen matrix material sheet with a thickness in the dry state
between 0.3 and 2.0 mm sheet which is then implanted or wrapped
upon the outside of the vascular or graft wall.
[0034] A further aspect of the present invention is "self fixation"
of the device delivering the drug or agent to the outer surface of
the vascular or graft wall. The collagen-device could be made more
adhesive to the vascular wall if in the final stage collagen is
combined with photoreactive groups such as FITS (fluorescein
isothiocyanate) or Bengal Rose both from Sigma Chemicals, St Louis,
Mo. Stimulation of the device with ultra violet light will activate
these photoreactive groups and will increase adhesion. Fibrin
sealant and acetylated collagen also have been found to increase
adhesion of collagen matrix material to the outside vascular
wall.
[0035] Early work showed a relationship between local vessel trauma
and expedited calcification. Recently, a study in humans has shown
that the matrix Gla-protein (protein .gamma.-carboxylated vitamin
K-dependent .gamma.-carboxylase) is constitutively expressed by
normal vascular smooth muscle cells and bone cells. High levels of
Gla-protein mRNA and non-.gamma.-carboxylated protein were found in
atherosclerotic vessel tissues. This .gamma.-carboxylated protein
is necessary to prevent or postpone beginning of vascular
calcification (Price, P. et al., "Warfarin causes rapid
calcification of the elastic lamellae in rat arteries and heart
valves," Atheroscler Thromb Vasc Biol, (1998) 18: 1400-1407). These
data indicate that calcification caused by injury must be actively
inhibited. Introduction of pharmaceuticals preventing calcium
accumulation helps to postpone calcification and helps prevent,
suppress or treat the vasculoproliferative processes. In one aspect
of this invention, local delivery of Vitamin K counteracts the
calcification effect associated with vessel injury by timely
activation of .gamma.-carboxylase (in this case Gla-protein) and
ensures other calcium-binding proteins function properly and do not
bind excess of calcium (Hermann, S. M. et al., "Polymorphisms of
the human matrix Gla-protein gene (MGP) vascular calcification and
myocardial infarction," Arterioscler Thromb Vasc Biol. (2000)
20:2836-2893. A mixture of Vitamin K and other anti-proliferative
drugs may be used.
[0036] The acute response, characterized by an inflammatory
reaction, is an attempt to limit disturbances in the homeostasis.
Hallmarks of this inflammatory reaction include leukocyte
accumulation, increased fibrin deposition and release of cytokines.
Addition of synthetic glucocorticoids like dexamethasone decreases
this inflammatory response and may eventually decrease the
vasculoproliferative process. Since the pharmacological mechanisms
of action of the antiproliferative agents and synthetic
glucocorticoids are different, agents with different "mechanisms of
action" may be expected to act synergistically. It may be useful,
therefore, to combine two or more of these agents.
[0037] This invention thus provides a method of preventing,
suppressing, or treating neointimal hyperplasia by extravascular,
(e.g., perivascular) local delivery of an effective amount of an
anti-vasculoproliferative agent with low water solubility (e.g.,
Rapamycin) alone or in combination with other antiproliferative
agents and adjuvants.
[0038] In one aspect, the present invention is a prosthetic device
that consists of a resorbable protein matrix combined with a drug,
placed on the outer surface of a blood vessel or graft. The device
then elutes the drug which inhibits smooth muscle cell
proliferation (anti-vasculoproliferative). Examples of such drugs
include Rapamycin, Paclitaxel, Tacrolimus, other cell cycle
inhibitors or similarly-functioning agents. A mixture of suitable
drugs and/or additives may be used. In addition to a resorbable
protein matrix and an antiproliferative agent, this implantable
device contains optionally, agents that inhibit collagen
accumulation in the vascular wall and pharmaceuticals that help
reduce calcification of the vascular wall.
[0039] Rapamycin is a particularly preferred drug for use with the
present invention. The Rapamycin [or other drug(s)] elutes from the
outside and diffuses through the vessel and/or graft wall to the
interior of the vein and/or artery and/or graft. Elution of
Rapamycin (or a similarly acting drug or a drug having similar
properties), into and through the vascular wall from the outside
takes place during the healing phase of the anastamotic sites and
the drug will prevent suppress/inhibit or treat smooth muscle cell
proliferation that accompanies such healing. Thus, in one aspect,
the present invention is a method of inhibiting the
vasculoproliferative response at the anastamotic ends of a vascular
access graft or shunt by the gradual elution or timed release of a
drug from outside to the vessel interior i.e., by transvascular
delivery using an extravascular source.
[0040] In another aspect the present invention is a prosthetic
device comprising a antiproliferative-imbibed, protein interior
layer and, optionally, an exterior support or skeletal structure or
layer. In one embodiment, the imbibed protein layer is collagen and
the exterior skeletal support material structure is a sheet of
PTFE. The antiproliferative drug, in that embodiment, is preferably
Rapamycin, or other similarly-functioning drugs.
[0041] Another embodiment of the present invention is a method of
inhibiting stenosis of hemodialysis access graft comprising the
method of placing the prosthetic device (described above) over a
graft or vascular structure and/or at the site of anastomosis and
anchoring the prosthetic device at the desired site (e.g., by
suturing).
BRIEF DESCRIPTION OF FIGURES
[0042] FIGS. 1A, 1B, 2A, and 2B illustrate preferred embodiments of
the present invention.
[0043] FIGS. 2A and 2B illustrate another embodiment of the present
invention in which an exterior support or skeletal structure are
employed.
[0044] FIGS. 3A-3C illustrate a self-interlocking embodiment of
this invention.
[0045] FIGS. 4A and 4B illustrate a second interlocking embodiment
of the present invention.
[0046] FIG. 5 Shows the basic device shown in FIGS. 1A-1B/2A-2B
include an exterior wire support or framework, which assists
retention of sleeve shape.
[0047] FIGS. 6-13 Illustrate various possible deployments of the
drug-eluting sleeve of the present invention in view of various
vessel reparative needs.
[0048] FIG. 14 Shows rates of release of collagen saturated with
tetracycline and rapamycin. Rapamycin was combined with a collagen
matrix material using four different formats. Numbers on y-axis
shows concentration of drug in micrograms per ml. Legend:
A=Collagen saturated with Tetracycline. B=Collagen Saturated with
Rapamycin. C=Rapamycin Dispersed throughout collagen. D=Collagen
conjugated with Rapamycin. E=Combination of dispersed and
conjugated forms of Rapamycin.
[0049] FIG. 15: Is a comparison of inhibition of growth of Smooth
Muscle Cells using collagen matrices combined with different
anti-proliferative agents. Numbers on .gamma.-axis denotes cell
numbers. Legend: A=Control; B=Collagen+Actinomycin D; and
C=Collagen+Rapamycin.
[0050] FIG. 16 Is a comparison of the effect of Rapamycin,
Tacrolimus and Paclitaxel (3 doses) on Human Smooth Muscle
Cells.
[0051] FIG. 17: Is a comparison of the effect of Rapamycin,
Tacrolimus and Paclitaxel (3 doses) on Human Endothelial Cells.
[0052] FIGS. 18A, 18B, 19A, 19B, and 20 illustrate some results
obtained using the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0053] In one aspect the present invention is a prosthetic device
adapted for extravascular drug or agent delivery comprising a drug
or agent-eluting matrix material combined with a drug(s) that can
prevent, suppress or treat vasculoproliferation.
[0054] Matrix Materials: Material for the matrix may be from
natural sources or may be synthetically manufactured or may be a
combination of the two. A device of this invention may employ a
biocompatible, biodegradable resorbable matrix material such as
collagen, fibrin or chitosan. A suitably biocompatible,
nonbiodegradable matrix may be also be used. Combination of
degradable and nonbiodegradable or two or more biodegradable
substances (e.g., collagen plus fibrin) or two or more
nonbiodegradable substances may be selected for the matrix
material. An important factor in the selection of a particular
matrix material is the porosity of the material and where
applicable, a controllable rate of biodegradation. The
characteristics of the matrix material is important because the
material creates a delivery depot or reservoir and control the
kinetics of agent delivery. The characteristics with respect to
thickness, porosity, rate of biodegradation etc. need not be
identical throughout the matrix. It is also conceivable that by
creating a polymer from the drug (for example, the
antiproliferative), the matrix and the drug are one and the same,
and, as the polymer degrades it releases the drug.
[0055] Collagen (Type I) is a preferred biocompatible biodegradable
resorbable material for the matrix of the drug eluting sleeve of
the present invention. The collagen source may be animal or human
or may be produced using recombinant DNA techniques. Other types of
collagen e.g., types II, III, V, XI singularly or in combination
with Type I may be used. Although collagen matrix in the form of a
sheet or membrane is the preferred embodiment of this invention,
other forms of collagen e.g., gel, fibrilla, sponge, tubular etc.,
may also be used. As is well known, the rate at which resorption of
the collagen occurs can be modified by cross-linking the
protein.
[0056] Therapeutic Agents: In order to prevent suppress or treat
the smooth muscle proliferative response that predominantly
contributes to the neointimal hyperplasia, therapeutic agents that
have significant antivasculoproliferative properties will be used
in this invention. It is to be understood that as presently
informed it is smooth muscle proliferation, which is believed to be
primarily responsible for the stenosis and luminal compromise
leading to graft failure. The present invention should not be
interpreted to require that failure mechanism for its operation.
Stated differently, applicants do not wish to be bound by any
theory of graft failure, which would tend to narrow the scope of
their invention. Examples of drugs with significant anti
proliferative effects include but are not limited to Rapamycin,
paclitaxel, other taxanes, tacrolimus, actinomycin D, angiopeptin,
vassenoids, flavoperidol, hormones such as estrogen, halofuginone,
matrix metalloprotienase inhibitors, ribosimes, interferons and
antisense compounds. Analogues of the parent compound e.g., those
of rapamycin, paclitaxel and tacrolimus may be used. Examples of
other therapeutic agents include anti-inflammatory compounds,
dexamethasone and other steroids, antiplatelet agents including
aspirin, clopidogrel, IIBIIIA antagonists, antithrombins,
anticoagulants including unfractionated and fractionated heparin,
statins, calcium channel blockers, protease inhibitors, alcohol,
botulin and genetic material. Vascular, bone marrow and stem cells
may also be used
[0057] These agents can be combined to the matrix singly or in
combination. Depending on the therapeutic agent, the agent can be
combined with the matrix using physical, chemical and/or biological
methods. A combination of techniques can be used. It will also be
appreciated that drug concentration need not be (and often will not
be) the same throughout the entire matrix.
[0058] It is to be understood that the process of elution of drug
from the matrix material (sleeve) to and through the vessel wall is
merely illustrative of one possible drug delivery process. For
example, a drug may be released by application of a stimulus or a
trigger e.g., light, temperature variation, pressure,
ultrasound-ionizing energy, electromagnetic or magnetic field.
Also, the drug may reside in the matrix as a pro-drug or in an
inactive form. Application of the stimulus referred to above
triggers conversion to the active form of the drug which is then
released. Illustrating this application, it is known that
Porphyrins and Psoralens are activated and may be released from a
matrix to which they are absorbed or bound, by application of
visible or ultraviolet light. Application of light modifies the
drug structure causing the association between the drug and the
protein reservoir or source to be disrupted. Thus, the drug is
released from its matrix or reservoir and elutes to and through the
vessel wall and into the vessel lumen in accordance with this
invention.
[0059] A device of this invention optionally includes agents that
accomplish other objectives e.g., that inhibit collagen
accumulation and help reduce calcification of the vascular wall.
Early work by Selye and colleagues showed a relationship between
local vessel trauma and expedited calcification. Recently, a study
in humans has shown that the matrix Gla-protein (protein
.gamma.-carboxylated vitamin K-dependent .gamma.-carboxylase) is
constitutively expressed by normal vascular smooth muscle cells and
bone cells. High levels of Gla-protein mRNA and
non-.gamma.-carboxylated protein were found in atherosclerotic
vessel tissues. This .gamma.-carboxylated protein is necessary to
prevent or postpone beginning of vascular calcification (Price P.
et al., "Warfarin causes rapid calcification of the elastic
lamellae in rat arteries and heart valves," Atheroscler Thromb.
Vasc. Biol. (1998); 18:1400-1407). These data indicate that
calcification caused by injury must be actively inhibited.
Introduction of pharmaceuticals preventing calcium accumulation
helps to postpone calcification and the restenotic processes. In
this invention, local delivery of Vitamin K counteracts the
calcification effect associated with vessel injury, by timely
activation of .gamma.-carboxylase (in this case Gla-protein) and
ensures other calcium-binding proteins function properly and do not
bind excess of calcium (Hermann S. M. et al., "Polymorphisms of the
human matrix Gla-protein gene (MGP) vascular calcification and
myocardial infarction," Arterioscler Thromb. Vasc. Biol. (2000);
20: 2836-93). A mixture of Vitamin K along with other
anti-proliferative drugs may be used.
[0060] The acute response to any injury, (in this instance,
surgical trauma) characterized by an inflammatory reaction, is an
attempt to limit disturbances in the homeostasis. Hallmarks of this
inflammatory reaction include leukocyte accumulation, increased
fibrin deposition and release of cytokines. Addition of synthetic
glucocorticoids like dexamethasone decreases this inflammatory
response and may eventually decrease the restenotic process. Since
the pharmacological mechanisms of action of the antiproliferative
agents and synthetic glucocorticoids are different, agents with
different "antirestenotic mechanisms" may be expected to act
synergistically. It may be useful, therefore, to combine two or
more of these agents.
[0061] Numerous other antiproliferative or anti-stenosis drugs and
other suitable therapeutics and adjuvants will likely occur to one
skilled in the art in light of the present disclosure.
[0062] Method of Making the Sleeve: In view of the above disclosure
several potential processes for making the prosthetic device and
for its application will occur to one skilled in the art.
[0063] Single or Uni Layer Device: In a preferred embodiment of
this invention, the protein matrix is a sheet or membrane of Type I
bovine collagen and the drug is Rapamycin. Collagen is a
particularly preferred example for the matrix because it has the
property of being biodegradable and reabsorbable. The durability of
the matrix reflects the time to complete reabsorption of the
collagen, the porosity influences the drug binding capacity of the
collagen matrix, both of these features can be controlled and
varied. As an example, a relatively flat sheet of collagen is
impregnated, absorbed, saturated, dispersed or immobilized with
Rapamycin. About 120 micrograms/cm.sup.2 (Range: 50 micrograms-2
milligrams/cm.sup.2) of Rapamycin is combined with the collagen
matrix material which in the dry form is in the form of a sheet
that is 0.3 to 2.0 mm thick. This drug combined collagen sheet
(sleeve), modified into a tube (cylinder) or other geometrical
shapes, is directly secured to the outside of the native vessel, at
the site of graft anastamosis and/or over the vein, artery or graft
itself. The device may be secured by sutures or staples. The suture
material itself may be combined with an anti vasculoproliferative
drug. In this aspect, the chosen antiproliferative agent permeates
through the vessel wall the rate of drug elution from the membrane
can be varied and can continue until the collagen matrix material
is completely resorbed. Tacrolimus, paclitaxel, other taxanes,
flavoperidol, antisense, analogues of Paclitaxel, Rapamycin and
tacrolimus, and other adjuvants well known to one skilled in the
art, may be used.
[0064] Double or Dual or Multi layer Device: In another aspect, the
present invention is a dual layered prosthetic device comprising an
antiproliferative-imbibed, inner matrix layer and, an external
support skeletal structure or layer. In this embodiment, the inner
matrix material is a sheet or membrane of type I collagen and the
exterior skeletal support material structure is a sheet of PTFE.
The antiproliferative drug, in this embodiment, is Rapamycin. The
sheet of collagen will be attached to the PTFE sheet using a
variety of techniques e.g., physically using sutures, adhesives,
staples or the two may be chemically bonded. The two sheath
composite would then be rolled to create either a tubular structure
or geometrical variations thereof. The composite device or sleeve
is then suitably trimmed so that it can be applied over the desired
site(s): artery, vein, graft anastomotic site etc., and the free
edges of the PTFE sleeve are attached to each other by adhesive,
sutures, staples etc. This stabilizes the entire device on the
outside of the vascular structure or graft. The drug then permeates
through the vascular or prosthetic material wall and while in the
wall the drug inhibits smooth cell proliferation, an integral part
of the healing response that follows surgical construction of the
graft.
[0065] Following placement on the outside of a vessel or prosthetic
surface, after a period of time the body absorbs the collagen
leaving its exterior support skeleton or structure intact. One
skilled in the art will appreciate that the body-resorbable aspect
of the protein layer chosen to imbibe the drug, is an optional
preferred practice of the present invention. The PTFE not being
bioabsorbable, tends to hold the resorbable protein layer in place
for a length of time sufficient for the drug to permeate through
the vascular or graft or prosthetic material wall. Besides its
value in supporting the drug eluting inner membrane or matrix
material there are other potential advantages of the external
layer. Although the desired effect of the drugs is their ability to
inhibit the smooth muscle cell proliferative response, it is this
proliferative response that contributes to the formation of a good
quality (firm) surgical scar. A weak scar at the site of surgical
anastamosis can potentially lead to graft disruption or aneurysm
formation. Having an external PTFE skeleton functions as an
additional reinforcement layer and prophylactically addresses the
treatment for problems related to a weak scar, graft disruption,
and/or aneurysm formation. The external PTFE layer serves to keep
the drug in close apposition with the outer aspect of the vessel or
graft wall and limits its diffusion to the surrounding tissues and
skin. It is also within the contemplation of the present invention
that the exterior skeletal or support aspect of the prosthetic
device could, itself, be biodegradable. Thus, a resorbable external
skeletal structure combined with a resorbable internal drug eluting
collagen layer, the two layers having the same or different rate of
degradability and resorption, would generate a healed vascular or
graft structure without the necessity of foreign material remaining
after the procedure. One skilled in the art would understand in
view of this disclosure that numerous other such materials are
likely to be usable in this invention. For example, Dacron.RTM.
polyester can also be a suitable material for the external support
structure.
[0066] A further object of the present invention is device
self-fixation to the outer surface of the vascular wall. The device
could be made more adhesive to the vascular wall if in the final
stage collagen is combined with photoreactive groups such as FITS
(fluorescein isothiocyanate) or Bengal Rose both from Sigma
Chemicals, St Louis, Mo., USA. Stimulation of the device with ultra
violet light activates the photoreactive groups and will increase
adhesion. Fibrin sealant and acetylated collagen have been found to
increase adhesion of collagen matrix material to the outside
vascular wall.
[0067] Another embodiment of the present invention is a method of
inhibiting stenosis of hemodialysis access graft comprising the
method of placing the prosthetic device (described above) over a
graft or vascular structure and/or at the site of anastomosis and
anchoring the prosthetic device at the desired site (e.g., by
suturing).
[0068] FIGS. 1A, 1B, 2A, and 2B illustrate preferred embodiments of
the present invention 1. In FIG. 1A there is shown a rectangular
sheet of a matrix material 2 having disbursed or distributed
therein an agent 3 of the present invention (shown by stippling).
FIG. 1B illustrates a further embodiment of the invention shown in
FIG. 1A in which a hole 4 has been created in the drug-containing
matrix material 3,2. It will be understood by one skilled in the
art that the diameter of hole 4 will be adjusted to accommodate the
outside diameter of any vascular or graft structure passing
therethrough. In one embodiment, the diameter of hole 4 is 6
millimeters.
[0069] FIGS. 2A and 2B illustrate a further embodiment to the
present invention in which an exterior support or skeletal
structure or means 5 is employed. Support 5 is exterior to matrix
material sheet 2 when sheet 2 is rolled or coiled into a
cylindrical shape. Exterior skeletal means such as polytetrafluoro
ethylene (PTFE) and dacron sheets are among the support materials
presently contemplated. Many other such exterior skeletal support
means will occur to one skilled in this art. As is shown, FIG. 2B
illustrates an embodiment to the invention in which a hole 4 (which
may vary in diameter) is employed.
[0070] FIGS. 3A, 3B, and 3C illustrate an embodiment of the
invention employing an interlocking design in which one edge of the
rectangular agent-eluting sheet or matrix material interlocks
adjacent the opposite edge. More specifically, FIG. 3A shows a
rectangular matrix material 2 having agent 3 (shown in stippling)
disposed or disbursed therein. Also shown on the sheet illustrated
in FIG. 3A are a series of v-shaped notches 6 located approximately
adjacent one edge 7 of the agent-containing matrix material.
Cooperating with notches 6 on the opposite edge 8 are a series of
projections 9. Projections 9 are arrow-head shaped. However, other
combinations of projection 9 and slots 6 certainly are contemplated
by this invention. Thus, assembly of a sleeve embodiment of the
present invention involves rolling edge 8 toward edge 7 (shown in
FIG. 3B) and inserting projections 9 into slots 6. As is shown in
FIG. 3C projections 9 have been inserted into slots 6 from the
inside of the tubular structure meaning that the points 10 of
projections 9 project from the inside to the outside of the
structure. As is shown, the following edges 11 of projections 9
cooperate with v-shaped slots 6 to lock the flat structure into a
cylindrical vascular-dimensioned sleeve 12. Vascular sleeve 12
further then defines a lumen 14. Lumen 14 is of a vascular
dimension such that the interior surface of sleeve 12 would be in
contact with the exterior surface of a vascular structure to which
sleeve 12 was attached. In this fashion, the drug or agent-eluting,
vascular-dimension sleeve is deployed over and around the vascular
structure with which this invention is to be used.
[0071] FIGS. 4A and 4B illustrate a second interlocking embodiment
of the present invention. In embodiment, a strip-form of the
present invention is utilized. Agent-eluting sleeve 16 comprises an
elongate drug or agent-eluting matrix material 17 (alone or in
conjunction with an external support means, not shown). Created in
matrix material 17 are two locks 18 located on opposite ends
thereof. Cooperating with lock 18 are windows 19 into which locks
18 are inserted such that sleeve 16 is deployed against and on the
exterior of the operant vascular structure. As is shown on FIG. 4B,
lock 18 may be inserted into window 19 from the inside toward the
outside. In an alternative embodiment lock 18 may be inserted into
window 19, from the outside toward the interior of the sleeve
structure. Also shown in FIG. 4A is a representative shunt opening
20 including two shunt contact wings or flaps 21.
[0072] FIG. 5 illustrates another embodiment to the present
invention in which an external wire support or framework means is
employed. External wire framework 20 surrounds a preferred
embodiment of the present invention i.e. a PTFE and drug-coated
collagen matrix material 22 disposed around vessel 24.
[0073] FIGS. 6-13 illustrate various arterio-venous fistuale. A
drug eluting sleeve or matrix material of the present invention 26
is shown to be implanted, wrapped or placed around the various
fistulae 32 shown in the several figures. In each of these figures
venous structures are designated 28 and arterial structures are
designated 30. Arrows 34 illustrate the direction of blood
flow.
[0074] FIGS. 10-13 illustrate a further embodiment of this
invention in which a graft e.g., a PTFE graft, 36 is used in
conjunction with the present invention. As is shown in FIG. 13,
graft 36 may itself include a matrix material with a drug or agent
36 (shown in stippling) of this invention.
[0075] A further application of the present sleeve involves
utilization of the interior drug-imbibing protein layer as a drug
source or drug reservoir. In that application the drug selected may
be replenished periodically, e.g., by puncturing the sleeve with a
needle and delivering additional drug thereto or creating a
reservoir for the drug within the sleeve from which it can be
gradually eluted.
EXAMPLES
[0076] The following examples are set forth to illustrate the
device and the method of preparing matrices for delivering
antiproliferative drug(s) and other therapeutics. The examples are
set forth for purpose of illustration and not intended in a
limiting sense.
Example 1
Inhibitory Effect of Different Antiproliferative Agents
[0077] Prefabricated collagen matrices were placed in different
antiproliferative drug solutions until complete saturation
occurred. The antiproliferative drugs were chosen to represent the
more active compounds capable of smooth muscle cell and fibroblast
inhibition without inhibiting collagenase and elastase enzymes.
(Collagenase and elastase enzymatically inhibit collagen
accumulation--one cause of restenosis). The collagen matrices were
saturated with these compounds at concentration of 25 .mu.g/ml
lyophilized, washed with 0.066 M phosphate buffer (pH 7.4) at
37.degree. C. for 24 hours and cut in the shape of a disc with
density of compound about 5 .mu.g per cm.sup.2. After washing,
sterile discs, 15 mm in diameter were placed in 24-well culture
plate and cells at a density of 5000 per cm.sup.2 were seeded. Five
days later cell number was measured and enzymatic activity was
evaluated in the aliquots of media via chromogenic substrates
hydrolysis and spectrophotometry. These data are presented in Table
1.
TABLE-US-00001 TABLE 1 Inhibitory effect of different
antiproliferative agents SMC Fibroblast Collagenase Elastase Agent
Inhibition % Inhibition % Activity % Activity % Control, plain 0 0
100 100 matrix Paclitaxel 88 .+-. 6 62 .+-. 11 98 .+-. 5 90 .+-. 4
Rapamycin 94 .+-. 5 90 .+-. 12 137 .+-. 8 142 .+-. 5 Cyclosporin A
61 .+-. 7 53 .+-. 7 104 .+-. 5 87 .+-. 7 Tetracycline free 11 .+-.
8 13 .+-. 5 56 .+-. 8 81 .+-. 4 base Methotrexate 32 .+-. 9 28 .+-.
6 23 .+-. 12 14 .+-. 3 Actinomycin D 44 .+-. 11 35 .+-. 8 55 .+-. 9
84 .+-. 11
[0078] In this comparative in vitro test, among tested agents,
Paclitaxel and Rapamycin performed similarly.
Example 2
Capacity of Different Types of Matrices to Bind Rapamycin
[0079] In the next in vitro study, the ability of different
matrices to bind Rapamycin was tested. A prefabricated (BioMend,
Sulzer Calcitek, Inc or Biopatch, Ethicon Inc, containing
collagen-alginate) collagen matrix with Rapamycin was prepared as
described in Example 1 at initial Rapamycin concentration of 250
.mu.g/ml. Prefabricated chitosan (using technique described in:
Almin, C., Chunlin, H., Juliang, B. et al "Antibiotic loaded
chitosan bar. In vitro, in vivo study of a possible treatment for
osteomyelitis," Clin Orthop pp. 239247 (September 1999) and fibrin
matrices (using technique mentioned in example 5) were also placed
in of 250 .mu.g/ml of rapamycin in DMSO solution until complete
saturation occurred. After solvent evaporation, the matrices
combined with drugs were washed with 0.066 M phosphate buffer (pH
7.4) at 37.degree. C. for 24 hours.
[0080] To compare matrix capacity, fluorescent Rapamycin derivate
loaded onto 1.88 cm.sup.2 matrix surface of the same thickness was
used. After incubation with 0.14 M NaCl solution, the residual
rapamycin was extracted with dimethylsulfoxide (DMSO) and yield was
measured using fluorescence spectroscopy. These data are presented
in Table 2.
TABLE-US-00002 TABLE 2 Matrix Capacity for Rapamycin Matrix
Rapamycin capacity (.mu.g per cm.sup.2) Collagen 124.5 .+-. 14.3
Collagen-alginate 131.1 .+-. 12.3 Chitosan 78.7 .+-. 8.9 Fibrin
145.8 .+-. 12.7
[0081] As expected, capacity of protein matrices was found to be
higher than the chitosan matrix, usefulness of fibrin or collagen
as therapeutic matrix for antiproliferative drug delivery may
depend on particular combination or additional components or
requirements of longevity of the matrix.
Example 3
Delivery Systems Using Liposomes
[0082] Liposomes represent a form of drug delivery system, and
offer controlled release of biologically active agents. They are
used in pharmaceutical formulations especially for water insoluble
drugs. Rapamycin is a typical example. Liposomal entrapment has
been shown to have considerable effect on the pharmacokinetics and
tissue distribution of administered drugs. The formulations tested
included nonionic liposomal formulation composed of glyceryl
dilaureate (Sigma Chemicals, St Louis, Mo.), cholesterol(Sigma
Chemicals, St. Louis, Mo.), and polyoxylene-10-stearyl (Sigma
Chemicals, St. Louis, Mo.) either at a weight ratio of 56:12:32
(Formulation 1) or nonionic 40% hydroalcoholic oil-in-water
liposomal emulsion containing isopropyl myristate (Sigma Chemicals,
St. Louis, Mo.) and mineral oil (Sigma Chemicals, St. Louis, Mo.)
(Formulation 2). Rapamycin was entrapped into each formulation at a
concentration of 250 .mu.g/ml in dimethylsulfoxide or isopropanol
and formed liposomes were applied on surface of prefabricated
collagen sheets to create maximal surface density of Rapamycin.
Samples were washed with 0.066 M phosphate buffer (pH 7.4) at
37.degree. C. for 24 hours. To compare matrix capacity, liposomes
loaded with fluorescent Rapamycin derivate placed onto 1.88
cm.sup.2 disc was used. After incubation with 0.14 M NaCI solution,
matrices with remaining Rapamycin were extracted with
dimethylsulfoxide (DMSO) and fluorescent yield was measured.
TABLE-US-00003 TABLE 3 Liposomal Delivery System Rapamycin Liposome
Type Binding Capacity .mu.g per cm.sup.2 Nonionic cholesterol
liposomes 117.4 .+-. 10.9 (Formulation1) Nonionic oil-in-water
emulsion 89.6 .+-. 7.5 (Formulation 2) Saturated collagen matrix
(DMSO) 124.5 .+-. 14.3 Saturated collagen matrix (isopropanol)
105.6 .+-. 9.7
[0083] Liposomal delivery systems do not have significant
advantages over saturated collagen matrix in ability to bind
Rapamycin. However the liposomal approach may be useful for other
antiproliferative drugs.
Example 4
Preparation of a Laminated Collagen Film
[0084] In order to prepare a textured, surface neutralized,
laminated collagen film an isotonic suspension of insoluble
fibrillar collagen was obtained. Three liters of chilled collagen
suspension at concentration of 5 to 18%, (preferred 12%) was
swollen overnight in 0.3-0.6 M acetic acid, (preferred 0.52 M), at
4.degree. C. The swollen suspension was dispersed with 3 liters of
crushed ice for 10-20 min, (preferred 12 min.) in a blender and
thereafter homogenized for 30 min in an Ultra-Turrax (Alfa,
Sweden). The resulting slurry was filtered through a series of
filters (Cellector, Bellco, UK) with pore sizes decreasing from 250
.mu.m to 20 .mu.m, mounted in filter holder (Millipore). After
degasation at 0.04-0.09 mbar, preferred 0.06 mbar, the slurry was
mixed with 2 liters of chilled 0.1-0.05 M NaOH, final pH adjusted
to 7.4.+-.0.3. The neutralized suspension can be stored at
4-6.degree. C. only for several hours prior to matrix formation.
This neutralized suspension serves as a foundation for preparation
of a saturated or dispersed form of a matrix containing rapamycin.
The neutralized slurry may be directly cast as a wet film with a
thickness of 3 mm on a flat hydrophobic surface at room
temperature. A dry film with a thickness of approximately 60-70
.mu.m is formed. Three to five ml of slurry cover an area of 10
cm.sup.2 area. On top of such a surface several layers may be
formed. The layers will serve as a basis for preparation of
saturated form of anti proliferative agent by immersing the
collagen film into solutions of rapamycin, Taxol or combinations
thereof. Simultaneous combination of neutralized slurry and
rapamycin or other agents in suspension may be used for preparation
of film with dispersed form of active ingredients.
[0085] An important factor in the preparation of the matrix
material is the porosity of the protein carrier from which the
device is to be formed. Porosity may be regulated by drying rate,
temperature, and the characteristics of the initial collagen.
Porosity is significant because it controls the kinetics of drug
release. It is desirable for the matrix to be sufficiently porous
to bind small molecules such as rapamycin (Molecular weight 914.2)
and durable enough to maintain the shape of device. Samples of
collagen matrix with effective pore size of 0.002 to 0.1 microns
were tested. Higher binding capacity (to bind rapamycin in
saturation experiments) was observed with the matrix having pore
size of 0.004 microns. In addition, collagen matrices with bigger
pore sizes are fragile. Since the binding capacity of the matrix to
the antiproliferative agent is critical for this application, three
different concentrations of rapamycin were used to prepare a
rapamycin -collagen matrix combination from commercially available
collagen prepared at optimal density of pores. The three different
concentrations labeled high, medium and low, were 120.+-.5
.mu.g/cm.sup.2, 60.+-.4 .mu.g/cm.sup.2, and 30.+-.3 .mu.g/cm.sup.2,
respectively. None of these matrices were fragile or had
non-uniform rapamycin distribution. Different densities permit
regulating kinetics of drug release.
Example 5
Preparation of an Implantable Fibrin Matrix Device Combined with an
Antiproliferative Agent
[0086] In general, to make a device based on a fibrin matrix loaded
with an antiproliferative agent, aqueous fibrinogen and thrombin
solutions are prepared as described below. Commercial fibrinogen
can be acquired from such vendors as Sigma, American Red Cross, or
can be prepared from plasma by well-known techniques.
Alternatively, fibrinogen prepared by recombinant methods is
suitable for use. Commercial active thrombin can be acquired from
Sigma or from Johnson and Johnson as thrombin, topical USP,
Thrombogen. To make the fibrinogen and thrombin solutions used to
prepare the matrix, the necessary components are measured, weighed
and dissolved in about 900 ml of deionized water. Tables 4 and 5
disclose preferable compositions used to prepare fibrinogen and
thrombin solutions to prefabricate matrix, respectively.
[0087] The glycerol in Table 4 used as a plasticizer. Other
plasticizers would also be suitable for the present invention. TRIS
buffer is used for pH adjustment. Suitable alternatives for TRIS
include HEPES, Tricine and other buffers with a pKa between 6.8 and
8.3. Triton X-100 is a non-ionic detergent and stabilizer and may
be substituted by other detergents and stabilizers. Caprylic acid
may be substituted by other agents that provide protection from
denaturation, for example, alginic acid.
TABLE-US-00004 TABLE 4 Fibrinogen Solution Composition Composition
Range Composition Preferred Component g/liter g/liter Fibrinogen
50-120 76 Glycerol 20-80 40.5 TRIS buffer 3-25 12.1 Caprylic Acid
10-35 18.7 Triton X-100 2-8 5.4 Heparin 0.5-6 2.38
TABLE-US-00005 TABLE 5 Thrombin composition Composition range
Composition preferred Component (g/liter) (g/liter) Thrombin
5,000-100,000 units 8,000 units Albumin 1-100 50 Factor XIII
1,000-5,000 units 2,500 units CaCl2 50-250 mg/liter 123 mg/liter
Troglitazone 3-24 8
[0088] Fibrinogen converted to fibrin is the most critical reagent
in the matrix because it controls the material properties of the
matrix, such as flexibility, pore size and fiber mass density.
These features determine how easily other molecules can diffuse
within the matrix and how long the matrix may remain intact before
it is resorbed.
[0089] In Table 5, albumin is a stabilizer of thrombin. Thrombin
controls the rate of fibrin matrix formation. The presence of
Factor XIII is preferred but not necessary. Factor XIII covalently
cross-links fibrin, making the matrix more stable. Calcium ions are
needed for activation of thrombin. Troglitozone (Sankyo, Japan) is
a thiazollidione derivate, which decreases collagen accumulation in
the vascular wall. (Yao L, Mizushige K, Murakami K et al.
Troglitozone decreases collagen accumulation in prediabetic stage
of a type II diabetic rat model. Heart 2000: 84: 209-210
[0090] It is preferable to completely dissolve each component
before adding the next component. If necessary, after the last
component is dissolved, the pH is adjusted to 7.0-7.4 and the
solution volume is adjusted to 1 liter with water. The solutions
are then degassed. Both solutions are dispensed by pump through
mixture chamber onto a non-stick, preferably hydrophobic, surface
to form a film approximately 2 mm thick. The film is then dried for
about 3 to 6 hours at temperature in the range of about 20.degree.
C. to 60.degree. C., at a pressure of about 30 Torr. Residual
moisture of the film is about 10%, preferably less than 3%, of the
total wet weight.
[0091] On this surface dry solid Rapamycin is added to create
density in the range of 100 to 500 .mu.g per cm.sup.2 of film. A
second layer of fibrin matrix is formed on top of this surface such
that the drug is sandwiched between the two layers of fibrin.
[0092] In one embodiment of the present invention, one would add
(and/or) an antiproliferative/anti restenotic agent like Rapamycin
or Taxol, an anti rejection drug like Rapamycin or tacrolimus, an
anti-inflammatory drug and/or an antisense oligonucleotide to
enhance antirestenotic effects. These solid materials would be
added to supplement the fibrin-Rapamycin sandwich complex described
above.
Example 6
Method of Cross Linking Chitosan Matrix
[0093] In order to increase binding capacity of a chitosan matrix
for antiproliferative drug, cross-linking of fiber is used. Fifty
ml of chilled chitosan suspension at concentration from 10% to 25%,
(preferred 12%) was gently and slowly mixed with 5 to 25 ml of
acrylic acid chloranhydride for 30 min. to acetylate this polymer.
After this time period, a solution of rapamycin in DMSO at
concentration of 250 .mu.g/ml was added, mixed vigorously, and
poured onto the chitosan matrix surface for spontaneous
cross-linking and formation of conjugated rapamycin. This approach,
because of the microporous structure of the chitozan, allows
increasing the binding capacity of the matrix from 15% to 45%.
Example 7
Incorporation of Rapamycin into Collagen Matrix by Dispersion,
Immobilization and Immobilization-Dispersion
[0094] Besides the technique of saturation, rapamycin was
incorporated into the collagen matrix by three different methods:
dispersion, immobilization, and immobilization-dispersion.
[0095] Dispersion technique: an aqueous slurry of water insoluble
collagen was prepared using non-crosslinked dry, highly purified,
lyophilized calfskin collagen obtained from Elastin Product Co.,
Inc. (Owensville, Mo.). This collagen and solubilizing buffer are
chilled to a temperature of 2-8.degree. C., preferred 4.degree. C.
and vigorously mixed to prepare collagen slurry containing 10-21%,
(preferred 12%) of collagen protein. Such slurry includes 9% of
plasticizer, glycerol 15% o rapamycin in DMSO at concentration of
250 .mu.g/ml and water. The solution had a viscosity of 50,000 cps.
Immediately after mixing with rapamycin, 8% glutaraldehyde is added
to the slurry (100-350 ml per liter of slurry). The aqueous slurry
must be homogenous and degassed, the pH is adjusted to 6.0-7.1. The
solution is constantly vigorously mixed and dispersed by pump onto
a non-stick surface to form a film approximately 2 mm thick. All
procedures are carried out at a temperature of 4.degree. C. The
film is then dried for about 3-7 hours at temperatures in the
vicinity of 45.degree. C., and a pressure of 15 Torr until its
residual moisture is less than about 10% of the total weight. The
drug solution application and drying steps are repeated three more
times.
[0096] II): Immobilization technique: The same collagen preparation
from Elastin Product Co. is used. One volume of 12% collagen slurry
is chilled and coupled with rapamycin via esterification of
antiproliferative drug. Esterification is carried out with 0.9 M
N-hydroxysuccynimide (Pierce Biochemical, Rockford, Ill.) in the
presence of 0.9 M N-dicyclohexylocarbodimide (Pierce Biochemical,
Rockford, Ill.) at 2-4.degree. C. for 2 days. Conjugates are
prepared by titration of active N-hydroxysuccynimide ester of
rapamycin in DMSO under the surface of stirred collagen suspension,
the pH of the reaction is maintained between 7.0 and 8.5, preferred
7.8. After drying, the films with conjugated rapamycin are washed
with 0.15 M NaCl containing 0.02 M sodium bicarbonate at a pH of
7.4. HPLC reveals no free rapamycin in the matrix. Rapamycin ester
reacts with amino- or hydroxyl-groups of aminoacid residues forming
a covalent linkage with collagen. After such immobilization,
Rapamycin is released as a result of in vivo or in vitro
degradation-erosion of the matrix. Nakano et al make reference to
collagen (SM-10500) degradation and resorption via natural
metabolic process in Rhesus monkeys during 6 months Ref: Nakano M,
Nakayama Y, Kohda A et al: Acute subcutaneous toxicity of SM-10500
in rats. Kisoto Rinsho (Clinical Report) 1995; 29: 1675-1699]
[0097] In order to study the rate of rapamycin release from the
matrix, samples are washed with 0.066 M phosphate buffer (pH 7.4)
at 37.degree. C. for 24 hours and cut to give a shape of disc with
area of 1.88 cm.sup.2, and placed into 24 well culture plate
containing 0.14 M NaCl, 0.05M Tris buffer, 0.5% of albumin, and 0.1
mg/ml collagenase, at pH 7.0. Collagenase is added to increase
erosion of collagen matrix and facilitate release of rapamycin.
Aliquots are collected at various time intervals from the
wells.
[0098] A combination of dispersed and conjugated forms is also
prepared. In all these forms, the content of rapamycin is 5.0 .mu.g
is per cm.sup.2. The samples are placed in wells and 1 ml of
elution media containing serum are added. Aliquots are taken every
hour.
[0099] The content of Rapamycin is measured according to the
procedure of Ferron et al. (Ferron G M, Conway W D, and Jusko W J.
Lipophilic benzamide and anilide derivatives as high-performance
liquid chromatography internal standard: application to sirolimus
(rapamycin) determination. J Chromatogr B Biomed Sci Appl 1997;
December 703: 243-251.) These measurements are made using batch
assay and, therefore, represent release rates at 0 ml/min flow
rate. The results are tabulated in Table 6 and graphically
illustrated in FIG. 14; concentrations of antiproliferative drug
are in .mu.g/ml.
[0100] These data show that different forms of drug imbedding and
drugs with different solubility have distinct kinetics. In the case
of comparatively soluble Tetracycline, after saturation of the
collagen matrix with the free base, peak release occurs in a short
period of time, whereas for less soluble rapamycin this peak is
postponed for several hours. It has been shown in experiments in
vitro, that collagen saturated with soluble antibiotics such as
gentamicin, cefotaxin, tetracycline or clindamycin delivers these
antibiotics at effective concentrations for 4 days. [Wachol-Drewek
Z, Pfeifer M, Scholl E. "Comparative investigation of drug delivery
of collagen implants saturated in antibiotic solutions and sponge
containing gentamicin." (Biomaterials 1996; 17: 1733-1738)].
TABLE-US-00006 TABLE 6 Rate of release of collagen saturated with
Tetracycline and Rapamycin. Rapamycin was combined with collagen
matrix using four different methods. Combination Collagen Collagen
Rapamycin Collagen of Saturated Saturated Dispersed Conjugated
Dispersed and Time With With Throughout With Conjugated (Hour)
Tetracycline Rapamycin Collagen Rapamycin Forms 1 0.06 0.01 0.01 0
0.01 2 0.4 0.05 0.03 0 0.02 3 0.96 0.09 0.06 0.01 0.07 4 0.54 0.15
0.08 0.02 0.09 5 0.15 0.19 0.12 0.05 0.17 6 0.08 0.28 0.18 0.07
0.26 7 0.02 0.57 0.19 0.11 0.31 8 0.01 0.44 0.29 0.13 0.32 9 0.01
0.24 0.41 0.19 0.34 10 -- 0.20 0.62 0.27 0.41 11 -- 0.19 0.61 0.31
0.78 12 -- 0.18 0.40 0.42 0.76 13 -- 0.15 0.32 0.45 0.79 14 -- 0.02
0.16 0.32 0.45 24 -- 0.11 0.24 0.42 Totally 0 0.003 0.23 0.53 0.39
Dissolved matrix
[0101] In other laboratories is also was shown in vivo, that,
collagen saturated with gentamycin at concentration of 3 .mu.g/g
and implanted into muscle tissue is capable of delivering
antibiotic into blood through day 28. However, concentration was
less than optimal. (Mehta S, Humphrey J S, Schenkman D I, et al.,
"Gentamycin distribution from a collagen carver." J Orthop. Res.,
1996; 14: 749-754.). It is theorized that knowing the low
concentration of collagenase in perivascular space and the low flow
of perivascular fluid (only a few milliliters per day) a matrix
material, saturated with rapamycin might produce in vivo delivery
kinetics, which will support effective local concentration of
antiproliferative drug for a period of several weeks to prevent and
combat progress of SMC proliferation. Inhibitory concentrations for
SMC would be in the range of 0.001 to 0.005 .mu.g/ml culture media.
Such levels are met or exceeded in vitro for 3 weeks. Moreover,
Rapamycin dispersed into collagen matrix may exhibit an
antiproliferative effect for a month or longer. Finally, conjugated
and combined forms may support treatment until complete matrix
erosion.
Example 8
Biological Activity of Rapamycin in the Rapamycin-Collagen
Matrix
[0102] The most important parameter when assessing the combination
of rapamycin and collagen is inhibition of smooth muscle cell (SMC)
growth. To evaluate this parameter SMC's at density of 5,000 cells
per cm.sup.2 are seeded onto control tissue culture surface and
testing matrices (Table 7). Cell growth curves are presented in
FIG. 15.
[0103] Actinomycin D is quickly released from the drug matrix and
suppresses cell growth for only a short period of time. A change of
media removes soluble Actinomycin and after several washes no
antibiotic is present in the media or in the matrix. As a result,
cells start proliferating as usual. Because of a slow gradual
release of rapamycin suppression of cell growth continued
throughout the observation period.
TABLE-US-00007 TABLE 7 Comparison of inhibition of growth of smooth
muscle cells using collagen matrices saturated with Actinomycin D
and Rapamycin Cell number Collagen + Days in Culture Control
Axtinomycin D Collagen + Rapamycin 0 5000 5000 5000 1 6430 .+-.
20.4 5230 .+-. 16.8 4800 .+-. 9 2 10240 .+-. 27.1 7350 .+-. 19.5
5040 .+-. 11.2 3 16340 .+-. 30.12 9400 .+-. 13.2 6230 .+-. 13.4 4
27100 .+-. 25.4 14280 .+-. 17.6 7400 .+-. 15.1 5 38450 .+-. 22.6
23540 .+-. 17.8 8000 .+-. 17.8 6 40000 .+-. 20.7 29300 .+-. 19.4
8550 .+-. 13.9 7 40100 .+-. 20.5 32090 .+-. 32.1 8500 .+-. 14.4
Example 9
[0104] Two different types of matrices, collagen and fibrin
combined with antiproliferative agents (singly or in combination)
along with Vitamin K are added to the cell culture medium in
different ratios. Cells are seeded at the same density, on day 5
numbers of viable cells are measured by Alamar blue assay. Data are
presented in Table 8.
TABLE-US-00008 TABLE 8 Inhibition of cell growth (%) Collagen
Collagen Fibrin Plus plus plus Collagen Rapamycin Rapamycin Fibrin
Rapamycin Matrix to Media plus Plus plus plus plus Ratio Rapamycin
Taxol Vitamin K Rapamycin Taxol 1:400 5 4 8 3 2 1:200 25 27 34 21
19 1:100 54 50 77 56 55 1:50 73 76 99 79 78 1:25 88 88 99 79 84
1:12.5 95 99 99 98 96 1:6.25 95 99 99 100 98
Example 10
Antiproliferative Effect of Combination of Rapamycin and Heparin
Combined to a Collagen Matrix
[0105] Antiproliferative effects of different components combined
within a matrix may exhibit a synergy. A combination of dispersed
Rapamycin, soluble and immobilized heparin are used. In order to
immobilize heparin 5 ml of chilled heparin solution at
concentration of 1 mg/ml to 10 mg/ml, (preferred 5 mg/ml) is mixed
with 5 to 20 ml, (preferred 11.4 ml) of acrylic acid chloranhydride
at the rate of approximately 1 .mu.l per min, (preferred 2.5 .mu.l
per min). After addition, mixture is agitated for 30 minutes at a
temperature of 4-8.degree. C. The heparinized collagen is
extensively washed with sodium phosphate buffered saline at pH 7.4.
A colorimetric assay with Eosin A is used to determine the
concentration of heparin immobilized on matrix. Using this method
between 0.01 mg/cm.sup.2 and 0.1 mg/cm.sup.2 may be covalently
linked to the matrix.
[0106] Such a formulation combined with Rapamycin has inhibitory
effect on SMC growth in culture if added in the form of suspension
into the media at ratio 1:100, whereas individual forms have lesser
effects; ratio of 1:25 for heparin alone to 1:65 for dispersed
rapamycin. Each of these drugs can inhibit restenosis via different
mechanisms, hence it is reasonable to expect synergistic effect
when used in combination. Heparin can also be used in matrix
saturated form in combination with antiproliferatives.
Example 11
[0107] Sustained local delivery of Dexamethasone in combination
with Rapamycin (or other antiproliferative agents) can be used to
simultaneously inhibit restenosis as well as inflammatory
reactions. Twenty percent (weight/weight) collagen slurry is
prepared, to which is added a 2% (weight/weight) suspension of
dexamethasone. This mixture is sprayed on to a plastic surface to
form the film. The final thickness of the film ranged from 1.92 to
2.14 mm (mean 2 mm). This sheet is flexible and mechanically
stable. The kinetics of dexamethasone elution from the c matrix
(collagen plus rapamycin) were characterized in an in vitro system.
Fifteen mm diameter sheets were placed in the wells and immersed in
2.5 ml of phosphate buffered solution. At time points ranging from
1 to seven days, concentration of dexamethasone in aliquots of
elution buffer were measured by spectrophotometry. Chemical
stability of the dexamethasone through the sheet formation, drying
storage and elution process was confirmed by HPLC. Cumulative in
vitro elution of dexamethasone is shown in Table 9.
[0108] More than 50% of the dexamethasone elution occurred within
the first three days, with a leveling off of the elution curves
after 6 days. Dexamethasone can prevent a severe inflammatory
response, which is maximal during this time period and can act
synergistically with rapamycin to reduce restenosis. In contrast to
a dexamethasone eluting stent, perivascular delivery does not
inhibit endothelial cell regeneration and acts directly on
fibroblasts and smooth muscle cells.
TABLE-US-00009 TABLE 9 Cumulative in-vitro elution of dexamethasone
from a collagen matrix. Eluted Dexamethasone Mass (micrograms) Time
(days) 0 0 211 .+-. 23 1 489 .+-. 31 2 605 .+-. 42 3 672 .+-. 38 4
725 .+-. 21 5 733 .+-. 18 6 745 .+-. 13 7
[0109] Combination of macro and micro porosity may increase
capacity of the device. Collagen and fibrin matrices were mixed to
obtain such a combination. In addition, good mechanical
characteristics of collagen improved stability of fibrin. To
prepare fibrin-Rapamycin loaded matrix, (Rapamycin density of 150
.mu.g/cm.sup.2) compositions disclosed in Tables 4 and 5 were used.
2. After formation of first dry layer of fibrin, second layer of
collagen, rapamycin and heparin was formed as described in example
4 (Rapamycin density of 128 .mu.g/cm.sup.2, heparin density of 5000
U/cm.sup.2). The collagen fibrin sheaths loaded with medicine
(thickness 2mm) were formed as tubular structures and externally
crosslinked using high concentration of glutaraldehyde (25%) for
one minute. After drying, spiral form of sleeve shown in FIG. 4 was
prepared. This sleeve was made planar on ten occasions, the spiral
shape was restored each time. The Rapamycin capacity of the final
sleeve was 143 .mu.g/cm.sup.2. In vitro elution of heparin
continues for 7 days.
[0110] Heparin concentration was measured as in example 10, buffer
for the dilution was replenished each day. The data are shown in
Table 10.
[0111] It is known that effective concentration of heparin to
inhibit SMC proliferation is in the range of 100 .mu.g/ml. In this
example, heparin can significantly inhibit SMC proliferation for at
least 4 days In addition diffusion of heparin form the sleeve can
prevent thrombotic events on the inner surface of the shunt and
damaged vessel wall for longer periods of time. Besides,
concentration of soluble heparin can be increased up to 20,000
units/cm.sup.2 without changing mechanical characteristics of the
matrix. Therefore, anti smooth muscle cell proliferation as well as
antithrombotic effect can be prolonged.
TABLE-US-00010 TABLE 10 Elution profile of heparin from a collagen
matrix combined with rapamycin and heparin. Time (days) Eluted
Heparin Mass (U/ml) 0 0 1 341 2 275 3 188 4 103 5 57 6 24
Examples 13 and 14
Comparison of In Vitro Effect of Rapamycin, Tacrolimus and
Paclitaxel on Human Smooth Muscle and Endothelial Cells
[0112] Human smooth muscle cells and endothelial cells (Clonetics,
USA) were seeded (100,000 cells) in 24 well plates overnight. Both
cell types were grown and maintained in OPTI-MEM (Gibco, Long
Island, N.Y.) and 5% fetal bovine serum at 37.degree. C. in a 5%
carbon dioxide and 95% atmospheric air. Cells were exposed to a
range of concentrations of Rapamycin (10-100 nM), Paclitaxel
(0.1-10 mM) and Tacrolimus (10-100 nM). Each cell type was allowed
to grow for 24 hours, last four hours in the presence of
[.sup.3H]-thymidine. Proliferation of cells was quantified as new
DNA synthesis using .sup.3H-thymidine uptake assay. After 72 hours
of culture, cells were washed twice with cold phosphate buffered
saline (PBS) and 1 ml of methanol was added to the contents of each
well, the plates were kept at 4.degree. C. for 60 minutes, cells
were then washed once with cold PBS and 500 microliter of 0.2 m
NaOH was added to each well and the plates were kept at 4.degree.
C. for 30 minutes. The contents of each well were transferred into
scintillation vials and liquid scintillation fluid was added to
quantify radioactivity using a liquid scintillation counter and
results expressed as counts per minute.
[0113] Results are shown in Tables 11 and 12 and corresponding
FIGS. 16 and 17 respectively. Rapamycin and Paclitaxel inhibit
proliferation of both human smooth muscle and endothelial cells
(new DNA synthesis). Tacrolimus appears to preferentially inhibit
new DNA synthesis in human smooth muscle cells, sparing endothelial
cells. This differential effect may be extremely important and can
be beneficially exploited if Tacrolimus were to be used for
inhibition of smooth muscle cell proliferation.
TABLE-US-00011 TABLE 11 Comparison of Effect of Rapamycin,
Tacrolimus and Paclitaxel (3 doses) on Human Smooth Muscle Cells
[.sup.3H] - thymidine uptake Assay Mean (.+-.SD) p Untreated
(Control) 17434 (1822) Rapamycin 6498 (245) <0.01 Tacrolimus
11995 (1850) <0.05 Paclitaxel 2421 (206) <0.001 Paclitaxel
2527 (195) <0.001 Paclitaxel 2710 (162) <0.001
TABLE-US-00012 TABLE 12 Comparison of Effect of Rapamycin,
Tacrolimus and Paclitaxel (3 doses) on Human Endothelial Cells
[.sup.3H] - thymidine uptake Assay Mean (.+-.SD) p Unttreated
(Control) 16342 (3039) Rapamycin 5787 (1323) <0.01 Tacrolimus
16073 (3008) ns Paclitaxel 2222 (228) <0.001 Paclitaxel 2648
(248) <0.001 Paclitaxel 3459 (272) <0.001
[0114] Animal Studies
[0115] A proof of principle study was performed using a porcine
model. A total of 6 pigs were studied, 2 were used as controls and
4 were treated. A 6 mm PTFE vascular graft was anastomosed between
the carotid artery on one side and the contralateral jugular vein,
this created an arterio venous (AV) loop that is similar in
construction to the human hemodialysis access loop. A collagen
sleeve combined with a known dose of Rapamycin (approximately 500
.mu.gm/cm.sup.2) was placed around the distal end of the PTFE
vascular graft just proximal to the venous anastomosis in the
treated group.
[0116] After 30 days an angiogram was performed to demonstrate
vessel and graft patency. The animals were euthanized and the
relevant segments dissected. The inhibitory effect of Rapamycin on
cell cycle progression, is believed to be via induction of cyclin
inhibitors. Hence, expression of p21 will increase in tissues
obtained from rapamycin treated animals but not from controls. In
other words, the presence of p21 is confirmation that that the
observed effect is attributable to Rapamycin. Tissues from treated
and untreated animals were obtained, RNA was prepared and reverse
transcribed to cDNA, which was amplified for house keeping gene,
b-actin and p21 by PCR.
[0117] Results
[0118] Both controls had luminal narrowing caused by severe
neo-intimal hyperplasia at the site of venous anastomosis (FIGS.
18A and 19A). All 4 treated animals had significantly higher
luminal patency of the vein and the graft, with minimal to absent
neo intimal hyperplasia (FIGS. 18B and 19B). Expression of p21 mRNA
was observed in venous tissue at the perianastamotic site obtained
from rapamycin treated animals (FIG. 20) but not from controls.
This demonstrates that the Rapamycin contained in the sleeve matrix
was responsible for the reduction/virtual abolition of neo intimal
hyperplasia (an expression of the vasculoproliferative response) an
effect mediated through rapamycin induced inhibition of cellular
proliferation.
* * * * *