|Title of Invention||
A DEVICE HAVING A SURFACE COVERED WITH A HYDROGEL INCORPORATING SARATIN
|Abstract||A medicament comprising Saratin with therapeutic agents for or site delivery for inhibiting platelet accumulation after vascular injures or endarterectomy. The invention furthermore provides medicail devices like catheters and stents incorporated with coatings of the medicament.|
|Full Text||Summary of the invention
The invention relates to the effect of a polypeptide called Saratin that significantly
decreases platelet adhesion and accumulation after vascular injuries such as
endarterectomy. The invention furthermore relates to the inhibition of vWF-
dependent binding of platelets to vascular wall collagens under conditions of
elevated shear and in more detail, to a novel medical use of Saratin as an
inhibitor of thrombosis wherein said polypeptide may be used locally as a topical
agent or as a coating for medical devices.
Field of the invention
The adhesion of blood cells especially platelets to the wall of injured blood
vessels is a well known phenomen in angioplasty and surgical procedures. Such
injuries may occure during various surgical and percutaneous therapies that have
been developed to reopen blocked channels, conduits, and other lumens, to
remove diseased tissue, and to implant substitute tissue, or components thereof.
Various types of intervention techniques have been developed which facilitate the
reduction or removal of the blockage in the blood vessel, allowing increased
blood flow through the vessel. One technique for treating stenosis or occlusion of
a blood vessel is percutaneous balloon angioplasty. A balloon catheter is
threaded through the patient's-arterial system and inserted into the narrowed or
blocked area, and the balloon is inflated to expand the constricted area.
Percutaneous transluminal angioplasty (PTCA) is the most widely angioplasty
method used to open obstructed atherosclerotic arteries.
Generally, angioplasty procedures produce excellent results obviating the need
for bypass surgery, but in about 30 - 40% of patients, an ostensibly successful
initial dilatation of the artery may be followed by a renarrowing of the vessel
(restenosis) some 3 to 9 months later. If this restenosis is severe, the patients
may require a second angioplasty procedure, often with implantation of a stent to
act as a scaffold in the vessel.
In other cases arterial reconstruction under by-pass surgery, which is a higher
risk procedure, may be required. With more than 800,000 PTCA procedures now
performed world-wide annually, the socio-economic implication of this 30 - 40%
restenosis rate has become a matter of serious concern to interventional
Restenosis is often the result of the balloon-mediated stretch and crush injury to
the arterial wall and the possibility that the guide wires of the catheters used in
such procedures during deployment cause injuries and can lead to proliferation of
the smooth muscle cells of the artery, resulting in reclosure of the artery
("restenosis") over the following months.
Because of the potential complications, related to restenosis and the fear of
dislodging an embolus from an ulcerative plaque and the severe resulting
consequences, the application of repetitive angioplasty in the carotid arteries are
severely limiting the options for minimally invasive intervention.
Attempts heretofore have been made to treat occlusions in the carotid arteries
leading to the brain by a other types of interventions.
Carotid Endarterectomy is a surgical method for removal of blockages from the
carotid arteries and is one of the most common vascular surgical procedures
performed in the United States. Results from multicenter trials have demonstrated
the efficacy of this procedure in the treatment of extracranial carotid disease in
both symptomatic and asymptomatic patients (JAMA 1995; 273:1421-28; N Engl.
J. Med. 1991; 325:445-53) and Endarterectomy procedures are used in the
treatment of occlusive vascular disease in other vascular beds (Vase. Surg.
In endarterectomy, the carotid artery is slit and plaque is removed from the vessel
irrthe slit area. In a surgery, the carotid bifiircation is exposed through an incision
in the neck ofthe patient and clamps afe-placed on either side of the occlusion to
isolate it, and an incision made to aperrthe artery. The occlusion is removed, the
isolated area irrigated and aspirated, and the artery sutured closed. The clamps
are removed to reestablish blood flow through the artery. The emboli and debris
contained within the clamps may cause considerable-problems after the carotid
artery is opened, allowing blood to flow into the previously isolated are.
While endarterectomy is an effective therapy, it often leaves the adventitia and a
significant area of thrombogenic subendothelium exposed. Despite the efficacy of
carotid endarterectomy, the operation can lead to complications including
thrombosis and the development of intimal hyperplasia causing complications
that undo the beneficial effect of the intervention, or create new problems.
Clinical studies have demonstrated a post carotid endarterectomy stroke rate of
1-10% in the immediate postoperative period, almost all of which is accounted for
by thrombus formation at the endarterectomy site with subsequent cerebral
embolization (Stroke 1984;15:950-55). Platelet accumulation may also result in
restenosis due to intimal hyperplasia development, which may occur within two
years postoperatively. Restenosis has been reported to occur in 10-20% of all
endarterectomized patients at 2-5 years postoperatively, most of which is due to
intimal hyperplasia, intimal thickening, and vessel diameter reduction as
documented by carotid duplex ultrasonic scanning (J. Vase. Surg. 1986;3:10-23).
The cellular and molecular response of the vessel wall to mechanically induced
trauma, surgical intervention, stent placement, placement of a vascular graft
(arterial, or arteriovenous graft e.g.,. dialysis graft) is a complex interaction of
inflammation, smooth muscle cell migration, proliferation and myofibroblast
transformation that occurs as soon as the trauma occurs (Futura;1997.p.289-
317). If the artery is severely damaged by disease, and perhaps hardened by
calcium deposition, intervention may also cause some degree of additional injury
with local de-endothelialisation and exposure of underlying extracellular matrix
components such as collagen and elastin. In some patients the excessive
recruitment of platelets and fibrinogen can then result in an acute thrombotic
Studies using endarterectomized rat carotid artery models (Neurosurg
19B5;16:773-79), as well as embolectomy balloon injury models (Lab. Invest
1983;49:327-33) have shown that as the endothelial ceils are steiped away during
vascular injury, platelets begin to adhere to the exposed subendothelium.
Spalloneet. al. (Netirostrrg 1985;16:773-79) using scanning electron microscopy
have shown that five minutes after a carotid endarterectomy in a rat, a monolayer
of platelets is formed over the injured area. Fifteen minutes- after the injury,
platelet aggregation and thrombus formation are-observed. Thirty minutes post
endartefeetomy, the site is covered with activated platelets and coated by fibrin
and red blood cells. Thrombus formation reaches its peak at three hours post
injury with a thick fibrin-platelet layer being observed. Platelets are an integral
component of this thrombus formation and hence thrombosis, but they also
appear to play a role in the development of intimal hyperplasia.
Studies using thrombocytopenic rats have further demonstrated a significant
decrease in intimal thickening following carotid artery injury as compared to
control rats (Proc. Natl. Acad. SCI USA 1989;86:8412-16). Once platelets adhere
to the exposed subendothelium of an injured vessel, they become activated and
release their granules. These granules contain vasoactive and thrombotic factors
(serotonin, ADP, fibrinogen, Von Willebrands factor, thromboxane A2), as well as
growth factors (platelet derived growth factor, transforming growth factor-beta,
and epidermal growth factor) (Circulation 1985;72:735-40). The exact
mechanisms by which platelets enhance the development of intimal hyperplasia
are not yet completely understood. Studies suggest that platelets provide
primarily a chemotactic stimulus for medial smooth muscle cell migration toward
the intima during the second phase of intimal hyperplasia development (Vase.
Surg. 1991;13:885-91). Other studies, using anti-PDGF antibodies, have
demonstrated the vital role that PDGF plays in neointimal smooth muscle cell
accumulation following a vascular injury (Science 1991;253:1129-32). Another
mechanism by which platelets may enhance the development of intimal
hyperplasia is via the activation of the coagulation cascade and the subsequent
accumulation of thrombin at the site of injury. Several studies have demonstrated
the myogenic effects of thrombin on smooth muscle cells (J. Clin. Invest.
1993;91:94-98, J. Vase. Surg. 1990;11:307-13). In addition, thrombin has been
shown to be a stimulus for platelet activation. Irrespective of the precise
mechanism, platelet adhesion and activation atihe site of a vascular injury play a
significant role in the development of thrombosis and intimal hyperplasia and
hence inhibition of platelet adhesion and activation may help, prevent or reduce
thrombosis rates and intimal hyperplasia development.
The adherence of platelets to the injured arterial wall is mediated in the first
instance by von WUiebrand factor (vWF), a muKimeric glycoprotein that is
released from endothelial cells and circulates in the plasma, where it functions as
a carrier protein for factor VIII (Annu. Tev. Biochem. 1998;67:395-424). Highly
multimerized vWF also circulates contained within alpha-granules of platelets,
from where it is released foUosniflg platelet activation (Annu. Tev. Biochem.
19^-67:395-424). Under elevated: steesr coraiitions, such as those encountered
in arteries at sites of atheromatous plaque or mechanical intervention, vWF may
bind, via its A3 domain, to surface-exposed collagen fibers (Biochemistry
1986;25(26):8357-8361, Blood 1987;70(5): 1577-1583, J. Biol. Chem.
1987;262(28):13835-13841). Collagen-bound vWF in turn then "tethers" platelets
via shear-dependent exposure of an epitope in the vWF-A1 domain, which
interacts with platelet GPIb/IX/V (Blood 1985;65(1):85-90, Blood 1985;65(4):823-
831, Br. J. Haematol 1986;63(4):681-691). Thus vWF acts as a bridge between
collagen and platelets and is a prerequisite for the adhesion of platelets to
collagen under flow (J. Lab. Clin. Med. 1974;83(2):296-300). Platelet rolling over
vWF results in weak adhesion, however, and additional, direct interactions
between collagen and other receptors on the platelet surface are required in
order to facilitate permanent platelet adhesion, activation and aggregation
(Thromb. Haemost 1997;78(1):434-438, Thromb. Haemost 1997;78(1):439-444).
Direct collagen receptors on platelets include GP VI (Blood 1987;69(6):1712-
1720, Thromb. Haemost 1999,81 (5):782-792, J.Clin. Invest. 1989;84(5):1440-
1445), GP la/lla (cfe/BO (J.Clin. Invest. 1989;84(5):1440-1445, Nature
1985;318(6045):470-472), and to a lesser extent GP IV (CD36) (J. Biol. Chem.
1989;264(13):7576-7583) and perhaps even p65 (J. Clin. Invest.
1997;100(3):514-521). In the absence of vWF-assisted platelet binding, these
receptors have proven to be too weak in mediating platelet recruitment to
collagen in flow (Br. J. Haematol 1986;63(4):681-691). Finally vWF, in
combination with fibrinogen, facilitates the cross-linking and further activation of
platelets via binding to platelet GP llb/llla (J. Clin. Invest 2000;105(6):783-791),
providing stability and strength to the developing thrombus.
With the advent of platelet GP llb/llla and ADP receptor antagonists great strides
forward in anti-aggregatory therapy have been made in recent years (Coronary
Art Dis 1999;10(8):553-560, J. Am, Coll. Surg. 2000;191(1):76-92). However,
these strategies afe not designed to inhibit the initial adhesion of platelets to
exposed collagen fibers, and despite the efficacy of GP llb/llla antagonists in
attenuating platelet-platelet interactions, platelets still adhere to the injured vessel
wall (Blood 1993;81(5):1263-1276, Circulation T995;91 (5): 1354-1362).
Furthermore, platetet^ctivaticai^Imc^txertainly extends beyond aggregation and
acute thrombosis, the progression of sub-acute and chronic intimaJ hyperplasia
being at least partially affected by mitogenic mediators such as platelet-derived
growth factor (PDGF), released by the activation of platelets. Indeed, the
inhibition of PDGF has been demonstrated to reduce infimal hyperplasia in
various animal species (Science 1991 ;253(5024):1129-1132, Circulation
The pathophysiological importance of vWF is suggested by the increase in
circulating vWF in patients with acute myocardial infarction (Thromb Haemost
2000;84:204-209, Circulation t998;98(4):294-299), with vWF levels being
positively correlated with poor subsequent prognosis (Circulation 1998;98(4):294-
299). In vivo studies have further shown that neutralizing anti-vWF antibodies
inhibit experimental thrombosis, confirming the essential role of vWF in thrombus
formation (Thromb. Haemost 1998;79(1):202-210). Furthermore, with the ever
more widespread use of angioplasty techniques in acute coronary syndromes,
which invariably results in damage to the vessel wall and exposure of collagen,
there is an increasing need for strategies that intervene pharmacologically as
early as possible during the platelet adhesion-activation-aggregation cascade.
The two main lines of therapy, which are currently being used in an attempt to
control platelet adhesion, activation and subsequent thrombosis and intimal
hyperplasia, are anti-platelet agents and anti-thrombotic administration. Although
drugs such as aspirin effectively block the synthesis of Thromboxane A2 through
inhibition of the cyclooxygenase pathway, they do not prevent the collagen-
induced platelet adhesion and activation, which stimulate the development of
intimal hyperplasia. The use of heparin as an antithrombotic agent is associated
with complications and limitations including a non predictable dose response,
need for dose laboratory monitoring, limited activity against clot bound thrombin,
multiple inhibitory sites, antithrombin III dependency, a risk of major bleeding, as
well as a need for continuous infusion. Clearly, an ideal therapeutic agent would
be one that produces site specific and localized effects without systemic
distribution or a generalized coagulopathy.
It appears that the vital steps which precipitate the cascade of events leading to
thrombosis and later intimal hyperplasia stem from the interaction between the
exposed subendothelial collagen at the site of vessel injury and a monolayer of
platelets which adhere to the exposed collagen. Hence a specific inhibitor of the
platelet to subendothefel coHagen adhesion may serve to prevent or at least
decrease the development of thrombus and intimal hyperplasia.
Several leech-derived substances have been reported to inhibit collagen-platelet
interactions (Blood 1995; 85I3):705-711, Platelets 2000; 11(2):83-86, J. Biol.
Chem. 1992; 267(10):6893-6898, J. Biol. Chem. 1992; 267(10):6899-6904, Blood
Coagol-Btbrinolysis 1991, 2(1): 179-184). Ctestabtese, an isopeptkdase with fibrin
depoiymerising activity isolated from Htrudo medicinaiis, has been reported to
inhibit platelet aggregation induced by various agonists, including collagen, but is
believed to bind directly to the platelet membrane (Platelets 2000; 11(2):83-86).
Leech antiplatelet protein (LAPP), a -13 kDa protein from the saliva of
Haementeria officinalis, inhibits platelet adhesion to collagen under static
conditions (J. Biol. Chem. 1992; 267(10):6899-6904, Thromb. Haemost 1999,
82(3): 1160-1163) and elevated flow (Arterioscler Thromb. Vase. Biol. 1995,
15(9): 1424-1431), with effects on both vWF- and platelet GP la/lla-mediated
binding to collagen (Thromb. Haemost 1999, 82(3): 1160-1163). Calin is a -65
kDa protein from Hirudo medicinalis for which a similar profile has emerged. Calin
also inhibits collagen-platelet interactions under both static and flow conditions
(Blood 1995; 85(3):705-711, Blood Coagul Fibrinolysis 1991, 2(1 ):179-184,
Thromb. Haemost 1999, 82(3): 1160-1163). Furthermore, both LAPP and Calin
are potent inhibitors of collagen-induced platelet aggregation, inhibiting
aggregation at concentrations similar to those which block vWF binding to
collagen (J. Biol. Chem. 1992; 267(10):6893-6898, Blood Coagul Fibrinolysis
1991, 2(1):179-184, Blood 1995, 85(3):712-719).
Both LAPP and Calin have been evaluated in models of thrombosis in vivo, with
mixed success. LAPP failed to reduce thrombus formation on collagen-coated
grafts in a baboon arterio-venous shunt despite the use of doses that inhibited
collagen-induced platelet aggregation (Arterioscler Thromb 1993, 13(11): 1593-
1601), whereas Calin dose-dependently inhibited thrombus formation in a
hamster model of venous thrombosis (Blood 1995, 85(3):712-719).
Non thrombogenic and anti thrombogenic coatings for stents and catheters are
known in the art. The non thrombogenic coatings and products are based on
modified and advanced polymers and examples are given in WO9301221 and
Anti thrombotic and anti restenosis coatings are in general biocompatible
coatings that may also serve as reservoirs for local drug delivery. The coatings
are mainly based on hydrogels and examples in the patent literature of methods
for preparing various types of hydrogels and coating medical devices include
W09211896, W09811828, WO0147572, EP0887369 and WO0139811.
The release profile of the therapeutic substances that are contained within the
coating can be adjusted for example by varying the thickness of polymer layers or
by selecting specific polymeric coatings that contribute selected physikochemical
properties (such as charge, hydrophobicity, hydrophilicity) and/or by preparing the
coating as different layers. The criteria for selection of the polymer and the
optimisation of release rate are understood by one of ordinary skill in the art.
Other coatings are described by Fischell Circulation, 1996, 94:1494-95), Topol et
al (Circulation, 1998, 98:1802-20) and McNair et al in device Technology, 1996,
The use- of stents, wires and catheters in the cardiovascularsystern is common
practice and vessel wall injury, embolization and the following restenosis are a
major concern of cardiologists during and after surgical intervention or
catheterizations. Alternative methods such as endarterectomy cause comparable
problems. Every procedure in which arteries are manipulated i.e. vascular
surgery ancLangioplasty, is susceptibie^to the development of intimal hyperplasia.
The utility of a method for preventing or decreasing the development of intimal
hyperplasia can not be over emphasized, and the benefits of a method capable of
doing this without producing systemic effects is even more encouraging.
Therefore the nesed for new and improved pharmaceuticals and methods for
inhibiting the earliest events in pathophysiology (e.g. platelet adhesion) is obvious
and contributions in this field are expected to substantially decrease morbidity
and mortality associated with angioplasitic or surgical procedures.
Description of the invention
In general, the present invention involves the introduction of the recently
described platelet adhesion inhibitor Saratin into or onto a selected location within
or on a lumen in a tissue, i.e. the vasculature or an organ under conditions such
that Saratin may be used locally as a topical agent or as an adherent coating on
the surface to prevent and inhibit an undesirable thrombotic and/or restenotic
response to vessel wall injury, including injury associated with angioplasty, stents,
dialysis graftsand other vascular grafts, and the treatment of benign hypertrophic
scar formation as well as the treatment and passivation of unstable
Saratin is a recently described (WO0056885) recombinant 12 kD, protein
originally isolated from a leech. The protein inhibits vWF-dependent binding of
platelets to arterial wall collagens under conditions of elevated shear and it is this
aspect of the invention that makes Saratin suitable to inhibit arterial thrombosis.
Another novel aspect is that Saratin can be used as a topical agent at the site of
injury with the benfrt of decreasing thrombosis and or intimal hyperplasia without
any systemic effects. This represents a modality with specific and localized
effects well suited for application by both surgeons and interventional radiologists
Saratin can be combined wife a variety of therapeutic agents for on-site delivery.
Examples for use in coronary-artery applications are antithrombotic agents, e.g.,
prostacyclin and salicylates, thrombolytic agents, e.g., streptokinase, urokinase,
tissue plasmirrogerractivator (TPA) and anisoylated plasminogen- streptokinase
activator comptex (APSAC), vasodilating agents, i.e., nitrates, calcium channel
blocking drugs, antF-proiiferative agents, i.e., colchicine and alkylating agents,
intercalating agents, growth modulating factors such as interleukins,
transformation growth factor-beta and congeners of platelet derived growth factor,
monoclonal -antibodies-directed=against growth factors, anti-inflammatory agents,
batrrstecoidal and non-steroidal, and-c#ier agents that can modulate vessel tone,
function, arteriosclerosis, and the healing response to vessel or organ injury post
intervention. Antibiotics can also be included in combinations or coatings
comprised by the invention. Moreover, a coating can be used to effect
pharmaceutical delivery focally within the vessel wall. By incorporation of the
active agent in a swellable polymer, the active agent will be released upon
swelling of the polymer.
In one embodiment the coating is made from a hydro-gel, such as poly-ethylene
oxide, albumin, hydrophilic poly-methacrylates and hydrophilic poly urethanes.
The present invention further provides the use of Saratin and derivatives via local
drug delivery devices/catheters or via stents and stent coatings and vascular
grafts and graft coating technologies, for example. The invention also provides
methods of administering Saratin in compositions that elute out regulated
quantities of the Saratin over time in a localized area.
In particular, one embodiment of the present invention relates to uses of catheter-
based devices to deliver Saratin locally. Saratin may as well be applied with or
without other therapeutic agents out of a polymer matrix into body tissues using
catheters. The basic requirements for the polymer material to be used in the
present method are biocompatibllity and drug release properties which can be
adapted to the specifc application.
The local controlled Saratin release may be achieved by permeation only,
iontophoresis only, electroporation only, or combined iontophoresis and
electroporation may be used to release and incorporate Saratin efficiently inside
the vessel lumen. Preferably, the catheter is able to perform procedures designed
to maintain a high concentration of drug in the selected vessel space such that
the results give an improved vessel coating with Saratin alone or with additional
The present invention is particularly applicable to the local delivery of Saratin
during and after interventional cardiology procedures such as angioplasty and
stent implantation and endarterectorny.
Detailed description of the invention
Suitable recombinant Saratirtfor use in the iiweHtion^was expressed and isolated
from Hansenuia polymorph® and was found to acts by blocking vWF binding to
collagen and effectively prevents the adhesion of platelets to collagen under
elevated shear. Saratin dose-dependently inhibited the binding of purified human
vWF to human type I and III collagens (IC50 = 0.23 ± 0.004 and 0.81 ± 0.04 ug ml'
1 respectively ) and to calf skin collagen (IC50 = 0.44 ± 0.008 ug ml-1).
Furthermore, saratin showed a similar inhibitory potency against the binding of
human, rodent and porcine plasma vWF to these collagens. In a flow chamber
under conditions of elevated shear (2700 s"1), saratin dose-dependently and
potently inhibited platelet aggregate formation on a collagen-coated surface (IC50
= 0.96 ± 0.25 ug ml"1), but at reduced shear (1300 s"1) a rightward shift in the
dose-response curve was noted (IC5o = 5.2 ± 1.4 ug ml"1). Surface plasmon
resonance analysis revealed both high and low affinity binding sites for saratin on
human collagen type III (Kd = 5 x 10"8 M and 2 x 10"6 M respectively), and
although low concentrations of Saratin, that inhibited platelet adhesion under
increased shear (i.e. saturation of high affinity binding sites) had no effect on
vWF-independent collagen-induced platelet aggregation, high concentrations (i.e.
saturation of low affinity binding sites) were found to inhibit platelet aggregation.
These data demonstrate that saratin is a potent inhibitor of vWF-dependent
platelet adhesion to collagen which forms the basis for the therapeutic potential
as an anti-thrombotic agent.
The studies further demonstrated that Saratin potently and dose-dependently
inhibits the binding of vWF not only to calf skin collagen, but to human collagen
types I and III, both of which are abundant in arterial wall sub-endothelial layers
and believed to be important in platelet-vessel wall interactions (Thromb Haemost
1997, 78(1):434-438). Since collagen-vWF-GP Ib/IX/V interactions only take
place at sufficiently elevated shear, it was an important aspect of this invention to
demonstrate the efficacy of saratin not only under static conditions, but also in-an
environment that more closely mimics the situation encountered in vivo. In a flow
chamber, where shear forces can be varied to simulate such an environment,
saratin cieariy inhibited platelet accumulation to coHagen, particularly at higher
shear. The rightward sfe&tjof-the. dose-response curve as a result of reducing
shear is nc4eworthy since the implication is that the efficacy of saratin in vivo may
be localized to areas of high shear, where disruption of laminar blood flow results
from changes to the endothelial surface presented to the blood, for example in
the presence of aterosclerotic plaque or after mechanical intervention.
Surface plasmon resonance studies with saratin revealed that collagen may
possess two independent binding sites for saratin, one of high affinity and one of
low affinity. Inhibition by saratin of vWF binding to collagen is explained by
saturation of the high affinity binding site, with IC50 values around 5 x 10"8 M, i.e.
equal to the dissociation constant for this site. Because vWF-independent
collagen-induced platelet aggregation was only inhibited at very high doses of
saratin, (above 100 uM), it seems that saturation of the low affinity collagen
binding site is associated with inhibition of direct collagen-collagen receptor
Another objective in this invention is the effect of Saratin to affect the local
environment of an endarterectomized vessel without requiring systemic
distribution and without altering platelet function or altering coagulation this
makes it an ideal modality for topical application during vascular surgical and
interventional radiological procedures.
In this spect of the invention the therapeutic Saratin effect was investigated using
a rat carotid endarterectomy (CEA) model described previously (J. Vase. Surg.
1998, 28:909-918). PLT activation is thought to be the initiating step in post CEA
thrombosis and restenosis due to IH. It has been observed, that the topical
application of Saratin on the lumenal surface will decrease the amount of platelet
adhesion to the endarterectomized artery and thus decrease post-op thrombosis
As a summary of theWeikneiital results the anti platelet adherence effect of
Saratin evaluated after two different postoperative times indicated that the
number of adherent platelets at 3 hours (Figure 4) and 24 hours (Figure 5) post
carotid endarterectomy was significantly reduced in the rats treated with Saratin
as compared to the control rats.
Platelet adhesion was reduced by 59% at 3 hours (64+ 17.2 vs155 ± 33.4 PLT
per grid P=0.05) and 77% at 24 hours (35 ± 11.3 vs 149 ± 36.6 PLT per grid P =.
0110) PlateJet adhesion was similar at 3 hours and 24 hours in the control groups
but did show a decrease in the Saratin treated group from 64 to 35 platelets per
Figures 6 and 7 show a typical representation of the endarterectomized surface
with and without topical Saratin using scanning electron microscopy at 2000 X
magnification. Figure 6 A, shows the control surface at 3 hours post carotid
endarterectomy, note the marked abunda«Ee~of cellular material , fibrin strands,
numerous red blood ceHs, and numerous platelets which are evident. Figure 6 B
shows a Saratin treated surface 3 hours post carotid endarterectomy. Note the
lack of cellular elements and the near barren collagen surface. Figure 7 A shows
a control surface at 24 hours post carotid endarterectomy, platelets are evident
as smalt white dots. Figure 7 B is a Saratin treated surface at 24 hours post
carotid endarterectomy. There is a clear reduction in platelet adhesion with
The application of topical Saratin following a carotid endarterectomy significantly
decreased the development of intimal hyperplasia as compared to the control
group. Percent lumenal stenosis as a measure of IH was significantly decreased
with Saratin application compared to controls This decrease in IH formation
correlated with the inhibition of PLT adherence. In terms of lumenal stenosis,
control rats showed a 29.8 ±6.8%, p=0.0042 lumenal stenosis vs. a 10.9 ± 1.8%
lumenal stenosis in the Saratin treated group (Figure 6 ). Saratin treated rats had
18.9% greater lumenal diameter than control rats. At two weeks post carotid
endarterectomy, 5 of the 15 control rats (15%) developed complete thrombus of
the carotid artery on histological analysis while 0 of 15 Saratin treated rats (0%)
developed carotid thrombosis. A likelihood ratio Chi Square analysis revealed an
odds ratio of 16.238 which suggests that the odds of the control group developing
occlusive thrombosis was 16 times greater than the rats treated with Saratin P =
No increased bleeding was encountered aJong the arterial suture line in the
Saratin group. Bleeding times and systemic platelet counts were not found to
change significantly in the Saratin treated rats as compared to control rats at 3
and 24 hours post endarterectomy.
The CEA model closely resembtes the human CEA operation and hence the
results propose a mechanism for decreasing the deleterious effects of platelet
adherence and aggregation at the endarterectomy site without affecting platelet
function systemicaHy or decreasing bemostasis. The simple, topical application of
Saratin during a rat CEA decreases platelet adherence, aggregation and
subsequent restenosis in tbexatr-oUdijed due to intimal hyperplasia.
Table 3 shows the results of the bleeding times and platelet counts. No
statistically significant differences weFe observed between pre-op and post-op
bleeding times. No stafisticaBy significant difference was- identified in the platelet
courrbdffierjences between the Saratin and control rats.
We have shown a significant decrease in platelet adhesion and accumulation
after a vascular injury similar to endarterectomy. The decreased platelet adhesion
is seen both immediately after endarterectomy (3 hours) as well as at 24 hours.
The effect at 24 hours is significant in that we believe this effect is not due to the
direct inhibitory effect of Saratin on the collagen (the serum half life of Saratin is
90 minutes), rather it is due to the initial inhibition of platelet aggregation and the
subsequent disruption of the platelet activation cascade. Once platelets are
initially inhibited from attaching to exposed collagen, the platelet cascade can not
proceed. Figures 4 and 5 demonstrate that the topical application of Saratin on
the recently injured vessel can inhibit platelet adhesion to a significant degree.
Saratin inhibited platelet adhesion by 60% and 75% at 3 and 24 hours
respectively. This inhibition is noted in the visible differences in cellular element
deposition between control and Saratin treated arteries at both 3 hours and 24
hours (Figures 7 and 8). This lack of cellular response we propose is due to the
inhibition of platelet adhesion. This represents a unique mode of therapy for the
inhibition of platelet adhesion to an injured vessel. Control rats had a significantly
reduced lumenal diameter at 2 weeks post carotid endarterectomy compared to
Saratin treated rats (Figure 9). The amount of intimal hyperplasia development
was significantly reduced in Saratin treated rats, which correlated with reduced
platelet adhesion and accumulation. The finding of decreased intimal hyperplasia
and thrombosis that are associated with decreased platelet adhesion provide for
clinically relevant endpoints and sequala of reduced platelet adhesion This
demonstrated that the site specific non systemic inhibition of platelet aggregation
and adhesion leads to a decreased thrombosis and occlusion rate and hence
reduces the incidence of post carotid endarterectomy related cerebrovascular
events. The level of intimal hyperplasia and lumenal stenosis improvement is
further demonstrated by the significant finding of a decreased thrombosis rate in
rats treated with Saratin. A full 33% of control rate showed thrombosis whereas
none of the Saratin treated carotid arteries were thrombosed. Of particular clinical
importance is the lack of systemic effects demonstrated by this agent. The local
application of Saratin did not affect systemic Weeding times or platelet counts.
Thisr implies that the decreased pJateJeLadheston andntbe subsequent decrease
in thrombosis and intimal hyperplasia ace the result of local effects. These new
findings related to Saratin are certainly important in that the-spectrum of clinical
applications for a modality that is capable of locaHy inhibiting the deleterious
effects of platelet adhesion and activation without disturbing the systemic
hemostatic mechanism, are many.
It has been pointed out earlier that various therapeutic interventions induce local
injuries which would ideally be treated immediately and locally.Left untreated the
injured cells initiate a series of processes involved in clotting, complement
activation, and cellular response to release of cytokines, induction of proliferation,
and other biologically active processes. It is difficult to stop these complex,
interrelated processes once they have begun.
In the present invention it is therefore an important aspect that Saratin is located
directly in the manipulated tissues. Another aspect of the local application is the
minimization of potential problems related to the systemic effects of the drugs
used for intervention.
Idealy Saratin treatment may be administered simultaneously with the appropriate
therapeutic intervention which may be achieved by incorporating Saratin into the
coating of a surgical device such as a ballon catheter or another device or part
thereof. Another aspect could also involve the direct coating of the injured vessels
Additionally, normal Saratin delivery means may be used in the invention as well,
such as free fluid form, including combinations with other therapeutic agents.
However, use of poh/mer/hydrogel matrices has certain advantages over free
fluid delivery. Delivery of an agent which has been incorporated into a polymer
matrix does not require additional lumens in the support catheter to convey the
free fluid drug solution into and out of the treatment site.
Additionally, the polymer matrices eliminate the risk of downstream leakage of
drug solution due to defective balloon sealing of vessel segments, thereby
avoiding the risk of exposure of non-target tissue to high concentrations of the
A general technical solution to the local application of Saratin either on a medical
device or as a coating to the injured, vessel is for example the incorporation of
Saratin into a polymer or hydrogel coating.
With respect to the polymer composition, the term "hydrogeF as used herein
includes synthetic polymers with pores or interstices of different sizes and
capacities and varying physicochemical properties especially with respect to the
charge or the hydmphific/hvjiBophobic nature of the gel matrix which may be
introduced during manufacture- of-the coating or coated device. A variety of
synthetic elastomers and naturally occurring polymeric materials are known to
those skilled in the art. Saratin can be incorporated in the matrix either during
polymer production or added after coating or molding of the polymer into the
desired shape. Additionally, many of a number of different polymeric materials
and methods of fabrication may be used to form the polymer matrices used in the
present invention. Examples of suitable polymer materials or combinations
include, but are not limited to, biocompatible and/or biodegradable polymers.
Several alkyl alkyl- and cyanoacrylates have been investigated for surgical use
and some isobutyl cyanoacrylates have been found especially suitable.
A typical hydrogel polymer may be produced from a monomer mixture
comprising 40-60 parts by weight of a purified monoester of a hydroxyalkyl alkyl
acrylate having a single olefinic double bond, 40-60 parts by weight of a
methacrylic monomer containing one olefinic double bond, and 0.001-5 parts by
weight of a polymerization initiator. Polymerization may be accomplished by the
conventional techniques of bulk polymerization, solution polymerization,
suspension polymerization or emulsion polymerization. The polymerization
technique used is dependent upon the volume of polymer required and the nature
of the final product being produced. A typical hydrogel product would be
described by a molar ratio of monoester to methacrylic monomer within the range
of 1:1 to 2.3:1, preferably 1.5:1, wherein the pore diameter of the polymer is
greater than 90 Angstroms.
As the monoester of a hydroxyalkyl acrylate having a single olefinic double bond,
acceptable compounds include, but are not limited to, 2-hydroxyethyl
methacrylate, glyceryl methacrylate, 2-hydroxypropyl methacrylate, glycidyl
methacrylate, 2-hydroxyethyl acrylate, and 2-hydroxypropyl acrylate. Acceptable
methacrylic monomers are methacrylic acid, methacrylamide 5 and
The polymerization initiator may depend on the method of polymerization or the
final intended use of the polymer. For example, where the polymer is to be
formed as a soM object, free radical initiators may be used.
Preferred initiators of that type include difunctional polyesters such as 2,5-
Dimethyl-2,5-bis(2ethylhexoylperoxy)hexane, or tertiarybutyl peroxypivHate.
Afterrratfvely, where the ultimate use of the polymer is as a coating applied in the
form of the monomer mixture and polymerized in situ, the initiator may be
radiation activated such as UV catalysts 2,2Azobis(2-methylpropionitrile) or
azobisbutyronitrile (AIBN). The initiators are not restricted to use in a particular
polymerization method or for a particular final product. For example, the free
radical initiators may be employed in coatings and the radiation activated
initiators may be employed in the formation of solid articles.
In addition to the substantially similar fractions of the monoester and methacrylic
monomer, the monomer mixture may be enhanced with trace amounts of a longer
chain alkyl acrylate or methacrylate ester comdnomer such as cyclohexyl
methacrylate, trimethylolpropane trimethacrylate or ethyleneglycol
dimethacrylate. Such additional comonomers enhance the polymer crosslinking
for situations where added polymer strength is desired. The trace amounts of
these comonomers are generally less than 0.1% by weight of the total monomer
The hydrogel polymers used in the present invention may be formed to produce
an article which is sufficiently crosslinked by intrinsic action that the resulting
article requires no additional crosslinking monomers.
Additional examples for biodegradable polymers are poly(lactides),
polyglycolides, polyanhydrides, polyorthoesters, polyactals, polydihydropyrans,
polycyanoacrylates and copolymers of these and polyethylene glycol. These can
take the form of co-polymer hydrogels or cross-linked polymer networks into
which drugs for enhanced local delivery can be incorporated either during
polymerization or, in the case of certain hydrogels, loaded subsequently.
Preferable matrices would be tailored according to the molecular characteristics
of the agent to control free diffusion outwards.
Multiple types of catheters and other medical devices coated with Saratin may be
constructed and used according to the individual therapeutic need they have
been designed for, or for the more specific purpose of delivering the Saratin
loaded polymer locally: The present invention has been tested with the ballon
catheter sold under the name Biue medical Devices BV GO available from Blue
medical but is not limited to this type of catheter.
Purified vWF and platelet binding to collagen under static conditions
vWF binding to collagen was investigated in microtiter plates, essentially as
described elsewhere (Blood 1995, 85(3):705-711). Various collagens, either
human collagen type I, type 111, or calf skin collagen (Sigma) were coated
overnight by pipeting 50 ul of collagen, dissolved in 50 mM acetic acid at 125 ug
ml"1 in 96-well microtiter plates in the presence of 200 ul PBS, to promote
renaturation. Following blocking non-coated sites with BSA, purified human vWF
(1.25 ug ml"1 for calfskin and collagen I coated plates; 0.625 ug ml"1 for collagen
III coated plates) or diluted normal plasma (human plasma: 1/80 on collagen I
and III, 1/40 on calfskin collagen; hamster, mouse and porcine plasma: 1/80 on
collagen III; 1/20 on collagen I and calf skin collagen) was added to the wells in
the presence of Saratin and incubated for 2 hours (diluted plasma) or 1 hour
(purified vWF). Binding of residual vWF to collagen was determined after a further
washing step using rabbit anti-vWF antiserum (Dako, Copenhagen, Denmark)
conjugated with horseradish peroxidase, which was developed and the resulting
light absorbance measured at 492 nm.
When microtiter plates were pre-incubated with either human collagen I, human
collagen III or calf skin collagen, the subsequent incubation of purified vWF in the
presence of saratin was associated with a dose-dependent inhibition in vWF-
collagen binding, with IC50 values of 0.23 ± 0.004, 0.81 ± 0.04 and 0.44 ± 0.008
ug ml"1 respectively (Figure 1). When purified vWF was replaced by diluted
normal human plasma the inhibitory potency of saratin was maintained (Table 1).
Saratin also inhibited the binding of vWF in diluted porcine, hamster and murine
plasma to the different collagens tested, with consistently highest efficacy against
collagen I (Table 1).
Platelet adhesion under-elevated shear
In view of the importance of shear forces in the control of vWF-dependent platelet
adhesion to collagen (J. Lab. Clin. Med. 1974, 83(2):296-3O0), flow chamber
perfusion studies were performed to examine the tahibiloiy acliorrof saratin under
similar flow conditions to those encountered in injured or diseased arteries. Caif
skin collagen was coated on to plastic coverslips as previously described (Blood
1995, 85 chamber witfi two coversiip holders, and chamber heights of 0.4, 0.6 or 1.0 mm
and a width of 1.0 cm, under pulsatile flow (rofler pump, Watson Marlow 6J03S,
VEL, Leuven, Belgium) at approximate shear rates of 2700, 1300 and 300 s"1
respectively. Anticoagulated whole blood (0.2 IU ml"1 low molecular weight
heparin, Clexane) was perfused over the collagen-coated coverslips for 5 min,
after which the rinsed coverslips were stained with GrQnwald-Giemsa and
assessed for platelet deposition by light microscopy and image analysis as
described (Blood 1995, 85(3):705-711), using surface coverage as a quantitative
At a shear rate of 2700 s"\ saratin dose-dependently inhibited platelet adhesion,
with an IC50 = 0.96 ± 0.25 ug ml"1 (Figure 2). However, at a more moderate shear
rate of 1300 s"\ a distinct rightward shift in the dose-response curve was
observed, with an IC50 = 5.2 ±1.4 pg ml*1 (Figure 2). At venous shear rates of
300 s"\ saratin was unable to inhibit the adhesion of perfused platelets up to 10
ug ml"1 (data not shown).
Binding Analysis by Surface Plasmon Resonance (SPR)
Protein interactions were identified and characterized by SPR using a BIAcore
3000 instrument (BIAcore, Freiburg, Germany). Coupling reagents were used
according to protocols developed by the supplier. Coupling to the CM 5 sensor
chip was done via activated carboxyfate groups to free amine groups of human
collagen type III (Sigma). The pH-scouting and the coupling chemistry was
performed under standard conditions (Anal Bfochem. 1991, 198(2):268-277, JAI
Press Ltd., 1992). For coupling the collagen was diluted to 0.125 ug ml"1 into 10
mM acetate buffer (pH 4.5), resulting in 331 resonance units (RU) immobilized
material. This matrix was used to study the binding of purified recombinant
saratin which was diluted into 20 mM Hepes (pH 7.4), 150 mM NaCI, 5 mM
EDTA, 0.005% Tween 20. AH binding experiments were performed at 25°C.
Titrations with Saratin were performed at concenfrations ranging from 7.8 nM to
10 uM . The resulting experimental RU plateau_values were plotted according to
Req/saratin concentration = (-K/vX Req) + (KAx R^a).
Titration of saratin onto the immobilized collagen surface resulted in
concentration-dependent binding: Farther., the maximal amoant~of sajafin bound
to collagen on the sensor surface resulted in a signal greater than the cafcutated
maximum signal for a 1:1 binding model. This points towards the existence of
more than one binding site for saratin on collagen type III, further substantiated
by the observation that fitting of the data to a 1:1 binding model according to
Langmuir gave no satisfactory results.
Scatchard analysis of the SPR data indicated the existence of two different
binding sites with significantly different affinities for saratin (Figure 3). Calculation
of the equilibrium constants resulted in a dissociation constant of 5 x 10'8 M for
the high affinity site and 2 x 10"6 M for the low affinity binding site.
The specificity of saratin on platelet function was further evaluated via
aggregation studies in platelet-rich plasma using collagen, ADP, ristocetin,
arachidonic add or the thromboxane mimetic U46619 as agonists. Citrated blood
(3.13%) from unmedicated, healthy volunteers was centrifuged (15 min, 100g) to
obtain platelet-rich plasma, to which saratin was added (final concentration 0-200
ug ml"1) for 1 rrrtn prior to induction of platelet aggregation with collagen (0.5 ug
ml"1), ADP (2.5 uM), ristocetin (0.9 mg ml"1), arachidonic acid (1.0 mM) or U46619
(1.3 uM). The maximum aggregation (amplitude) over a 5 min period was
observed for each agonist
Saratin failed to inhibit maximal aggregation to all agonists tested, including
collagen, at end concentrations up to 40 ug ml"1 (Table 2). Saratin was, however,
able to partially inhibit collagen-induced platelet aggregation at 100 ug ml"1 (not
shown), with complete inhibition at 200 ug ml"1, although aggregation to ADP was
unaffected, even at 200 ug ml"1 saratin.
Rat CEA model
A rat CEA model that employed an open technique with arteriotomy, direct
removal of intana and portions of media and suture closure of the artery was
used. One group of rats received Saratin white the other group served as
controls. Endpoint measurements included, 1) PLT adhesion, 2) Thrombosis
rate, 3) Intimal hyperplasia development. Saratin was applied directly to the
endaraterectomized surface of the carotid artery prior to arterial closure. Electron
micrographs (2000 X mag) of prepared carotid arteries were used for quantitative
analysis of PLT aggregation. Total PLT numbers were counted using a
standardized overlay grid. IH and thrombosis were assessed by computer
assisted morphometric analysis of elastin stained carotid artery sections with
direct measurement of IH area.
Carotid endarterectomy operation
Animals were sedated with isoflurane in a bell jar, weighed, and then
anesthetized with a combination of ketamine (100 mg/kg) and acepromazine
maleate (1 mg/kg) intraperitoneally. After adequate anesthesia was confirmed with
lack of response to hind paw stimulation, 4 cc of normal saline was injected
subcutaneously in the region of the upper middle back to serve as a fluid bolus
(10cc/kg) to compensate for any intra-operative blood loss. The neck area was
then shaved and preped with 7% isopropyl alcohol. Using sterile technique and a
dissecting microscope (x 40,sz40 Olympus, Olympus America Inc., Melville, NY)
a midline cervical incision was made. The superficial muscles were divided and
the dissection carried down to the level of the right carotid artery. The cervical
nerves in the region of the artery were dissected free to preserve pharyngeal
function and prevent post-operative respiratory compromise.
After adequate carotid artery exposure, proximal and distal control at the
bifurcation, approximately 1.5-cm apart, was obtained using 3-0 silk suture
tourniquets. Using a corneal blade an arteriotomy was made and extended to
6mm in length with micro- scissors. Using a 27-gauge needle, the intima was
transversely scored across the vessel in two parallel lines, approximately 2mm
apart. The intima and medial layer were removed with micro- forceps. Saratin
group rats received 5ul Saratin solution applied directly to the endarterectomizad
surface. The arteriotomy was closed with a running 10-0 monofilament nylon
suture (MS/9, Ethilon, Ethicon Inc., Somerville, NJ) beginning at the distal end.
The distal tourniquet was removed first" to assess for suture line hemostasis
followed by removal of the proximal tourniquet Time of Saratin application was 5
minutes, which represented the time of arteriotomy closure. Any suture line
bleeding was gently tamponaded with a sterile cotton tip applicator until
hemostasis was achieved. The endarterectomized carotid artery was assessed
with a hand held Doppler to confirm patency. The superficial muscle layer and
skin were then closed with a running 3-0 absorbable suture.
In the platelet adhesion subgroups, the rats were re anesthetized and the
endarterectomized carotid arteries were harvested and fixed in a 4%
glutaldehyde solution at 3 hours or 24 hours post carotid endarterectomy. During
the harvesting procedure, the carotid segments were opened longitudinally along
the site of the suture closure therefore exposing the endarterectomized area. The
arteries were then postfixed with osmium tetroxide, dehydrated in a graded
alcohol series, critical point dried with C02 (1072 psi and 31.1°C), coated with
gold palladium, and placed in the scanning electron microscope (JEOL JSM
5410, JEOL, USA Peabody, MA). The endarterectomized areas were scanned at
2000 X magnification and photographic images were obtained. The photographic
images were matched and arranged in a collage allowing visualization of a
greater area than was possible with a single field of the scanning electron
microscope monitor. Once the collage of photographs was assembled, it was
covered with a transparent overlay grid. The same overlay grid was used for all
specimen photographs, 116 squares were used to count the total number of
platelets in each photograph. The number 116 is the maximum number of
squares that could be consistently counted on all of the photograph collages.
Platelet counts were performed by two blinded observers..
At two weeks post carotid endarterectomy, rats in the intimal hyperplasia group
were anesthetized and the endarterectomized carotid artery exposed. The
abdomen was opened in the midline and the distal aorta and inferior vena cava
exposed The vena cava was transected and the distal aorta cannulated with a
20-^gafage calhelei to rnfixse norraaJ safee at 1O0 mm Hg until the vena cava
effluent ran clear. Next, 10% buffered formalin was infused-at 100 mm Hg in an
equal volume to complete the perfusion-fixation technique. A one-centimeter
section of the operated carotid artery was dissected free and placed in 10%
formalin until further processing for histology. The arteries were paraffin blocked,
sectioned, and elastin stained with Verhoeffs and Van Gieson's stain. Multiple
sections were taken at intervals of 3 micrometers each continuing along the
distance of the continuous 10-0 nylon suture arteriotomy closure to standardize
the region of sectioning. The elastin-stained slides were photographed using a
KODAK DC 120 Zoom digital camera (Eastman KODAK Company, Rochester,
NY). Any thrombosed sections were noted at this time. Non thrombosed images
were downloaded into a computer and the lumenal areas of the carotids were
analyzed using the National Institutes of Health (Bethesda, MD),lmageJ Software
program, Version 0.99i. This software package allowed us to delineate the inner
area of the intimal hyperplasia and thus obtain an accurate measure of the cross-
sectional area of the vessel lumen. Also, the outer area of intimal hyperplasia was
determined. The difference between the two areas (outer area of intimal
hyperplasia minus the actual lumen) was determined as the absolute area of
intimal hyperplasia. Because the arterial cross section had individual variations
of shape, the values were expressed as a ratio of the absolute area of intimal
hyperplasia to the outer limit of intimal hyperplasia, and was reported as a
percent lumen stenosis. This ratio represents the proportion of the lumen area
occupied by intimal hyperplasia and allowed for comparison of the arterial cross
sections of varying size(4). Minimal variability was seen between measurements
with two blinded observers.
Bleeding times-and platelet counts
In order to evaluate the effects of Saratin on systemic platelet counts and
bleeding times, the platelet counts and bleeding times of 12 rats were
determined. If it was necessary to obtain a preoperative blood sample from each
rat of approximately 1-1.5cc's, which represents a significant portion of a rats total
blood volume. In view of this, it is reasonable to expect a decrease in the post
operative platelet counts of-both control and Saratin rats. In order to assess the
effects of Saratin of platelet counts, we determined the difference in platelet
counts for each rat by subtracting the preoperative platelet count from the
postoperative platelet count to obtain a difference scone. Differences in platelet
ceunis-were analyzed using a 2_X 2 factorial arrangement of treatments ANOVA
model, which demonstrated no statistical significance in the platelet count
differences between control and Saratin treated rats. All rats had their platelet
counts and bleeding times determined preoperative^. Six rats underwent a
carotid endarterectomy and were harvested 3 hours post op with bleeding times
and platelet counts measured, and the remaining 6 rats were harvested 24 hours
post op with bleeding times and platelet counts measured. Three of the rats in
each time group received topical Saratin, and the remaining 3 rats served as
Bleeding times were determined by transecting the distal 2 mm of the rat's tail
and submerging approximately 4 em's of the tail into a solution of phosphate
buffered solution at 37°C. The amount of time elapsed from tail transection to
cessation of bleeding was measured and was assigned as the bleeding time.
Platelet counts were determined by drawing a 1-1.5cc sample of blood from the
internal jugular vein which was analyzed in a Coulter STKS blood analyzer and
the results were expressed x 103.
Means ± standard errors are reported. Unpaired t-test analysis were performed
using the Stat View program (SAS Institute Inc. Cary, NC 27513) version 5.0 to
compare Saratin treated and control groups for numbers of adherent platelets,
percent lumenai stenosis and bleeding times. A likelihood ratio Chi Square
analysis was used to evaluate the odds ratio for development of thrombosis two
weeks post carotid endarterectomy. A 2 X 2 factorial arrangement of treatments
ANOVA model was used to evaluate the differences between preoperative and
postoperative platelet counts.
Sprague-Dawley rats (350 - 400g) were organized into carotid endarterectomy
groups based on two mairr objects. 1) Evaluation of platelet adhesion and 2)
Evaluation of lumen stenosis due to intimal hyperplasia, and thrombosis rate.
Within these two objectives, rats were divided into control ancLSacatin treated
animals. All rats underwent a-carotid endarterectomy (see below), Saratin treated
rats received a topical application of 5ul solution of Saratin on the lumenai
surface of the carotid artery immediately after removal of the intimaVmedia layer.
The platelet adhesion group consisted of electron microscopy evaluation at 3
hours post carotid endarterectomy (n = 17) and 24 hours post carotid
endarterectomy (n = 19). The intimal hyperplasia group (n = 25) were harvested
two weeks post carotid endarterectomy.
Preparation of Saratin coated hydrogel catheter
Surfaces of PA-12 based materials have been activated by emersing the devices
in a solution of 2 mol-% macro-initiator (poly(octadecen-alt-malein acetic acid
anhydrid) with a per-ester (11-16 mol-%) dissolved in isopropanol and 0,5 mol
ethylenglykol-dimethacrylat (EGDMA) /1 mol macro-initiator. After drying the
macro-initiator-/EGDMA coating. The coating has been annealed another 5 bis
10 min at 120 °C which helped to improve fixation of the macro-initiator to the
surface thus improving crosslinking.
For optimising coating conditions hydrogel coatings generated on a PA-12 carrier
sheet have been used. Prior to coating, the sheets have been washed with iso-
propanol or aceton and dried.
Ballon catheter type Blue Medical Devises GO (RX PTCA catheter) consiting of
Vestamid (PA-12) have been used for coating with hydrogel. Aqueous solutions
of 5 mol-% acrylic acid 100ml and 0,2 bis 0,8 mol-% methylen-bis-acrylic have
been mixed and used to coat sheets or catheters. After polymerisation the
coated device has been washed with water and for another 24 hrs in PBS buffer.
Following this, the polymer is annealed in the oven at 60-80°C for 0,5 to 3 hours.
Thickness of dried hydrogel coatings was in a range of 1 to 4 um, the swelling of
hydrogel in aqueous solution ranged from 10 to 50 g hydrogel wet / 1g hydrogel
When immersed (30 min) in a buffered PBS buffer pH 7,4 solution of Saratin a
concentration of 50 ngml has been used.
Organic solvents have been used to prepare the coating solutions when sprayed
on a ballon catheter using standard small scale spray coating equipment like that
available from EFD.
Figure 1. Effect of saratin on purified human vWF binding to human collagen
types I (circles) & 111 (squares), and calf skin collagen (triangles). IC50 with type I =
0.23 ± 0.004 ug ml"1, type III = 0.81 ± 0.04 ug ml"1 & calf skin collagen = 0.44 ±
0.008 ug ml"1.
Figure 2. Effect of shear on the inhibition by saratin of platelet aggregate
formation on human collagen type III in a flow chamber in vitro. Circles indicate a
shear rate of 2700 s"1 (IC50 = 0.96 ± 0.25 ug ml"1), whereas squares indicate a
shear rate of 1300 s"1 (IC50 = 5.2 ± 1.4 ug ml"1).
Figure 3. Scatchard analysis of saratin binding to immobilized human collagen as
detected by surface plasmon resonance, indicating the presence of a high affinity
(Kd = 5 x 10"8 M, solid line) and a low affinity (Kd = 2 x 10"6 M, broken line) binding
site for saratin on collagen III.
Figure 4. Number of platelets adherent to the exposed subendothelial surface 3
post carotid endarteFectomy, comparing Saratin application (n = 7) and control
groups (n = 10). Data are means ± SE. The Saratin group received 5ul of Sasatin
solution applied topically to the exposed subendothelial surface. Star indicates a
P value of 0.05.
Figure 5. Number of platelets adherent to the exposed subendothelial surface
24 hours post carotid endarterectomy comparing Saratin application (n = 9) and
control groups ( n = 1X)). Data are means ± SE. Platelets were counted using the
scanning electron microscope. The Saratin group received 5ul of Saratin solution
applied topically to the exposed subendotfqelial surface. Star indicates a P value
Figure 6. Electron micrograph (2000 X) of an endarterectomized rat carotid
artery 3 hours post carotid endarterectomy. A, control surface. B, surface
receiving topical Saratin (5ul). The control surface shows abundant ceHular
elements including fibrin strands, red blood cells, and platelets. The Saratin
treated surface shows a marked decrease in cellular elements.
Figure 7. Electron micrograph (2000X) of an endarterectomized rat carotid artery
24 hours post carotid endarterectomy. A, control surface. B, surface receiving
topical Saratin (5ul). The control surface shows numerous red blood cells and
platelets. The Saratin treated surface shows a marked decrease in platelet
Figure 8. Percent lumenal stenosis secondary to intimal hyperplasia 2 weeks
post carotid endarterectomy. Saratin (n = 15), and a control group (n = 10) are
shown. Saratin group received 5ul of Saratin applied topically to the exposed
subendothelial surface. Star indicates a P value of 0.004.
Figure 9. Cross sections through carotid arteries showing reduction of intimal
hyperplasia in Saratin treated arteries (B) as compared to the untreated control
(A) in the rat. 1H= intimal hyperplasia
Table 1. IC50 concentrations of saratin necessary to inhibit platelet adhesion in
PRP from various species binding to human collagen types I & III, and calf skin
Table 2. Effect of saratin on the maximum platelet aggregation (%) in PRP
induced by various agonists, with end concentrations of reagents as indicated.
Table 3. Preoperative and postoperative bleeding times and platelet counts.
1, A medicament comprising polypeptide Saratin with therapeutic
agents for on site delivery for Inhibiting platelet accumulation
after vascuiar injuries or endarterectomy by administrator of a
therapeutically effective amount of Saratin which prevents
platelet adhesion, thereby inhibiting thrombosis and restenosis.
2, The medicament as claimed in claim 1, wherein the vascuiar
Injury Is associated with atherosclerosis, cardiac transplant
vasculopathy, coronary restenosis following coronary
intervention, balloon angioplasty, stent placement, rotabiation,
endarterectomy, including carotid enoartereccomy, dialysis
graft shunts and other graft anastomoses, unstable angina,
acute myocardial infarction, stroke, benign hypertrophy, or
benign prostatic hypertrophy.
3, The medicament as claimed in claim 1, wherein the Saratin >s
administered locally via a catheter or wherein Saratin is
incorporated into endoluminal caving of a catheter which is
directed locally to the tissue.
4. The medicament as claimed in claim 1, wherein Saratin is
Incorporated Into a locally administered polymer that permits
local sustained release of Saratin.
5. The medicament as claimed in claim 5, wherein the polymer
formulated Saratin is administered locally via a catheter,
6. The medicament as claimed In claim 1, wherein Saratin Is
incorporated into a stent or stent coating which is placed
locally on or in the tissue.
7. The medicament as claimed in claim 1, wherein Saratin is
incorporated into an endovascular graft or an endovascular
graft coating which is placed locally on or in the tissue.
8. A device with a surface cover with a hydrogel incorporating an
amount of biologically active Saratin therein for iocal delivery
therefrom and means associated with the coating to provide an
9. A catheter device, characterized in that, said device comprises
a polymeric material containing Saratin.
A medicament comprising Saratin with therapeutic agents for or site
delivery for inhibiting platelet accumulation after vascular injures or
endarterectomy. The invention furthermore provides medicail devices
like catheters and stents incorporated with coatings of the
|Indian Patent Application Number||331/KOLNP/2003|
|PG Journal Number||48/2011|
|Date of Filing||20-Mar-2003|
|Name of Patentee||MERCK PATENT GMBH|
|Applicant Address||FRANKRURTER STRASSE 250, 64293 DARMSTADT|
|PCT International Classification Number||A61K 38/00|
|PCT International Application Number||PCT/EP2001/09746|
|PCT International Filing date||2001-08-23|