Note: Descriptions are shown in the official language in which they were submitted.
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IRRIGATION SOLUTION AND METHOD FOR INHIBITION OF TUMOR CELL
ADHESION, PAIN AND INFLAMMATION
FIELD OF THE INVENTTON
The present invention relates to methods of inhibiting tumor cell adhesion
and/or
invasion and/or local tumor cell metastasis while simultaneously treating
pain, and/or
inflammation, and/or smooth muscle spasm and/or restenosis during surgical
procedures
using perioperative, local delivery of a combination of therapeutic agents.
BACKGROUND OF THE INVENTION
Endoscopy is a surgical procedure in which a camera, attached to a remote
light
source and video monitor, is inserted into a body cavity (e.g., joint,
peritoneal cavity,
bladder, thorax, etc.) through a small portal incision in the overlying slcin
and body wall.
Through similar portal incisions, surgical instruments may be placed in a body
cavity,
their use guided by arthroscopic visualization. As endoscopists' slcills have
improved, an
increasing number of operative procedures, once performed by "open" surgical
technique,
now can be accomplished endoscopically. Such procedures include, for example,
appendectomies, cholecystectomies and cardiac surgery. As a result of widening
surgical
indications and the development of small diameter endoscopes, pediatric
endoscopy has
become routine.
Throughout each endoscopic procedure, physiologic irrigation fluid (e.g.,
normal
saline or lactated Ringer's) is flushed continuously through the joint,
distending the body
cavity removing operative debris, thereby providing clearer visualization.
U.S. Patent
No. 4,504,493 to Marshall discloses an isomolar solution of glycerol in water
fox a non-
conductive and optically clear irrigation solution for arthroscopy.
Trrigation is also used in othex procedures, such as cardiovasculax and
general
vascular diagnostic and therapeutic procedures, urologic pxocedures and the
treatment of
burns and any operative wounds. Tn each case, a physiologic fluid is used to
irrigate a
wound or body cavity or passage. Conventional physiologic irrigation fluids do
not
inhibit tumor cell adhesion and/ox invasion and/or local tumor cell
metastasis, nor do they
provide analgesic, anti-inflammatory, anti-spasm and anti-restenotic effects.
Cell adhesion molecules are important in cell-cell interaction and
interactions
between cells and components of the extracellular matrix. While these adhesion
molecules are fundamental to diverse biological processes and regulate
intracellular
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signaling events, these molecules are particularly important in the earliest
stages of tumor
metastasis that require adhesion and/or invasion of tumor cells. Specific cell
adhesion
molecules and proteinases have been associated with the attachment and
implantation of
free tumor cells which occur during metastasis in a wide variety of human
malignancies,
including breast, prostate, liver, ovarian and bladder cancer. The diverse
adhesion
molecules, which mediate the adhesion interactions, are comprised of cellular
surface
receptor-ligand pairs. The cellular surface receptors constitute a group of
transmembrane
proteins that can be classified as members of related biological families or
superfamilies
based upon homologous structure and shared functional characteristics.
Adhesion of
cancer cells at secondary sites is known to be regulated by several families
of adhesion
proteins, including the CD44 proteoglycans, integrins and selectins. The
primary
functions of these receptors are to mediate cellular binding to the specific
structural
proteins that comprise the extracellular matrix (ECM) and to recognize
membrane bound
ligands mediating cell-cell adhesion. Cellular adhesion and invasion can also
be
facilitated by proteinases, such as the metalloproteinases (MMPs) and their
natural
inhibitors, tissue inhibitors of metalloproteinases (TIMPs).
Surgical trauma has been found to increase the frequency of tumor implantation
at
the sites of surgical injury and wound healing. One of the consequences of
surgical
trauma to malignant tissues is the dissemination of free tumor cells locally
at the
operative site. For example, patients with pancreatic cancer often exhibit
peritoneal
dissemination and hepatic metastasis after undergoing surgery. Adhesive
molecules and
receptors mediating the attachment of free-floating tumor cells and
implantation at
surgical sites and at sites of wound healing are frequently present on
numerous other
normal cell types. Surgical trauma also stimulates and potentiates the
production and
release of proteinases from tumor cells, inflammatory cells, and components of
the
extracellular matrix. Thus, a pharmaceutical approach aimed at interfering
with the
function of cell adhesion molecules to decrease tumor cell adhesion and
inhibit the
production and activation of proteinases is to deliver the therapeutic agents
only to the
tissues at risk for local tumor metastasis. Since the process of free tumor-
cell attachment
and invasion into the extracellular matrix or other cells during surgery is a
dynamic
process involving specific interactions temporally related to the operative
trauma, the
optimal time for pharmaceutical intervention is at the time of surgery.
Discovery of the
role of multiple adhesive proteins, adhesion receptors, and proteinases in the
metastatic
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spread of cancer enables development of pharmaceutical compositions that block
the
attaclunent of tumor cells to extracellular matrix proteins andlor inhibit
tumor cell
invasion during surgical procedures.
Alleviating pain and suffering in postoperative patients is an area of special
focus
in clinical medicine, especially with the growing number of outpatient
operations
performed each year. The most widely used agents, cyclooxygenase inhibitors
(e.g.,
ibuprofen) and opioids (e.g., morphine, fentanyl), have significant side
effects including
gastrointestinal irritation/bleeding and respiratory depression. The high
incidence of
nausea and vomiting related to opioids is especially problematic in the
postoperative
period. Therapeutic agents aimed at treating postoperative pain while avoiding
detrimental side effects are not easily developed because the molecular
targets for these
agents are distributed widely throughout the body and mediate diverse
physiological
actions. Despite the significant clinical need to inhibit pain and
inflammation, as well as
vasospasm, smooth muscle spasm and restenosis, methods for the delivery of
inhibitors of
pain, inflammation, spasm and restenosis at effective dosages while minimizing
adverse
systemic side effects have not been developed. As an example, conventional
(i.e.,
intravenous, oral, subcutaneous or intramusculax) methods of administration of
opiates in
therapeutic doses frequently is associated with significant adverse side
effects, including
severe respiratory depression, changes in mood, mental clouding, profound
nausea and
vomiting.
Prior studies have demonstrated the ability of endogenous agents, such as
serotonin (5-hydroxytryptamine, sometimes referred to herein as "5-HT"),
bradykinin and
histamine, to produce pain and inflammation. Sicuteri, F., et al., "Serotonin-
Bradylcinin
Potentiation in the Pain Receptors in Man," Life Sci. 4:309-316 (1965);
Rosenthal, S.R.,
"Histamine as the Chemical Mediator for Cutaneous Pain," J. Invest. De~mat.
69:98-105
(1977); Richardson, B.P., et al., "Identification of Serotonin M-Receptor
Subtypes and
their Specific Bloclcade by a New Class of Drugs," Nature 316:126-131 (1985);
Whalley,
E.T., et al., "The Effect of Kinin Agonists and Antagonists," Naunyn-Schmiedeb
Af~ch.
Phar~macol. 36: 652-57 (1987); Lang, E., et al., "Chemo-Sensitivity of Fine
Afferents
from Rat Skin In Vitf~o," J. Neuyo~hysiol. 63: 887-901 (1990).
For example, 5-HT applied to a human blister base (denuded skin) has been
demonstrated to cause pain that can be inhibited by 5-HT3 receptor
antagonists.
Richardson et al. (1985). Similarly, peripherally applied bradykinin produces
pain that
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can be blocked by bradykinin receptor antagonists. Sicutexi et al., 1965;
Whalley et al.,
1987; Dray, A., et al., "Bradykinin and Inflammatory Pain," Trends Neurosci.
16: 99-104
(1993). Peripherally applied histamine produces vasodilation, itching and pain
that can
be inhibited by histamine receptor antagonists. Rosenthal, 1977; Douglas,
W.W.,
"Histamine and 5-Hydroxytryptamine (Serotonin) a,nd their Antagonists", in
Goodman,
L.S., et al., ed., The Pharmacological Basis of Therapeutics, MacMillan
Publishing
Company, New Yorlc, pp. 605-638 (1985); Rumore, M.M., et aL, "Analgesic
Effects of
Antihistaminics," Life Sci. 36:403-416 (1985). Combinations of these three
agonists
(5-HT, bradykinin and histamine) applied together have been demonstrated to
display a
synergistic pain-causing effect, producing a long-lasting and intense pain
signal. Sicuteri
et al., 1965; Richardson et al., 1985; Kessler, W., et al., "Excitation of
Cutaneous
Afferent Nerve Endings In Vitro by a Combination of Inflammatory Mediators and
Conditioning Effect of Substance P," Exp. Brain Res. 91:467-476 (1992).
In the body, 5-HT is located in platelets and in central neurons, histamine is
found
in mast cells, a~zd bradykinin is produced from a larger precursor molecule
during tissue
trauma, pH changes and temperature changes. Because 5-HT can be released in
large
amounts from platelets at sites of tissue injury, producing plasma levels 20-
fold greater
than resting levels (Ashton, J.H., et al., "Serotonin as a Mediator of Cyclic
Flow
Variations in Stenosed Canine Coronary Arteries," Circulation 73:572-578
(1986)), it is
possible that endogenous 5-HT plays a role in producing postoperative pain,
hyperalgesia
and inflammation. In fact, activated platelets have been shown to excite
peripheral
nociceptors in vitro. Ringlcamp, M., et al., "Activated Human Platelets in
Plasma Excite
Nociceptors in Rat Skin, In Vitro," Neurosci. Lett. 170:103-106 (1994).
Similarly,
histamine and bradykinin also are released into tissues during trauma. Kimura,
E., et al.,
"Changes in Bradykinin Level in Coronary Sinus Blood After the Experimental
Occlusion of a Coronary Artery," Am Heart J. 85:635-647 (1973); Douglas, 1985;
Dray
et al. (1993).
In addition, prostaglandins also are known to cause pain and inflammation.
Cyclooxygenase inhibitors, e.g., ibuprofen, are commonly used in non-surgical
and
post-operative settings to block the production of prostaglandins, thereby
reducing
prostaglandin-mediated pain and inflammation. Flower, R.J., et al., "Analgesic-
Antipyretics and Anti-Inflammatory Agents; Drugs Employed in the Treatment of
Gout,"
in Goodman, L.S., et al., ed., The Pharmacological Basis of Therapeutics,
MacMillan
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CA 02502363 2005-04-14
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Publishing Company, New York, pp. 674-715 (1985): Gyclooxygenase inhibitors
are
associated with some adverse systemic side effects when applied
conventionally. For
example, indomethacin or ketorolac have well recognized gastrointestinal and
renal
adverse side effects.
As discussed, 5-HT, histamine, bxadykinin and prostaglandins cause pain and
inflammation. The various receptors through which these agents mediate their
effects on
peripheral tissues have been known and/or debated for the past two decades.
Most
studies have been performed in rats or other animal models. However, there axe
differences in pharmacology and receptor sequences between human and animal
species.
There have been no studies conclusively demonstrating the importance of 5-HT,
bradykinin or histamine in producing postoperative pain in humans.
° Furthermore, antagonists of these mediators currently are not used
for
postoperative pain treatment. A class of drugs, termed 5-HT and norepinephrine
uptake
antagonists, which includes amitriptyline, has been used orally with moderate
success for
chronic pain conditions. However, the mechanisms of chronic versus acute pain
states
axe thought to be considerably different. In fact, two studies in the acute
pain setting
using amitriptyline perioperatively have shown no pain-relieving effect of
amitriptyline.
Levine, J.D., et al., "Desipramine Enhances Opiate Postoperative Analgesia,"
Paih 27:45-
49 (1986); Kerrick, J.M., et al., "Low-Dose Amitriptyline as an Adjunct to
Opioids for
Postoperative Orthopedic Pain: a Placebo-Controlled Trial Period," Pain 52:325-
30
(1993). In both studies the drug was given orally. The second study noted that
oral
amitriptyline actually produced a lower overall sense of well being in
postoperative
patients, which may be due to the drug's affinity for multiple amine receptors
in the brain.
Amitriptyline, in addition to blocldng the uptake of 5-HT and norepinephrine,
is a
potent 5-HT receptor antagonist. Therefore, the lack of efficacy in reducing
postoperative pain in the previously mentioned studies would appear to
conflict with the
proposal of a role for endogenous 5-HT in acute pain. There axe a number of
reasons for
the lack of acute pain relief found with amitriptyline in these two studies.
(1) The first
study (Levine et al., 1986) used amitriptyline preoperatively for one week up
until the
night prior to surgery whereas the second study (Kerrick et al., 1993) only
used
amitriptyline postoperatively. Therefore, no amitriptyline was present in the
operative
site tissues during the actual tissue injury phase, the time at which 5-HT is
purported to
be released. (2) Amitriptyline is known to be extensively metabolized by the
liver. With
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oral administration, the concentration of amitriptyline in the operative site
tissues may not
have been sufficiently high for a long enough time period to inhibit the
activity of
postoperatively released 5-HT in the second study. (3) Since multiple
inflammatory
mediators exist, and studies have demonstrated synergism between the
inflammatory
mediators, blocking only one agent (5-HT) may not sufficiently inhibit the
inflammatory
response to tissue injury.
There have been a few studies demonstrating the ability of extremely high
concentrations (1% - 3% solutions -- i.e., 10 - 30 mg per milliliter) of
histamine) (H1)
receptor antagonists to act as local anesthetics for surgical procedures. This
anesthetic
effect is not believed to be mediated via H1 receptors but, rather, due to a
non-specific
interaction with neuronal membrane sodium channels (similar to the action of
lidocaine).
Given the side effects (e.g., sedation) associated with these high
"anesthetic"
concentrations of histamine receptor antagonists, local administration of
histamine
receptor antagonists currently is not used in the perioperative setting.
SUMMARY OF THE INVENTION
In one aspect, the present invention is directed towards preventing and
treating
tumor cell adhesion, and/or invasion and/or local metastasis in patients
undergoing
surgical procedures. The invention describes methods and pharmaceutical
compositions
based upon a combination of agents that will inhibit tumor cell attachment
and/or
invasion, and/or Iocal metastasis during swgical procedures, and
simultaneously inhibit
pain, and/or inflammation, and/or smooth muscle spasm and/or restenosis
associated with
the operation. According to one aspect of the invention, a method is provided
for
reducing or preventing tumor cell adhesion, andlor invasion and/or local
metastasis,
which comprises administering directly to the operative site a composition
which
includes a combination of two or more metabolically active agents, at least
one of which
is an anti-tumor adhesion or anti-invasion or anti-local metastasis agent, in
a
pharmaceutically effective carrier for delivery in an irrigation fluid.
Metabolically active
agents include, but are not limited to, all compounds that act directly or
indirectly to
modulate or alter the biological, biochemical or biophysical state of a cell,
including
agents that alter the electrical potential of the plasma membrane, the binding
of ligands to
a receptor, the enzymatic activity or expression level of cellular receptors,
enzyme
inhibitors or activators, inhibitors of protein-protein interactions, RNA-
protein
interactions, or DNA-protein interactions. For example, functional receptor
antagonists
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may include monoclonal antibodies that competitively inhibit receptor binding
sites,
soluble receptors that reduce the free ligand concentration available to
activate a receptor,
inhibitors of receptor signaling pathways, activators and inhibitors of
intracellular or
extracellular enzymes and agents that modulate the binding of transcription
factors to
DNA.
Specifically, the present invention provides a pharmacological method of
treating
tumor cell adhesion and/or invasion and/or local metastasis using a
combination of agents
delivered locally and periprocedurally to achieve maximal therapeutic benefit.
The use of
a combination of agents overcomes the limitations of existing therapeutic
approaches
which rely on the use of a single agent to block the multifactorial tumor cell
adhesion and
invasion processes which are modulated by numerous proinflammatory cytokines
and
receptors that are linked to the inflammatory response arising from surgical
procedures.
Proinflammatory cytokines have been reported to stimulate the expression of
adhesion
receptors and metalloproteinases present on both tumor cells and normal cells,
thereby
increasing their adhesive and invasive properties. A combination of an anti-
adhesion
agent and an anti-inflammatory agent, for example, may provide an additive or
synergistic benefit to the pharmaceutical composition.
This invention utilizes the approach of combining agents that act
simultaneously
on molecular targets associated with tumor cell adhesion, and/or tumor cell
invasion,
and/or metastasis, and/or inflammation, and/or pain, and/or smooth muscle
spasm, and/or
restenosis and delivering these agents to the operative site throughout the
surgical
procedure. These combinations of agents will be able to achieve a preemptive
inhibition
of tumor cell adhesion and/or invasion during the entire period of the
surgical procedure
while also preemptively inhibiting pain, and/or inflammation, and/or smooth
muscle
spasm and/or restenosis. Inhibition of a single adhesion receptor-ligand
interaction
through systemic delivery is likely to lead to adverse reactions since these
molecules also
function to deliver signals to normal cells. For example, CD44 receptor
binding plays a
physiological role in the immune response of normal T- cells and other
hematopoietic
cells and, therefore, inhibition of CD44 binding could lead to undesirable
effects on the
immune system.
Specifically, the present invention provides pharmaceutical compositions of
metabolically active agents that are based on a combination of at least two
agents that act
simultaneously on distinct molecular targets. At least one agent is an anti-
tumor cell
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adhesion or anti-invasion or anti-local metastasis agent that directly reduces
or prevents
the attachment of tumor cells to either the extracellular matrix ox to other
cells (both
normal and tumor). Suitable agents for inclusion in the pharmaceutical
composition will
be fiuther characterized by a requirement for pharmacological selectivity and
specificity
which limits interactions of the agent to a single family (or class) of
receptors or a single
enzyme family (e.g., CD44, integrins, selectins, protein tyrosine kinases,
MMPs and
MAP Kinases). A particular suitable agent may interact specifically with one
or more
receptor subtypes (or isotypes) while exhibiting specificity for the receptor
family. Each
suitable agent will associate with its molecular target through a specific
molecular
mechanism which can be characterized by a defined stoichiometry (typically
1:1) for the
ligand-receptor (or inhibitor-enzyme) complex and which can be characterized
by its
equilibrium binding or kinetic constant.
At least one second agent belongs to a class of receptor antagonists, enzyme
inhibitors or cytokines that possess anti-pain, anti-inflammatory, anti-spasm,
and/or anti-
restenosis activity. The optimal drug combination includes at least one agent
drawn from
a class of anti-tumor cell adhesion and/or anti-invasion agents which include
receptor
antagonists that act to inhibit and reduce the activity ox the expression of a
cell adhesion
receptor molecular target (e.g., integrin receptor antagonists, CD44 receptor
antagonists
and selectin receptor antagonists) or a proteinase inhibitor.
In addition, other suitable agents for inclusion in an optimal drug
combination that
are functional anti-tumor cell adhesion agents, which act to inhibit cellular
signal
transduction pathways, activated by cell adhesion receptors. The molecular
targets that
comprise these pathways include membrane associated and intracellularly
localized
enzymes that transduce and integrate the signals produced by activated cell
surface
, adhesion receptors. As examples, these agents include inhibitors of the
enzymes
belonging to the following families: protein tyrosine kinases, protein lcinase
C, and
mitogen-activated protein kinases (MAPK). Such enzyme inhibitors may interact
with a
particular membex or members of the kinase family through specific mechanisms
with are
characterized by both a defined stoichiometry (typically 1:1 ) of the enzyme-
inhibitor
complex and which can be characterized by equilibrium inhibition constants.
Despite a number of experimental studies, an effective treatment for
preventing
tumor cell adhesion and/or invasion during surgical procedures has not been
developed
previously. It is the object of the present invention to provide a method for
the
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preemptive treatment of tumor cell adhesion and/or invasion and/or local
metastasis
during surgical procedures employing irrigation fluids for the continuous
delivery of
therapeutic agents. Existing methods of delivery do not provide a means to
initiate
delivery of pharnlaceutical agents directly to the operative site and maintain
drug levels at
or above therapeutically effective concentrations throughout the surgical
procedure. Due
to the use of large volumes of irrigation fluid during certain surgical
procedures (e.g.,
Iaparoscopic surgery), inclusion of the agents in the irrigation fluid
provides a method to
prevent dilution of the agents and maintain therapeutic levels. Problems
associated with
systemic delivery of therapeutic peptides, proteins and small molecules as
potential anti-
tumor cell adhesion/invasion, and anti-metastasis agents, including toxicity,
pharmaceutical stability and poor pharmacokinetic profiles, may be reduced or
obviated
by local delivery of these therapeutic agents in an irrigation fluid directly
to the operative
site.
A multiple drug combination can be delivered via irrigation locally and
periprocedurally (i.e., intra-operative delivery alone or intra-operative
delivery together
with pre-operative and/or post-operative delivery). This invention also
provides for an
anti-tumor adhesion and/or mt-invasion and/or local metastasis agent to be
delivered in
combination with one or more analgesic and/or anti-inflammatory and/or anti-
spasm
and/or anti-restenosis agents.
A significant advantage of the invention is derived from the rapid onset of
pharmacological action. Direct, local delivery of the anti-tumor adhesion
agents initiated
at the outset of the surgical procedure and continued during the surgical
procedure (i.e.,
perioperatively) has the potential to inhibit one of the earliest steps of
metastasis, the
initial adhesion process. The immediate and constant delivery of
therapeutically effective
concentrations of anti-adhesion and/or invasion agents is expected to
preemptively inhibit
the initial attachment of tumor cells to both the extracellular matrix and
other cells and
subsequent invasion into these tissues. Studies have found that the highest
rate of tumor
implantation occurred when tumor cells were injected immediately after
surgery,
indicative of the critical timing for therapeutic inhibition of the adhesion
process.
The method of delivery and pharmaceutical composition of the present invention
provide distinct advantages which include: 1) a combination drug therapy
directed to the
multifactorial causes of tumor cell adhesion/invasion/local metastasis,
inflammation,
pain, smooth muscle spasm and restenosis during surgical procedures, 2) local
delivery of
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the drug combination.achieves an instantaneous therapeutic concentration of
anti-tumor
adhesion agents at the operative site, 3) continuous delivery of therapeutic
agents in an
irrigation solution provides a constant drug concentration in a
therapeutically effective
range at the operative site during a surgical procedure, 4) local delivery
permits a
reduction in total drug dose and dosing frequency compared to systemic
delivery, and
5) direct, Local delivery to the surgical site enables use of novel,
pharmaceutically active
agents, such as certain peptides and antibodies, which cannot be delivered
systemically.
The present invention further provides a solution constituting a mixture of
multiple agents in low concentrations directed at inhibiting locally the
mediators of pain,
inflammation, spasm and restenosis in a physiologic electrolyte carrier fluid.
The
invention also provides a method for perioperative delivery of the irrigation
solution
containing these agents directly to a surgical site, where it works locally at
the receptor
and enzyme levels to preemptively limit pain, inflammation, spasm and
restenosis at the
site. Due to the local perioperative delivery method of the present invention,
a desired
therapeutic effect can be achieved with lower doses of agents than are
necessary when
employing other methods of delivery (i.e., intravenous, intramuscular,
subcutaneous and
oral). The anti-painlanti-inflammation agents in the solution include
agents.selected from
the following classes of receptor antagonists and agonists and enzyme
activators and
inhibitors, each class acting through a differing molecular mechanism of
action for pain
and inflammation inhibition: (1) serotonin receptor antagonists; (2) serotonin
receptor
agonists; (3) histamine receptor antagonists; (4) bradykinin receptor
antagonists;
(5) lcallilcrein inhibitors; (6) tachykinin receptor antagonists, including
neurokininl and
neurolcinin2 receptor subtype antagonists; (7) calcitonin gene-related peptide
(CGRP)
receptor antagonists; (8) interleukin receptor antagonists; (9) inhibitors of
enzymes active
in the synthetic pathway for arachidonic acid metabolites, including (a)
phospholipase
inhibitors, including PLA2 isoforrn inhibitors and PLC, isoform ~inhibitors,
(b) cyclooxygenase inhibitors, and (c) lipooxygenase inhibitors; (10)
prostanoid receptor
antagonists including eicosanoid EP-1 and EP-4 receptor subtype antagonists
and
thromboxane receptor subtype antagonists; (11) leukotriene receptor
antagonists
30~ including leulcotriene Bq. receptor subtype antagonists and leuleotriene
Dq. receptor
subtype antagonists; (12) opioid receptor agonists, including ~.-opioid, 8-
opioid, and
~c-opioid receptor subtype agonists; (13) purinoceptor agonists and
antagonists including
PBX receptor antagonists and P2y receptor agonists; and (14) adenosine
triphosphate
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(ATP)-sensitive potassium channel openers. Each of the above agents functions
either as
an anti-inflammatory agent andlor as an anti-nociceptive, i.e., anti-pain or
analgesic,
agent. The selection of agents from these classes of compounds is tailored for
the
particular application.
Several preferred embodiments of the solution of the present invention also
include anti-spasm agents for particular applications. Fox example, anti-spasm
agents
may be included alone or in combination with anti-painlanti-inflammation
agents in
solutions used for vascular procedures to limit vasospasm, and anti-spasm
agents may be
included for urologic procedures to limit spasm in the urinary tract and
bladder wall. For
such applications, anti-spasm agents are utilized in the solution. For
example, an anti-
pain/anti-inflammation agent that also serves as an anti-spasm agent may be
included.
Suitable anti-inflannnatory/anti-pain agents that also act as anti-spasm
agents include
serotonin receptor antagonists, tachykinin receptor antagonists, and ATP-
sensitive
potassium channel openers. Other agents ~ which may be' utilized in the
solution
specifically for their anti-spasm properties include calcium channel
antagonists,
endothelin receptor antagonists and the nitric oxide donors (enzyme
activators).
Specific preferred embodiments of the solution of the present invention for
use in
cardiovascular and general vascular procedures include anti-restenosis agents,
which
most preferably are used in combination with anti-spasm agents. Suitable anti-
restenosis
agents include: (1) antiplatelet agents including: (a) thrombin inhibitors and
receptor
antagonists, (b) adenosine disphosphate (ADP) receptor antagonists (also known
as
purinoceptorl receptor antagonists), (c) thromboxane inhibitors and receptor
antagonists
and (d) platelet membrane glycoprotein receptor antagonists; (2) inhibitors of
cell
adhesion molecules, including (a) selectin inhibitors and (b) integrin
inhibitors;
(3) anti-chemotactic agents; (4) interleukin receptor antagonists (which also
serve as
anti-pain/anti-inflammation agents); and (5) intracellular signaling
inhibitors including:
(a) protein lcinase C (PKC) inhibitors and protein tyrosine kinase inhibitors,
(b) modulators of intracellular protein tyrosine phosphatases, (c) inhibitors
of src
homology (SH2) domains, and (d) calcium channel antagonists. Such agents are
useful
in preventing restenosis of arteries treated by angioplasty, rotational
atherectomy or other
cardiovascular or general vascular therapeutic or diagnostic procedure.
The present invention also provides a method for manufacturing a medicament
compounded as a dilute irrigation solution for use in continuously iiTigating
an operative
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site or wound during an operative procedure. The method entails dissolving in
a
physiologic electrolyte carrier fluid at least one inhibitor of tumor cell
adhesion, invasion
and/or metastasis and preferably a plurality of anti-pain/anti-inflammatory
agents, and for
some applications anti-spasm agents and/or anti-restenosis agents, each agent
included at
a concentration of preferably no more than 100,000 nanomolar, and more
preferably no
more than 10,000 nanomolar.
The method of the present invention provides for the delivery of a dilute
combination of at least one inhibitor of tumor cell adhesion, invasion and/or
metastasis
and preferably multiple receptor antagonists and agonists and enzyme
inhibitors and
activators directly to a wound or operative site, during therapeutic or
diagnostic
procedures for the inhibition of pain, inflammation, spasm and restenosis.
Since the
active ingredients in the solution are being locally applied directly to the
operative tissues
in a continuous fashion, the drugs may be used efficaciously at extremely low
doses
relative to those doses required for therapeutic effect when the same drugs
are delivered
orally, intramuscularly, subcutaneously or intravenously. As used herein, the
term "local"
encompasses application of a drug, in and around a wound or other operative
site, and
excludes oral, subcutaneous, intravenous and intramuscular administration. The
term
"continuous" as used herein encompasses uninterrupted application, repeated
application
at frequent intervals (e.g., repeated intravascular boluses at frequent
intervals
intraprocedurally), and applications which are uninterrupted except for brief
cessations
such as to permit the introduction of other drugs or agents or procedural
equipment, such
that a substantially constant predetermined concentration is maintained
locally at the
wound or operative site.
The advantages of low dose applications of agents are three-fold. The most
important is the absence of systemic side effects which often limit the
usefulness of these
agents. Additionally, the agents selected for particular applications in the
solutions of the
present invention are highly specific with regard to the mediators on which
they work.
This specificity is maintained by the low dosages utilized. Finally, the cost
of these
active agents per operative procedure is low.
The advantages of local administration of the agents via luminal irrigation or
other
fluid application are the following: (1) local administration guarantees a
lcnown
concentration at the target site, regardless of interpatient variability in
metabolism, blood
flow, etc.; (2) because of the direct mode of delivery, a therapeutic
concentration is
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obtained instantaneously and, thus, improved dosage control is provided; and
(3) local
administration of the active agents directly to a wound or operative site also
substantially
reduces degradation of the agents through extracellular processes, e.g., first-
and
second-pass metabolism, that would otherwise occur if the agents were given
orally,
intravenously, subcutaneously or intramuscularly. This is particularly true
for those
active agents that are peptides, which are metabolized rapidly. Thus, local
administration
permits the use of compounds or agents which otherwise could not be employed
therapeutically. For example, some agents in the following classes are
peptidic:
bradykinin receptor antagonists; tachykinin receptor antagonists; opioid
receptor agonists;
CGRP receptor antagonists; and interleukin receptor antagonists. Local,
continuous
delivery to the wound or operative site minimizes drug degradation or
metabolism while
also providing for the continuous replacement of that portion of the agent
that may be
degraded, to ensure that a local therapeutic concentration, sufficient to
maintain receptor
occupancy, is maintained throughout the duration of the operative procedure.
Local administration of the solution perioperatively throughout a surgical
procedure in accordance with the present invention produces a preemptive
analgesic,
anti-inflammatory, anti-spasmodic or anti-restenotic effect. As used herein,
the term
"perioperative" encompasses application intraprocedurally, pre- and
intraprocedurally,
intra- and postprocedurally, and pre-, intra- and postprocedurally. To
maximize the
preemptive anti-inflammatory, analgesic (for certain applications),
antispasmodic (for
certain applications) and antirestenotic (for certain applications) effects,
the solutions of
the present invention are most preferably applied pre-, intra- and
postoperatively. By
occupying the target receptors or inactivating or activating targeted enzymes
prior to the
initiation of significant operative trauma locally, the agents of the present
solution
modulate specific pathways to preemptively inhibit the targeted pathologic
process. If
inflammatory mediators and processes are preemptively inhibited in accordance
with the
present invention before they can exert tissue damage, the benefit is moxe
substantial than
if given after the damage has been initiated.
Inhibiting more than one tumor cell adhesion, invasion and/or metastasis,
inflammatory, spasm or restenosis mediator by application of the multiple
agent solution
of the present invention is useful to dramatically reduce the degree of cell
adhesion,
invasion and/or metastasis, inflammation, pain, and spasm, and theoretically
should
reduce restenosis. The irrigation solutions of the present invention include
combinations
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of drugs, each solution acting on multiple receptors or enzymes. The drug
agents are thus
simultaneously effective against a combination of pathologic processes,
including tumor
cell adhesion, invasion and/or metastasis, pain and inflammation, vasospasm,
smooth
muscle spasm and restenosis. The action of these agents is considered to be
synergistic,
in that the multiple receptor antagousts and inhibitory agonists of the
present invention
provide a disproportionately increased efficacy in combination relative to the
efficacy of
the individual agents. The synergistic action of several of the agents of the
present
invention are discussed, by way of example, below in the detailed descriptions
of those
agents.
In addition to arthroscopy, the solution of the present invention may also be
applied locally to any human body cavity or passage, operative wound,
traumatic wound
(e.g., burns) or in any operative/interventional procedure in which irrigation
can be
performed. These procedures include, but 'are not limited to, urological
procedures,
cardiovascular and general vascular diagnostic and therapeutic procedures,
endoscopic
procedures and oral, dental and periodontal procedures. As used hereafter, the
term
"wound", unless otherwise specified, is intended to include surgical wounds,
operative/interventional sites, traumatic wounds and burns.
Used perioperatively, the solution should result in a clinically significant
decrease
in operative site pain and inflammation relative to currently-used irrigation
fluids, thereby
decreasing the patient's postoperative analgesic (i.e., opiate) requirement
and, where
appropriate, allowing earlier patient mobilization of the operative site. No
extra effort on
the part of the surgeon and operating room personnel is required to use the
present
solution relative to conventional irrigation fluids.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will now be described in greater detail, by way of
example,
with reference to the accompanying drawings in which:
FIGURE 1 provides a schematic overview of a generic vascular cell showing
molecular targets and flow of signaling information leading to contraction,
secretion
and/or proliferation. The integration of extrinsic signals through receptors,
ion channels
and other membrane proteins axe common to platelets, neutrophils, endothelial
cells and
smooth muscle cells. Representative examples of molecular targets are included
for
major groups of molecules which are therapeutic targets of drugs included in
the
solutions of the present invention.
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FIGURE 2 provides a detailed diagram of the signaling pathways illustrating
"crosstalk" between G-protein coupled receptor (GPCR) pathways and receptor
tyrosine
lcinase (RTK) pathways in a vascular smooth muscle cell. Only representative
proteins in
each pathway have been shown to simplify the flow of information. Activation
of
GPCRs leads to increases in intracellular calcium and increased protein kinase
C (PKC)
activity and subsequent smooth muscle contraction or spasm. In addition,
"crosstalk" to
the RTK signaling pathway occurs through activation of PYK2 (a newly
discovered
protein tyrosine kinase) and PTK-X (an undefined protein tyrosine kinase),
triggering
proliferation. Conversely, while activation of RTKs directly initiates
proliferation,
"crosstalk" to the GPCR pathway occurs at the level of PKC activity and
calcium levels.
LGR designates ligand-gated receptor, and MAPK designates mitogen-activated
protein
kinase. These interactions define the basis for synergistic interactions
between molecular
targets mediating spasm and restenosis. The GPCR signaling pathway also
mediates
signal transduction (FIGURES 3 and 7) leading to pain transmission in other
cell types
(e.g., neurons).
FIGURE 3 provides a diagram of the G-Protein Coupled Receptor (GPCR)
pathway. Specific molecular sites of action for some drugs in a preferred
arthroscopic
solution of the present invention are identified.
FIGURE 4 provides a diagram of the G-Protein Coupled Receptor (GPCR)
pathway including the signaling proteins responsible for "crosstalk" with the
Growth
Factor Receptor signaling pathway. Specific molecular sites of action for some
drugs in a
preferred cardiovascular and general vascular solution of the present
invention are
identified. (See also FIGURE 5.)
FIGURE 5 provides a diagram of the Growth Factor Receptor signaling pathway
including the signaling proteins responsible for "crosstalk" with the G-
Protein Coupled
Receptor signaling pathway. Specific molecular sites of action for some drugs
in a
preferred cardiovascular and general vascular solution of the present
invention are
identified. (See also FIGURE 4.)
FIGURE 6 provides a diagram of the G-Protein Coupled Receptor pathway
including the signaling proteins responsible for "crosstallc" with the Growth
Factor
Receptor signaling pathway. Specific molecular sites of action for some drugs
in a
preferred urologic solution are identified.
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FIGURE 7 provides a diagram of the G-Protein Coupled Receptor pathway.
Specific molecular sites of action for some drugs in a preferred general
surgical wound
solution of the present invention are identified.
FIGURE 8 provides a diagram of the mechanism of action of nitric oxide (NO)
donor drugs and NO causing relaxation of vascular smooth muscle.
Physiologically,
certain hormones and transmitters can activate a form of NO synthase in the
endothelial
cell through elevated intracellular calcium resulting in increased synthesis
of NO. NO
donors may generate NO extracellularly or be metabolized to NO within the
smooth
muscle cell. Extracellular NO can diffuse across the endothelial cell or
directly enter the
smooth muscle cell. The primary target of NO is the soluble guanylate cyclase
(GC),
leading to activation of a cGMP-dependent protein kinase (PING) and subsequent
extrusion of calcium from the smooth muscle cell via a membrane pump. NO also
hyperpolarizes the cell by opening potassium channels which in turn cause
closure of
voltage-sensitive calcium channels. Thus, the synergistic interactions of
calcium channel
antagonists, potassium channel openers and NO donors are evident from the
above signal
transduction pathway.
FIGURES 9, l0A and lOB provide charts of the percent of vasoconstriction
versus time in control arteries, in the proximal segment of subject arteries,
and in the
distal segment of subject axteries, respectively, for the animal study
described in
EXAMPLE VII herein demonstrating the effect on vasoconstriction of infusion
with
histamine and serotonin antagonists, used in the solutions of the present
invention, during
balloon angioplasty.
FIGURES 11 and 12 provide charts of plasma extravasation versus dosage of
amitriptyline, used in the solutions of the present invention, delivered
intravenously and
intra-articularly, respectively, to knee joints in which extravasation has
been induced by
introduction of 5-hydroxytryptamine in the animal study described in EXAMPLE
VIII
herein.
FIGURES 13, 14 and 15 provide charts of mean vasoconstriction (negative
values) ox vasodilation (positive values), ~1 standard error of the mean for
the proximal
(FIGURE 13), mid (FIGURE 14) and distal (FIGURE 15) segments of arteries
treated
with saline (N=4) or with a solution formulated in accordance with the present
invention
(N=7), at the immediate and 15 minute post-rotational atherectomy time points
in the
animal study of Example XIII described herein.
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DETAILED DESCRIPTTON OF THE PREFERRED EMBODIMENT
In accordance with the present invention, methods and solutions are provided
for
reducing or preventing tumor cell adhesion, and/or invasion and/or local
metastasis,
which comprises administering directly to the operative site a composition
which
includes a combination of two or more metabolically active agents, at least
one of which
is an anti-tumor adhesion or anti-invasion or anti-local metastasis agent, in
a
pharmaceutically effective carrier for delivery in an irrigation fluid.
I. INHIBITORS OF CELL ADHESION AND INVASION MOLECULES
A. METALLOPROTE1NASE ANTAGONISTS AND TISSUE
. INHIBITORS OF METALLOPROTE1NASES (TIMP)
Matrix metalloproteinases (MMPs) are a family of zinc-dependent neutral
endopeptidases whose enzymatic activity is capable of controlled degradation
of the
extracellulax matrix (ECM). This activity serves as a key control factor in
the adherence,
invasive capacity, metastasis, growth, and angiogenesis of tumors. In humans,
cloning
and sequencing have identified 17 members of this family. These members are
divided
into subgroups of collagenases, gelatinases, stromelysins and stromelysin-like
MMPs,
membrane-type-MMPs, and novel MMPs.
The MMPs share common structural and functional characteristics. All families
to date have at least three domains: a prodomain which contains a conserved
cysteine
residue and which is lost on enzyme activation; a catalytic domain of 106 to
119 residues
which contains conserved structural metal-binding sites; and a highly
conserved zinc-
binding active site domain of S2 to 58 amino acids.
Cells do not generally constitutively express MMPs, but rather their
expression is
rapidly induced in response to exogenous signals, cytolcines or growth factors
and altered
cell-matrix and cell-cell interactions. Exceptions to this rule are MMP-8 and
MMP-9 that
are stored in secretory granules of neutrophils and eosinophils, and MMP-
7'that is stored
in secretory epithelial cells.
Expression of MMPs is primarily regulated at the level of transcription, and
their
proteolytic activity is regulated by zymogen activation and inhibition of
activity.
Extracellulax signals such as cytolcines (IL-1, tumor necrosis factor, etc.)
and others
activate the AP-1 transcription factor complex composed of members of Jun and
Fos
proteins, which bind to the AP-1 cis-element and activate transcription of the
corresponding MMP gene. Most MMPs are secreted as latent precursors
(zymogens),
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which are proteolytically activated in the extracellular space with the
exception of
MMP-11 and MT1-MMP, which are activated prior to their secretion by Golgi-
associated
furin-like proteases.
The activity of MMPs in the extracellular space is controlled by the tissue
inhibitors of matrix metalloproteinases (TIMPs), a family of natural
inhibitors. Thus far,
four members of this family have been identified (TIMP-1, 2, 3, 4) and well
characterized. These TIMPs have similar inhibitory activities against most of
the MMPs,
but differ with respect to their interactions with proMMPs, solubility,
regulation of
expression, and tissue specific expression. TIMP-3 unlike other TIMPs inhibits
the
activity of TNF-alpha-converting enzyme (TACE), suggesting a role in
modulating
inflammation. TGF-beta, IL-1, IL-6, TNF-alpha, EGF, and glucocorticoids
regulate
TIMP-1 at the transcriptional level. TIMP-2 is expressed constitutively and
TIMP-4
expression is restricted. TIMP-3 expression is up-regulated by mitogens,
phorbol esters
and TGF-beta.
The MMPs have been shown to facilitate tumor cell invasion and metastasis by
at
least three mechanisms. In a physiological sense, first, the MMPs can modulate
tumor
cell adhesion. This process is necessary for cells to attach and move through
the ECM.
Cell transfection studies altering the ratio of MMP-2 to TIMP-2 have
demonstrated
variation in the adhesive phenotype of tumor cells. Next, the proteinase
action of MMPs
can degrade ECM macromolecules such as collagens, laminins, and proteoglycans
allowing tumor cell invasion. Finally, MMPs can act on ECM components or other
proteins to activate biological functionality. For example, laminin-5 is
degraded by
MMP-2 to produce a soluble chemotactic fragment.
The classic role of MMPs to degrade ECM components, and in particular the
basement membrane is considered critical for tumor cell invasion.
Experimentally, the
necessity for protease action in tumor cell invasion has been demonstrated
using the
chemoinvasion assay. Abundant expression of distinct MMPs occurs in invasive
primary
tumors or their metastases. The level of MMP-1 expression correlates with a
poor
prognosis of colorectal and esophageal cancer and the expression of MMP-2 and
MMP-3
are closely related to lymph node metastasis. Expression of MMP-2, TIMP-2 and
MT1-MMP correlates with poor prognosis in bladder cancer. MMP-7 knockout mice
show reduction in intestinal tumorigenesis and MMP-2 def cient mice show
reduced
angiogenesis and tumor progression. Infiltration of inflammatory cells is also
~a
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prominent feature of many malignant tumors, and they can be a source of MMPs,
contributing to tumor cell invasion. These inflammatory cells also produce
cytokines,
which can enhance MMP expression by tumor and stromal cells leading to further
tumor
cell invasion.
The TIMPs also play a role in tumor cell invasion. The ability of TIMPs 1, 2,
3,
and 4 to inhibit tumor growth has been shown by over-expressing them in
various human
and rodent models. In melanoma cells, over-expression of TIMP-2 inhibits the
growth of
tumors implanted in the skin of SCID mice. This growth inhibition seems
independent of
angiogenesis but dependent on the collagen matrix. Current concepts on the
role of
TIMPs in cancer now focus on inhibition of tumor invasion, metastasis, and
regulation of
ECM homeostasis.
Based upon in vitro, in vivo, i~ situ, and clinical studies, inhibition of MMP
activity can inhibit adhesion, invasion, and metastasis of neoplastic cells
and effectively
suppress tumor growth and metastasis. Prevention of tumor growth can be
accomplished
by inhibition of MMP expression or activity at multiple steps in the synthetic
pathway of
MMPs.
Inhibition of MMP transcription can be accomplished by blocking signaling
pathways regulating MMP transcription directly or by inhibiting transcription
factors
responsible for activating transcription of MMP genes. Inhibition of signaling
pathways
involved in AP-1 activation, the MAPKs (ERI~1,2,SAPK/JNI~ or p38) can markedly
inhibit the expression of MMPs and the invasion of malignant cells in vitro.
Expression
of dominant negative transcription factors, which requires an effective method
of gene
therapy in vivo, may be used to inhibit MMP production. For example,
expression of
dominant negative c-Jun has been shown to inhibit the induction of MMPs 1, 2,
3, 10 and
14 as well as the in vitro invasion of keratinocytes. Retinoids as inhibitors
of AP-1
activity could also be used to prevent MMP gene expression in selected human
carcinomas.
Another method for inhibiting expression of specific MMPs is to use antisense
RNAs. Antisense oligonucleotides for MMP-7 have been shown to inhibit invasion
of
colon carcinoma cells in vit~~o and i~ vivo. An MMP-9 antisense-ribozyme
expression
construct has been shown to inhibit expression of MMP-9 and the invasion of
rat
osteocarcinoma cells.
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The most extensively studied area of inhibiting tumor cell invasion via
inhibition
of MMPs has been the development of compounds to block MMP activity. Synthetic
MMP inhibitors (MMPIs), such as hydroxyamate inhibitors, are small peptide
analogs of
fibrillar collagens, which specifcally interact with the zinc in the catalytic
site of MMPs
and inhibit their activity. Currently, several of these agents are in clinical
trials for
treating invasive tumors. The first class to be developed was the broad-
spectrum MMPIs,
which nonspecifically inhibit multiple MMPs. Examples are batimastat (BB-94)
with
poor oral bioavailability requiring local administration and marimastat (BB-
2516) which
can be orally administered. Results of pre-clinical studies show these agents
to inhibit
tumor invasion, metastasis, and growth. Next, development of selective agents
for
targeting of certain MMPs was accomplished. Since peptide-based MMP inhibitors
are
costly to produce and generally have poor bioavailability, non-peptidic
inhibitors have
been developed through rational design based on X-ray crystallographic
information of
the MMP active site. Examples of non-peptidic inhibitors are CGS27023A
(Novartis),
AG3340 (Agouron), and the fenbufen derivative BAY12-9566 (Bayer). In addition,
tetracyclines devoid of their anti-microbial effect have been developed to
block host
derived MMPs. This class of chemically modified nonmicrobial tetracyclines
(CMTs)
has been shown to block the expression of MMP 2, 3 and 8, by multiple
mechanisms and
have also been shown to induce apoptosis of malignant cells. Most recently,
bisphosphonates have been identified as MMP inhibitors and the aggrecanase-I
inhibitors, SE206, BB-16, and XS309 have been shown to have good potency.
Finally,
the ability of TIMPs to potently and specifically inhibit MMP activity has
raised interest
in their use. TIMPs 1, 2, 3, and 4 can inhibit invasion of malignant cells ih
vitro and
in vivo.
In one aspect, the present invention provides for administering directly to an
operative site via irrigation solution one or more MMP inhibitors in
combination with at
least one additional anti-pain and/or anti-inflammatory andlor anti-spasm
and/or anti-
restenosis agent that would be effective in preventing or inhibiting tumor
cell adhesion,
invasion, and metastasis. Inhibitors and antagonists may act at the level of
MMP
transcription, translation, secretion, activation, or degradation.
One slcilled in the art will appreciate that the choice of a MMP inhibitor as
a
therapeutic agent for a patient will, in part, determine the amounts of the
agents to be
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administered for any particular treatment protocol and that these amounts can
be readily
determined.
MMP inhibitors suitable for use in the present invention are listed in the
Table
below.
TABLE 1
METALLOPROTEINASE ANTAGONISTS AND TIMPS
Agent Therapeutic PreferredMost Preferred
Concentrations Concentrations
Batimastat 0.1-10,000 100
Marimastat 0.1-10,000 100
.
CGS27032A 1-100,000 1000
AG3340 0.1-10,000 100
Bayl2-9566 10-1,000,000 10,000
CMT 1 0.1-1000 mg/kg 100 mg/kg
Minocycline 300-500,000 300,000
SE206 10-1,000,000 10,000
BB 16 10-1,000,000 10,000
XS309 100-10,000,000 100,000
Antisense 0.5-5,000 5,000
RNA
TIMPS 1 0.1-10,000 1,000
B. CD44 RECEPTOR ANTAGONISTS AND INHIBITORS OF CD44
SIGNALING
CD44 is a transmembrane glycoprotein that comprises a family of surface
receptors composed of numerous variant isoforms. The CD44 receptor is found in
a wide
variety of tissues including the central nervous system, lung, epidermis,
liver, colon and
pancreas. This family of related cell surface adhesion molecules are
multifunctional
proteins expressed in both normal and malignant tissues. CD44 is encoded by a
single
gene containing 20 exons, 10 of which (vl-v10) are variant exons inserted by
alternative
splicing. The standard isoform of CD44 (CD44s), which is ubiquitously
expressed, does
not contain sequences encoded by these variant exons. Numerous variant
isoforms of
CD44 (CD44v) which contain different combinations of exons (vl-v10) inserted
into the
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extracellular domain are restricted in their expression to proliferating
epithelial cells,
activated lymphocytes and malignant cells.
Recent studies have demonstrated that the CD44 protein is the primary receptor
for hyaluronate in humans. Since CD44 is the principle transmembrane receptor
for this
extracellular matrix glycosa~ninoglycan, CD44 has also been termed the
hyaluronate
receptor. It can also bind some other extracellular matrix Iigands
(chondroitin sulphate,
heparin sulphate, and fibronectin) with lower affinity.
This CD44 receptor-ligand interaction is required for many normal cellular
processes including cellular adhesion (aggregation and migration), hyaluronate
degradation, lymphocyte activation, lymphocyte homing into inflammatory sites,
and
release of cytokines. Additional specialized functions include assembly of a
pericellular
matrix during chondrogenesis and wound healing. Interactions between CD44
receptor
and hyaluronic acid (HA) deliver important signals to normal and transformed
CD44
bearing cells. In common with many other cell surface receptors, cells that
express CD44
regulate expression of this receptor in response to ligand binding.
The CD44 molecule is an 85kD glycosylated molecule with N-terminal sequence
homology to cartilage link proteins. Isoforins of CD44 of vaxying sizes have
been
described on many cell types. Variations in the size of CD44 isoforms occur
due to
glycosylation differences and to the addition of chondroitin sulfate molecules
to CD44.
The 85kD form has been identified in serum, plasma and synovial fluid.
Alternative
splicing creates a hematopoietic form of the molecule, CD44H, and additional
subtypes.
Alternative splicing results in changes in protein glycosylation and provides
a mechanism
for the regulation of binding activity of CD44. Thus, the CD44 molecule, by
existing in
several isoforms and due to its wide cellular distribution and functional
association with
other adhesion molecules, is a multifunctional proinflamrnatory molecule
involved in
immune cell activation as well as metastasis of certain tumor cell types.
The predominant isoform found on human leukocytes is CD44H. Both
hyaluronate and CD44 mAb binding to monocytes induces IL-1 release. On T
cells,
hyaluronate and CD44 mAb ligation of CD44 have disparate effects; CD44 mAbs
augment T cell triggering while hyaluronate suppresses T cell triggering.
Finally, CD44
mAbs and polyclonal anti-CD44 serum have been shown to inhibit the binding of
lymphocytes to endothelial venules in inflammatory sites such as synovium.
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The physiological functions of CD44 indicate that it plays a role in the
metastatic
spread of tumors. CD44 properties, such as hyaluronate-mediated adhesion,
cellular
motility, and adhesion to lymphoid tissue, axe among several that are required
by invasive
and metastatic tumor cells. Ih vivo experiments indicate that tumor cells
transfected to
overexpress specific CD44 isoforms display enhanced metastatic potential. Of
the
numerous CD44 isoforms, specific isoforms enhance tumor cell metastasis more
efficiently than others do. Metastatic potential can be conferred to non-
metastasizing cell
lines by transfection with a variant of CD44 and high levels of CD44 are
associated with
several types of malignant tumors.
A number of studies have reported that human tumors display specific
alterations
in CD44 isoform expression, and have further shown that the extent of clinical
disease in
specific tumors correlates with the CD44 isoform expression pattern. As an
example, it
was found that expression of a splice variant of CD44 was required for
adenocarcinoma
cells to metastasize and a monoclonal antibody (mAb) against this particular
CD44
variant prevented metastasis. In addition, a wide range of epithelial and
mesenchymal
malignancies express high levels of CD44H and a variety of variant isoforms of
CD44.
Specifically, reports have documented CD44 variant expression as a common
feature of
epithelial ovarian cancer.
Many studies have investigated the pattern of CD44 distribution in tumors. The
presence of the CD44 standard and the CD44-9v isoform on the surface of
gastric cancer
cells was found to be significantly related to a higher tumor-induced
mortality and a
shorter survival time for patients. Intestinal-type gastric carcinomas
predominantly
express the CD44-6v isoform that provides the ability for these tumor cells to
metastasize. There is substantial evidence that integrin receptors and
different isoforins
of the CD44 receptor are altered following the malignant transformation of
colonic
mucosa into adenomas and invasive carcinomas and which are correlated with
their
metastatic potential. Expression of the CD44-6v isoform has been associated
with
adverse prognosis of colorectal cancer due to the development of tumor
metastasis and
adhesion. These data suggest that CD44 plays an important role in tumor
metastasis and
that a variety of molecules that inhibit CD44 receptor interaction with its
natural ligands
could be used for therapy.
At the cellular level, recent studies have characterized cytoplasmic signal
transduction pathways for CD44 in activated endothelial cells that involve
tyrosine
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kinases. HA binding activity of CD44 is linked to cellular activation in some
cell types.
Addition of degradation products of hyaluronan (3 to 10 disaccharides) to
cells induces
rapid tyrosine phosphorylation of multiple proteins, which is sustained.
Increased
phosphorylation of the CD44 receptor was also observed. Pretreatment of cells
with
either anti-CD44-xeceptor antibody or a non-selective inhibitor of tyrosine
kinase activity
inhibited both the induced protein tyrosine phosphorylation and the
proliferation
response. Protein kinase C (PKC) activity was increased two-to-three fold in
the
membranes of treated cells. Furthermore, it was shown that PI~C activation
triggered the
cytoplasmic kinase cascade involving Raf 1 kinase, MAP kinase (MEK-1), and
extracellular signal-regulated kinase (ERIC-1). ERK is a dual-specificity
kinase that
controls expression of proteins relevant to tumorigenesis, as well as tumor
cell.
proliferation, and motility.
In summary, phosphorylation of the CD44 receptor results in an increase in
tyrosine phosphorylation, leading to the activation of a cytoplasmic cascade,
early
response gene activation and cellular proliferation. Thus, an alternative,
indirect
approach to inhibit CD44 activation and signaling would be to employ
inhibitors of
CD44-activated cellular signaling pathways, rather than utilizing direct CD44
receptor
antagonists. Tyrosine-kinase inhibitors, PI~C inhibitors (e.g., calphostin) or
ERK
inhibitors could be employed as agents that would block CD44-mediated
signaling
proteins and provide novel targets for therapeutic agents.
One of the critical events in tumor growth and metastasis is the interaction
between tumor cells and host tissue stroma that is mediated by different
combinations of
adhesion receptors on different tumor cell types. Several lines of evidence
indicate that
interaction between the CD44 receptor expressed on tumor cells and tissue
stromal HA
can enhance growth and invasiveness of certain tumors. Transformation of colon
mucosa
to carcinoma is associated with overexpression of several CD44 alternative
splice
variants. The importance of CD44-hyaluronate interaction in tumor development
is made
clear by the disruption of CD44-hyaluronan interaction by soluble recombinant
CD44,
which was shown to inhibit tumor formation by lymphoma, and melanoma cells
transfected with CD44. The differential inhibitory effect of soluble wild-type
and mutant
CD44-Ig fusion proteins on melanoma growth i~ vivo was demonstrated by local
administration of a mutant, nonhyaluronate binding, CD44-Ig fusion protein
which had
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no effect on subcutaneous melanoma growth in mice whereas the infusion of wild-
type
CD44-Ig blocked tumor development.
The . present invention provides for administering a CD44 inhibitor or CD44
receptor antagonist in combination with at least one additional anti-pain
and/or anti
s inflammatory and/or anti-spasm and/or anti-restenosis agent which would be
effective in
preventing or inhibiting tumor cell adhesion and metastasis while inhibiting
one or more
of the other undesirable processes associated with surgical procedures.
Inhibitors and
antagonists may act directly by physical association and binding to the cell
surface
receptor to prevent binding of activating ligands or may act as indirect
inhibitors, by
inhibiting distinct molecules associated with CD44 signal transduction.
Direct receptor antagonists include monoclonal or polyclonal antibodies made
against variants of CD44, or fragments thereof, which can be used as
inhibitors of HA
binding to CD44 and can be employed as therapeutic agents. Anti-CD44
monoclonal
antibodies that can be employed include mAbs that recognize epitopes present
on all
isoforms of CD44 or suitable mAbs that recognize only post-translationally
modified
forms of CD44. Isoform (variant)-specific anti-CD44 mAbs have been described
previously (F12, A1G3, A3D8) in U.S. Patent No. 5,863,540 and the mAbs IM7 and
BU52. Other monoclonal antibodies suitable for the present invention include
anti-CD44
mAbs directed against unique CD44 isoforms that are produced as a result of
alternative
splicing with ten variable exons.
The present invention discloses the use of a recombinantly produced, chimeric
soluble CD44 (CSCD44) protein, in which the extracellular domain, or a portion
thereof,
of a CD44 receptor is covalently linked to a constant domain of an IgG
molecule which
may be used as an anti-tumor, anti-cell adhesion arid anti-metastasis agent.
In particular,
and by way of first example, a chimeric polypeptide (recombinant chimera)
comprising
the extracellular domain of the CD44 receptor extracellular polypeptide
coupled to the
CH2 and CH3 regions of a mouse IgG1 heavy chain polypeptide could be employed.
A
second example is a chimeric fusion construct comprised of the HA ligand
binding
domain of the CD44 receptor with portions of the Fc antibody (termed Fc fusion
soluble
receptors) that have been created for CD44 receptors. The molecular form of
the active
soluble CD44 receptor can be either monomeric or dimeric. Methods have been
established to test soluble CD44 antibody binding to the variant isoforms of
CD44
expressed on cell surfaces.
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A second method for inhibition of CD44-hyaluronan receptor-ligand binding is
to
employ hyaluronan oligosaccharides of defined size (hyaluronan oligomers) to
inhibit
tumor formation by saturating the CD44 receptors with excess ligand. One study
found
that hyaluronan oligomers injected at concentrations as low as 1 mg/ml
inhibited
melanoma growth. Thus, the local delivery of these oligomers can inhibit CD44
interactions with its natural substrate and provide a suitable method for the
control of
local tumor development.
One skilled in the art will appreciate that the choice of a specific anti-CD44
monoclonal antibody or CD44 peptide sequence as a therapeutic agent for a
patient will,
in part, determine the amounts of the agents to be administered for any
particular
treatment protocol and that these amounts can be readily determined. Those
skilled in the
art will appreciate that the amounts of the peptide, protein or antibody to be
administered
for a particular treatment protocol may be a function of a particular CD44
domain
associated with a pathologic clinical condition, and may be a function of a
particular cell
type mediating metastasis.
CD44 receptox antagonists suitable for use as anti-adhesion and/or anti-
invasion
and/or anti-metastasis agents in the surgical application of the current
invention, delivered
in combination with anti-pain and/or anti-inflammatory and/or ant-spasm and or
anti-
restenosis agents, are listed in the Table below.
TABLE 2
CD44 RECEPTOR ANTAGONISTS
Therapeutic Preferred Most Preferred
Agent Concentrations (~,g/ml) Concentrations (~.g/ml)
anti-CD44 mAb IM7 0.1-1000 100
anti-CD44 mAb BU52 0.1-1000 100
~
anti-CD44 mAb F12 0.1-1000 100
anti-CD44 mAb A1G3 0.1-1000 100
anti-CD44 mAb A3D8 0.1-1000 100
chimeric rhCD44:Fc 0.1-1000 100
anti-CD44 Fab 0.1-1000 100
hyaluronan oligomers0.1-1000 1000
soluble CD44 receptor1.0-10000 1000
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C. INTEGRIN RECEPTOR ANTAGONISTS AND INHIBITORS OF
INTEGRIN SIGNALING
Integrins are a superfamily of cell surface receptors involved in cell-cell
and cell-
matrix adhesion. These receptors play a fundamental role in the regulation of
numerous
cellular functions such as cell differentiation and migration, maintenance of
tissue
architecture, blood clot formation and retraction programmed cell death. In
addition, the
integrins have been implicated in cancer cell differentiation, tumor
progression and
metastasis due to their important role in mediating tumor cell interactions
with the
extracellular matrix and endothelial cells. Integrin expression and function
are altered in
many malignant cells, although changes in integrin expression vary between
different
tumor types. Specific integrin receptor subtypes on tumor cells are capable of
mediating
attachment to many adhesive proteins that are components of the extracellular
matrix,
activated platelets and endothelial cells at sites of surgical trauma.
The integrins are a family of heterodimeric, transmembrane a(3 receptors that
are
expressed on a wide variety of cells. The receptor family includes at least 14
known
a-subunits and 8 (3-subunits that associate with each other to form numerous
subtype
combinations that demonstrate differing ligand specificities. The a-subunit
appeaxs to be
a critical determinant of Iigand specificity and integrins containing av
demonstrate
specificity for vitronectin, a5 for fibronectin, and a3 for collagen/laminin.
Numerous
integrin heterodimer subunit combinations have been found on a variety of
tumor cells
that promote their binding to fibronectin, laminin, vitronectin, fibrinogen,
collagen, and
thrombospondin. Vascular cell adhesion molecule-1 (VCAM-1) is the endothelial
cell
ligand for two leukocyte integrins, a4~31 and a4~37. Mucosal addressin cell
adhesion
molecule 1 (MadCAM-1) is also recognized as a ligand for a4(37 integrin.
Interactions between fibronectin and several subtypes of integrin receptors
that
bind to it play particularly important roles at several stages of tumor
development and
influence the process of malignant metastasis. Fibronectin is an extracellular
glycoprotein consisting of repeating units of amino acids. A single gene
encodes it and
alternative splicing allows formation of multiple isoforms. Specific protein
domains
enable the molecule to interact with a variety of cells through both integrin
and non-
integrin receptors. Fibronectin has at least two independent cell adhesion
regions with
different receptor specificities. The cell adhesive domain in the central
portion of
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fibronectin is comprised of at least two minimal amino acid sequences, an Arg-
Gly-Asp
(RGD) sequence and a Pro-His-Ser-Arg-Asn (PHSRN) sequence. The a5(31
fibronectin-
specific integrin binds to the central RGDIPHSRN site. The a4(31 integrin
binds to the
IIICS site. Peptide and antibody inhibitors of fibronectin and integrin
functions have
been shown to be effective inhibitors of metastasis, and are potentially
important agents
for the control of tumor-cell adhesion.
Integrin-activated pathways mediated by intracellular kinases and the adapter
proteins are particularly important for integrin receptor-mediated anchorage
dependence.
Recently, signal transduction studies in a variety of cell types demonstrated
that integrin
receptor binding to extracellular matrix proteins rapidly generates
intracellular signals via
enhanced tyrosine phosphorylation and have established the role of the focal
adhesion
lcinase (FAK) protein-tyrosine kinase (PTK) in linking integrin receptors to
intracellular
signaling pathways. FAK associates with several different cytoplasmic
signaling proteins
such as Src-family PTKs and several SH2-domain proteins (Shc, Grb2 and PI 3-
kinase).
This enables FAK to function within a network of integrin-stimulated signaling
pathways
leading to the activation of targets such as the ERK and JNK/mitogen-activated
protein
kinase pathways. These signaling mechanisms provide a rationale for the
therapeutic
potential of tyrosine kinase inhibitors as anti-adhesion agents based upon
their action as
direct inhibitors of integrin receptor-mediated signaling at a point proximal
to the
receptor in the signal transduction cascade.
A peptide sequence motif responsible for the cell attachment in the
fibronectin
ligand fox the integrin receptor (axginine-glycine-aspartic acid) has provided
a basis for
the development of therapeutic agents since the integrin-binding activity of
adhesion
proteins can be reproduced by short synthetic peptides containing the RGD
sequence.
Peptides containing the RGD sequence have been shown to inhibit tumor cell
attachment
i~ vit~~o. Agents that bind selectively to only one or a few of the RGD-
directed integrins
have been prepared by cyclizing peptides with selected sequences flanking the
RGD
motif and by synthesizing RGD mimics. The development of a series of peptide
analogs
of the RGD sequence of fibronectin have been synthesized and their anti-
metastatic
effects in mice and inhibitory effects on tumor cell invasion i~ vitro have
been examined.
Cellular and animal studies have shown that anti-adhesion peptides and
polypeptides are useful for the prevention of peritoneal dissemination of
certain forms of
cancer. For example, a study using the peptidic integrin antagonists tyrosyl-
isoleucyl-
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glycyl-Beryl-arginine (YIGSR), acetyl-arginyl-glycyl-aspartyl-serinamide (Ac-
RGDS-
NH2) and arginyl-glycyl-aspartic acid (RGD) examined the effect on the
adhesion and
invasiveness of gastric cancer cell lines using a peritoneal-seeding cell
Line,
OCUM-2MD3. It was reported that both x2(31 and x3(31 integrin expression was
markedly increased on OCUM-2MD3 cells and the ability of these cells to bind
to the
extracellulax matrix was also significantly higher than that of control cells.
The adhesion
polypeptides,. YIGSR and RGD, and two RGD derivatives significantly inhibited
the
adhesion and invasiveness of OCUM-2MD3 cells to the submesothelial ECM in a
cell-
specific manner. The survival of nude mice with peritoneal dissemination given
YIGSR
or RGD sequences intraperitoneally was Longer than that of untreated mice. A
' therapeutic approach based upon interfering with tumor cell attachment with
integrin-
binding peptides has been shown to be an effective anti-metastatic strategy in
animal
experiments and the frequency of tumor implantation at sites of surgical
trauma is also
reduced if cells are preexposed to fibrinogen, Iaminin or~ peptides containing
the RGDS
motif.
These studies and others suggest that inhibition of specific integrin receptor-
ligand interactions by use of synthetic fibronectin fragments, peptide analogs
and mimics
containing the RGD sequence, or antibodies represents a useful approach for
the
development of clinically useful antagonists for subtype-specific integrin
receptors. A
number of these peptides, such as those disclosed within U.S. Patent No.
5,627,263, have
been found to be more effective in inhibiting tumor cell adhesion and tumor
metastasis
than the original RGDS peptide, and are disclosed for use in the present
invention in
combination with other agents. In particular, the pxesent invention provides
for the use of
peptides and peptide analogs which are specific for fibronectin-binding
integrins, in
particular the as (31 integrin. In addition, specific agents for inhibition of
binding to a2,
a6 and oc~ integrin subunits as part of ~i 1 integrin heterodimers are also
provided.
An additional class of inhibitors is the naturally occurring disintegrins,
which
include a family of small, highly homologous, cysteine-rich polypeptides
purified from
snake venoms, which contain the RGD sequence. Disintegrins can competitively
inhibit
integrin-ligand interactions and have been shown to inhibit cell adhesion
interactions in a
variety of systems. Specific snake venom-derived disintegrins have been shown
to be
2000 times more potent than short, synthetic, linear RGD-containing peptides
in blocking
fibrinogen-dependent platelet aggregation. The greater potency of the
disintegrin
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molecules stems from amino acids surrounding the RGD sequence and intrachain
disulfide bridges that constrain the RGD sequence in an appropriate
conformation.
Specific disintegrins block the adhesive functions of integrin Bell surface
receptors and
thereby inhibit integrin-dependent cell reactions in a variety of cell types
and tissues. The
disintegrin kistrin was shown by affinity crosslinking to specifically bind
with high
affinity to aV(i3 and not to a5~i1 or other abundant integrins. The related
disintegrin
echistatin specifically inhibited lasl-labeled kistrin binding to aV(33, while
a structurally
distinct disintegrin, decorsin had 1000-fold lower affinity.
Two recently isolated disintegrins from the venom of Chinese viper
(Agkist~odon
ussu~iensis), ussuristatin 1 (US-1) and 2 (US-2) have been characterized as
potent
inhibitors of cell adhesion. Both polypeptides are composed of 71 amino acids
and are
characterized by sequences that show strong similarities to other
disintegrins. US-1 had a
typical Arg-Gly-Asp (RGD) sequence, which is responsible for blocking the
binding of
fibrinogen to the receptor. In US-2, the corresponding sequence was Lys-Gly-
Asp
(KGD). US-2 also inhibited platelet aggregation, but. the ICsos were about ten
times
higher. US-1 also dose-dependently inhibited the adhesion of human melanoma
cells to
fibrinogen and fibronectin with an ICSO = 17-33 nM, while US-2 did not inhibit
the cell
adhesion to fbronectin.
An additional heterodimeric disintegrin, EC3 (Mr = 14,762), isolated from
Echis
ca~~inatus venom is a potent antagonist of a4 integrins. Each subunit contains
67 residues
and displays a high degree of homology to other disintegrins. EC3 inhibited
adhesion of
cells expressing a4[31 and a4~37 integrins to natural ligands such as vascular
cell
adhesion molecule 1 (VCAM-1) and mucosal addressin cell adhesion molecule 1
(MadCAM-1) with ICSO = 6-30 nM, adhesion of K562 cells (a5~31) to fibronectin
with
ICSO = 150 nM, and the adhesion of aIIb~33 Chinese hamster ovary cells to
fibrinogen
with ICSO = 500 nM.
Examples of integrin antagonists suitable for the present invention are listed
below. As an example, for each of the listed agents, the preferred and most
preferred
concentrations of an irrigation solution containing the listed agent are
provided, such
concentrations expected to be therapeutically effective.
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TABLE 3
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF INTEGR1N
RECEPTOR ANTAGONISTS
Compounds Therapeutic PreferredMost Preferred
Concentrations Concentrations
(~,M) (~M)
RGD 5-50000 5000
GRGDSP 5-50000 5000
YIGSR 5-50000 5000
GACRGDCLGA 0.1-10000 100
GRGDSPK 0.1-1000 10
GRGDTP 0.1-1000 10
SDGRG 0.1-1000 10
RGDSPASSKP 0.1-1000 10
cyclo(RGD-D-Phe-Val)0.1-1000 10
Trimesyl(DRGDS)3 0.1-1000 10
US-1 0.03-3000 3
kistrin 0.1-1000 10
EC-3 0.1-1000 1
S D. SELECTIN RECEPTOR ANTAGONISTS AND INHIBITORS OF
SELECTIN SIGNALING
Adhesion of malignant cells to the vascular endothelial surface involves a
family
of adhesion receptors similar to those involved in the recruitment of
inflammatory cells to
tissue sites. Selectins are involved in a cascade of sequential molecular
steps following
endothelial cell (EC) activation. The selectin family consists of E-selectin
(ELAM-1),
P-selectin (GMP-140), and L-selectin (LECAM-1). The carbohydrate determinants,
sialyl Lewis A (SLe~) and sialyl Lewis X (SLeX), which are frequently
expressed on
human cancer cells, serve as ligands for E-selectin, which is expressed on
vascular
endothelial cells. These carbohydrate determinants are involved in the
adhesion of cancer
~ 5 cells to vascular endothelium and thus contribute to metastasis of cancer.
The selectins
are inducible endothelial-expressed adhesion molecules involved in leul{ocyte
recruitment. The initial adhesion mediated by these molecules triggers
activation of
integrin molecules through the action of several cytokines. The degree of
expression of
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the carbohydrate ligands at the surface of cancer cells is well correlated
with the
frequency of metastasis. Monoclonal antibodies directed to Slea or SIeX
blocked adhesion
of tumor cells (leukemia, colon carcinoma, and histiocytic lymphoma cells) to
EC and
platelets. Also selectin expression is frequently up-regulated on breast
carcinoma
endothelium.
P-selectin, also known as GMP-I40 or PADGEM, is a membrane glycoprotein
located in secretory granules of resting (unstimulated) platelets and
endothelium. When
mediators activate these cells, P-selectin is rapidly redistributed to the
plasma membrane.
The selectins constitute a family of structurally and functionally related
molecules.
Structural motifs common to each of these molecules include an N-terminal
lectin-like
domain followed by an EGF-like region, a series of consensus repeats related
to those in
complement-binding proteins, a transmembrane domain, and a short cytoplasmic
tail.
P-selectin is the receptor for neutrophils and monocytes when it is expressed
on activated
platelets and endothelium. This property facilitates rapid adhesion of
leukocytes to
endothelium at regions of tissue injury as well as platelet-leukocyte
interactions at sites of
inflammation and hemorrhage. Studies also have found that P-selectin binds to
tumor
cells in a variety of tissue sections and that it binds to the cell surface of
a number of cell
lines derived from carcinomas. Acting in concert with other adhesion
molecules,
P-selectin participates in tumor metastasis and is thus a target for drugs
that block
adhesion receptor function.
Studies supporting a role for selectins in metastasis are based upon the
adhesion
of human colon carcinoma cell lines to selectins using an in vitro flow model.
Recombinant forms of P-selectin and Chinese hamster ovary cells expressing P-
selectin
supported attachment and rolling of KM12-L4 colon carcinoma cells and this
effect was
abolished by pretreatment of the KM12-L4 cells with neuraminidase. KM12-L4
cells
interact with P-selectin through a PSGL-I-independent adhesion pathway. An
E-selectin-IgG chimera was also found to support sialylated moiety-dependent
adhesion
of colon carcinoma cells under fluid flow. Thus, sialylated moieties are
involved in the
selectin mediated adhesion of human cancer cells to IL-1-simulated endothelium
under
flow conditions. Furthermore, the efficiency of E-selectin-mediated binding of
human
colon carcinoma cells to human and mouse EC has been found to be correlated
with the
metastatic potential of the cancer cells.
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Recent studies evaluated the role of E-selectin-sialyl Lewis x (Sle")/ sialyl
Lewis a
(Slew) interacxion in mediating in vitro adhesion of two human colon cancer
cell lines,
HT-29 and COLD 201, to human umbilical cord endothelial cells (HUVEC). Colon
cancer cell lines had a strong expression of carbohydrate epitopes and it was
established
that adhesion of HT-29 and COLO 201 cells to IL-1-stimulated HUVEC could be
inhibited by a monoclonal antibody directed against E-selectin. Prior
incubation of cells
with two different antibodies directed against Sle" and antibodies directed
against related
Lewis epitopes, LeX and Lea, had no significant effect on adhesion. Three
antibodies
directed against Slea differed in their capacity to inhibit the adhesion of HT-
29 and
COLO 201 cells. Thus, the Slea epitope is important for adhesion of colon
cancer cells
mediated predominantly by E-selectin.
Additional studies have demonstrated that pretreatment of HUVEC with tiunor
necrosis factor-a (TNF-alpha) augmented the binding of the COLO 205 carcinoma
cell
line. The increased adherence was both concentration-and time-dependent with
maximal
tumor cell attachment found at 4 hr. This result was consistent with increased
expression
of the adhesion molecule E-selectin on the endothelium. Incubation of TNF-
stimulated
HUVECs with BB1 l, an anti-E-selectin mAb, prior to the addition of COLD 205
resulted
in complete inhibition of adherence of the tumor cells. These studies confirm
the utility
of specific antibodies directed against E-selectins to inhibit tumor cell
attachment to
endothelial cell targets. Other studies have found that pretreatment with
either an
anti-P-selectin or an anti-L-selectin monoclonal antibody (i.e., MAb PB 1.3
and MAb
DREG-200), or a sialyl LewisX-containing oligosaccharide (Sle"-OS) are
effective in
inhibiting cell adhesion.
Similar in vitro and ivc vivo studies have established a role for selectins in
the
process of metastasis using SW1990 cells derived from a human pancreatic
malignancy.
SW1990 cells also strongly express Slea and SLeX antigens, CD44H, and (31
integrin.
These cells exhibit binding activity to IL-1-activated HUVECS and human
peritoneal
mesothelial cells. The adhesion leading to implantation of cancer cells to
endothelial
cells was inhibited by treatment with the antibodies against Slea and against
(31 integrin.
In animal studies, treatments with the antibodies against Slea and (31
integrin each
inhibited the development of liver metastasis in nude mice with S W 1990 cells
and
prolonged their survival.
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Elucidation of the signal transduction properties of the selectins has
revealed a
common mechanism shared by other cell adhesion receptors. Studies using HT-29
human colon carcinoma cells, which adhere specifically to an E-selectin-IgG
chimera,
have shown that, upon adhesion to E-selectin, there is an increase in the
amount of
tyrosine phosphorylation of several proteins in HT-29 cell lysates. This
effect is specific
for adhesion to E-selectin, since addition of an E-selectin blocking
monoclonal antibody
caused a significant decrease in tyrosine phosphorylation relative to E-
selectin alone. A
lcinase assay showed a dose-dependent and significant decrease in c-src
activity upon
adhesion to E-selectin, which correlates with an increase in phosphorylation
of Tyr 527,
the negative regulatory tyrosine. These studies identify a signaling pathway
involving the
E-selectin ligand on HT-29 cells and c-src.
Other studies have demonstrated that lymphocyte binding to P-selectin induces
tyrosine phosphorylation of distinct proteins. Activation appears to occur at
the initial
contact with surface adhesion molecules. The P-selectin effect was time
dependent,
demonstrating an early response after 10 min and a maximum effect at 30 min.
Identified
proteins included pp125 focal adhesion kinase (FAK) and paxillin. Treatment
with a
tyrosine kinase inhibitor, genistein, or with the protein kinase C inhibitor,
staurosporine,
resulted in decreased pp 125 FAIL phosphorylation. These signaling mechanisms
provide
a rationale for the therapeutic potential of tyrosine kinase inhibitors as
anti-adhesion
agents based upon their action as direct inhibitors of selectin receptor-
mediated signaling.
The rapid signaling properties of the selectin receptors underscore the
requirement for
delivery of the therapeutic agent at the beginning of the surgical procedure
to provide a
preemptive inhibitory effect.
This invention provides a method for inhibiting the spread of metastatic tumor
cells by administering to a patient a pharmaceutical composition containing a
therapeutically effective amount of an anti-E-selectin specific mAb or
antibody fragment,
and/or an anti-P-selectin specific mAb or antibody fragment, and/or an anti-L-
selectin
specific mAb or antibody fragment, and/or an anti-sialyl Lewisa (Slew) mAb in
combination with either one or more anti-inflammatory and/or anti-pain and/or
anti-
spasm andlor anti-restenosis agents.
Studies have also found that heparin inhibited the binding of selectins to
their
carbohydrate ligands. Heparin (enoxaparin) inhibition of selectin interactions
is expected
to decrease selectin-related tumor cell adhesion.
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Examples of selectin antagonists suitable for the present invention are listed
below. For each of the listed agents, the preferred and most preferred
concentrations of
an irrigation solution containing the listed agent are provided, such
concentrations
expected to be therapeutically effective.
TABLE 4
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF SELECTIN
RECEPTOR ANTAGONISTS
Agent Therapeutic Preferred Most Preferred
Concentrations (~g/ml) Concentrations (~g/ml)
anti-SLeX mAb PB1.3 0.1-1000 100
anti-SLea mAb DREG-200 0.1-1000 100
SIeX-OS 0.1-1000 100
anti-E-selectin mAb BB11 0.1-10000 1000
anti-P-selectin IgG 0.1-10000 1000
anti-E-selectin IgG 1.0-10000 1000
enoxapaxin 0.1-1000 100
From the molecular and cellular mechanisms of action defined for the classes
of
agents identified herein, these agents are expected to exhibit tumor cell anti-
adhesion,
anti-invasion and/or anti-metastatic actions when applied perioperatively in
an irrigation
solution. These agents are expected to be effective drugs when delivered in an
irrigation
solution during surgical procedures involving identified or occult
malignancies. The
method of delivery and the compositions for inclusion in the irrigation are
expected to be
useful in procedures involving the peritoneal, abdominal, thoracic, pleural,
mediastinal,
urogenital, epidural, intra-thecal, and joint cavities including, but not
limited to,
operations indicated for oncological treatment of ovarian, gastric, pancreatic
and colon
cancer. Each metabolically active anti-tumor adhesion agent may be delivered
in
combination with one or more other anti-pain and/or anti-inflammatory andlor
anti-spasm
and/or anti-restenosis agents. Suitable anti-tumor agents for inclusion in the
pharmaceutical composition will be further characterized by a requirement for
pharmacological selectivity and specificity which limits interactions of the
agent to a
single family (or class) of receptors or a single enzyme family (e.g., CD44,
integrins,
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selectins, protein tyrosine kinases, MMPs and MAP I~inases). A particular
suitable agent
may interact specifically with one or more receptor subtypes (or isotypes)
while
exhibiting specificity for the receptor family. Each suitable agent will
associate with its
molecular target through a specific molecular mechanism which can be
characterized by a
defined stoicluometry (typically 1:1 ) for the ligand-receptor (or inhibitor-
enzyme)
complex and which can be characterized by its equilibrium binding or kinetic
constant.
These agents may include small molecule drugs, natural or synthetic peptides
or peptoids,
polypeptides, proteins, recombinant chimeric proteins, monoclonal or
polyclonal
antibodies, oligonucleotides or gene therapy vectors (viral and nonviral).
For example, an agent such as an integrin receptor antagonist can exert its
actions
on any cells associated with the fluid spaces of the peritoneal cavity and
structures
comprising the cavity that are involved in normal function or are present due
to a
pathological condition. These cells and structures include, but are not
limited to:
epithelial cells; mesothelial cells; inflammatory cells including lymphocytes,
macrophages, mast cells, monocytes, eosinophils; and other cells including
endothelial
cells, smooth muscle cells, and fibroblasts; and all combinations of the
above.
The present invention also provides for formulations of the active therapeutic
agents) which may be delivered in a formulation useful for introduction and
administration to the operative site that would enhance the delivery, uptake,
stability or
pharmacolcinetics of the combination of anti-adhesion, anti-invasion and/or
anti-
metastatic agents and other agents. Such a formulation could include, but is
not limited
to, microparticles, microspheres or nanoparticles composed of proteins,
carbohydrates,
synthetic organic compounds, or inorganic compounds. Examples of formulation
molecules include, but are not limited to, lipids capable of forming liposomes
or other
ordered lipid structures, cationic lipids, hydrophilic polymers, polycations
(e.g.,
protamine, spermidine and polylysine), peptide or synthetic liga~zds and
antibodies
capable of targeting materials to specific cell types, gels, slow release
matrices, soluble
and insoluble particles, as well as other formulation elements known to those
slcilled in
the art.
The present invention provides for the delivery of a combination of anti-tumor
adhesion and/or anti-invasion and/or anti-local metastatic drugs, present
either as multiple
pharmaceutically active substances within a homogeneous (e.g., a single
encapsulated
microsphere) or as a discrete mixture of individual delivery vehicles (e.g., a
group of
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microspheres encapsulating one or more agents). Examples of formulation
molecules
include, but are not limited to, hydrophilic polymers, polycations (e.g.,
protamine,
spermidine, polylysine and chitosan), peptidic or synthetic ligands and
antibodies capable
of targeting materials to specific cell types, gels, slow release matrices,
soluble and
insoluble particles, as well as formulation elements not listed.
The present invention provides for the local delivery of a combination of
tumor
cell anti-adhesion drugs) via an irrigation solution, such irrigation solution
containing the
drugs which are present at therapeutically effective low concentrations and
enabling the
drugs to be delivered directly to the desired tissue. The drug-containing
irrigation
solution may be employed intra-operatively, intra- and pre-operatively, intra-
and post-
operatively or pre-, intra- and post-operatively in connection with a surgical
procedure.
Conventional methods used for drug delivery have required systemic (e.g.,
oral,
intramuscular, intravenous, subcutaneous) administration which necessitates
higher
concentrations of drugs (and higher total doses) to be administered to the
patient in order
to achieve significant therapeutic concentrations at the pathologically
affected tissue.
Systemic administration also results in high concentrations in tissues other
than the
targeted tissue which is undesirable and, depending on the dose, may result in
adverse
side effects. These systemic methods subject the drug to second-pass
metabolism and
rapid degradation, thereby limiting the duration of the effective therapeutic
concentration.
Since the combinations of tumor cell anti-adhesion, anti-invasion and anti-
metastasis
agents (with or without one or more anti-pain and/or anti-inflammatory and/or
anti-spasm
and/or anti-restenosis agents) are administered directly to the operative site
by irrigation,
vascular perfusion is not required to carry the drug to the targeted tissue.
This significant
advantage allows for the local delivery of a lower therapeutically effective
total dose for a
variety of tumor cell anti-adhesion, anti-local metastasis drugs than would
otherwise be
possible via other routes of administration.
In other aspects, the irrigation solutions of the present invention comprise a
dilute
solution of at least one inhibitor of tumor cell adhesion, invasion and/or
metastasis and
preferably multiple pain/inflammation inhibitory agents, anti-spasm agents and
anti-
restenosis agents in a physiologic carrier. The carrier is a liquid, which as
used herein is
intended to encompass biocompatible solvents, suspensions, polymerizable and
non-polymerizable gels, pastes and salves. Preferably the carrier is an
aqueous solution
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which may include physiologic electrolytes, such as normal saline or lactated
Ringer's
solution.
The anti-inflammation/anti-pain agents are selected from the group consisting
of:
(1) serotonin receptor antagonists; (2) serotonin receptor agonists; (3)
histamine receptor
antagonists; (4) bradykinin receptor antagonists; (5) kallikrein inhibitors;
(6) tachykinin
receptor antagonists, including neurokinin, and neurokinin2 receptor subtype
antagonists;
(7) calcitonin gene-related peptide (CGRP) receptor antagonists; (8)
interleukin receptor
antagonists; (9) inhibitors of enzymes active in the synthetic pathway for
arachidonic acid
metabolites, including (a) phospholipase inhibitors, including PLA2 isoform
inhibitors
and PLC, isoform inhibitors (b) cyclooxygenase inhibitors, and (c)
lipooxygenase
inhibitors; (10) prostanoid receptor antagonists including eicosanoid EP-l and
EP-4
receptor subtype antagonists and thromboxane receptor subtype antagonists;
(11) leukotriene receptor antagonists including leukotriene B4 receptor
subtype
antagonists and leukotriene D4 receptor subtype antagonists; (12) opioid
receptor
agonists, including p.-opioid, 8-opioid, and ~c-opioid receptor subtype
agonists;
(13) purinoceptor agonists and antagonists including P2X receptor antagonists
and P2y
receptor agonists; and (14) adenosine triphosphate (ATP)-sensitive potassium
channel
openers.
Suitable anti-inflammatory/anti-pain agents which also act as anti-spasm
agents
include serotonin receptor antagonists, tachykinin receptor antagonists, ATP-
sensitive
potassium channel openers and calcium channel antagonists. Other agents which
may be
utilized in the solution specifically for their anti-spasm properties
including endothelin
receptor antagonists, calcium channel antagonists and the nitric oxide donors
(enzyme
activators).
Specific preferred embodiments of the solution of the present invention for
use in
cardiovascular and general vascular procedures include anti-restenosis agents,
which
most preferably are used in combination with anti-spasm agents. Suitable anti-
restenosis
agents include: (1) antiplatelet agents including: (a) thrombin inhibitors and
receptor
antagonists, (b) adenosine disphosphate (ADP) receptor antagonists (also known
as
purinoceptorl receptor antagonists), (c) thromboxane inhibitors and receptor
antagonists
and (d) platelet membrane glycoprotein receptor antagonists; (2) inhibitors of
cell
adhesion molecules, including (a) selectin inhibitors and (b) integrin
inhibitors;
(3) anti-chemotactic agents; (4) interleukin receptor antagonists (which also
serve as
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anti-pain/anti-inflammation agents); and (5) intracellular signaling
inhibitors including:
(a) protein kinase C (PKC) inhibitors and protein tyrosine phosphatases, (b)
modulators
of intracellular protein tyrosine lcinase inhibitors, (c) inhibitors of src
homology2 (SH2)
domains, and (d) calcium channel antagonists. Such agents are useful in
preventing
restenosis of arteries treated by angioplasty, rotational atherectomy or other
cardiovascular or general vascular therapeutic procedure.
In each of the surgical solutions of the present invention, the agents are
included
in low concentrations and are delivered locally in low doses relative to
concentrations and
doses required with conventional methods of drug administration to achieve the
desired
therapeutic effect. It is impossible to obtain an equivalent therapeutic
effect by delivering
similarly dosed agents via other (i.e., intravenous, subcutaneous,
intramuscular or oral)
routes of drug administration since drugs given systemically are subject to
first- and
second-pass metabolism. The concentration of each agent is determined in part
based on
its dissociation constant, Kd. As used herein, the term dissociation constant
is intended to
encompass both the equilibrium dissociation constant for its respective
agonist-receptor
or antagonist-receptor interaction and the equilibrium inhibitory constant for
its
respective activator-enzyme or inhibitor-enzyme interaction. Each agent is
preferably
included at a low concentration of 0.1 to 10,000 times Kd nanomolar, except
for
cyclooxygenase inhibitors, which may be required at larger concentrations
depending on
the particular inhibitor selected. Preferably, each agent is included at a
concentration of
1.0 to 1,000 times Kd nanomolar and most preferably at approximately 100 times
Kd
nanomolar. These concentrations are adjusted as needed to account for dilution
in the
absence of metabolic transformation at the local delivery site. The exact
agents selected
for use in the solution, and the concentration of the agents, varies in
accordance with the
particular application, as described below.
A solution in accordance with the present invention can include just a single
or
multiple inhibitors) of tumor cell adhesion, invasion and/or metastasis,
pain/inflammation inhibitory agent(s), a single or multiple anti-spasm
agent(s), a
combination of inhibitors of tumor cell adhesion, invasion and/or metastasis,
anti-spasm
and pain/inflammation inhibitory agents, or anti-restenosis agents from the
enumerated
classes, at low concentration. However, due to the aforementioned synergistic
effect of
multiple agents, and the desire to broadly block pain and inflammation, ,
spasm and
restenosis, it is preferred that multiple agents be utilized.
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The surgical solutions constitute a novel therapeutic approach by combining
multiple pharmacologic agents acting' at distinct receptor and enzyme
molecular targets.
To date, pharmacologic strategies have focused on the development of highly
specif c
drugs that axe selective for individual receptor subtypes and enzyme isoforms
that
mediate responses to individual signaling neurotransmitters and hormones. As
an
example, endothelin peptides are some of the most potent vasoconstrictors
known.
Selective antagonists that are specific for subtypes of endothelin (ET)
receptors are being
sought by several pharmaceutical companies for use in the treatment of
numerous
disorders involving elevated endothelin levels in the body. Recognizing the
potential role
of the receptor subtype ETA in hypertension, these drug companies specifically
are
targeting the development of selective antagonists to the ETA receptor subtype
for the
anticipated treatment of coronary vasospasm. This standard pharmacologic
strategy,
although well accepted, is not optimal since many other vasoconstrictor agents
(e.g.,
serotonin, prostaglandin, eicosanoid, etc.) simultaneously may be responsible
for
initiating and maintaining a vasospastic episode (see FIGURES 2 and 4).
Furthermore,
despite inactivation of a single receptor subtype or enzyme, activation of
other receptor
subtypes or enzymes and the resultant signal transmission often can trigger a
cascade
effect. This explains the significant difficulty in employing a single
receptor-specific
drug to block a pathophysiologic process in which multiple transmitters play a
role.
Therefore, targeting only a specific individual receptor subtype, such as ETA,
is likely to
be ineffective.
In contrast to the standard approach to pharmacologic therapy, the therapeutic
approach of the present surgical solutions is based on the rationale that a
combination of
drugs acting simultaneously on distinct molecular targets is required to
inhibit the full
spectrum of events that underlie the development of a pathophysiologic state.
Furthermore, instead of targeting a specific receptor subtype alone, the
surgical solutions
are composed of drugs that target common molecular mechanisms operating in
different
cellular physiologic processes involved in the development of pain,
inflammation,
vasospasm, smooth muscle spasm and restenosis (see FIGURE 1). In this way, the
cascading of additional receptors and enzymes in the nociceptive,
inflammatory,
spasmodic and restenotic pathways is minimized by the surgical solutions. In
these
pathophysiologic pathways, the surgical solutions inhibit the cascade effect
both
"upstream" and "downstream".
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An example of "upstream" inhibition is the cyclooxygenase antagonists in the
setting of pain and inflammation. The cyclooxygenase enzymes (COXz and COX2)
catalyze the conversion of arachidonic acid to prostaglandin H which is an
intermediate
in the biosynthesis of inflammatory and nociceptive mediators including
prostaglandins,
leukotrienes, and thromboxa.nes. The cyclooxygenase inhibitors block
"upstream" the
formation of these inflammatory and nociceptive mediators. This strategy
precludes the
need to block the interactions of the seven described subtypes of prostanoid
receptors
with ~ their natural ligands. A similar "upstream" inhibitor included in the
surgical
solutions is aprotinin, a kallikrein inhibitor. The enzyme kallikrein, a
serine protease,
cleaves the high molecular weight kininogens in plasma to produce
bradylcinins,
important mediators of pain and inflammation. By inhibition of kallikrein,
aprotinin
effectively inhibits the synthesis of bradykinins, thereby providing an
effective
"upstream" inhibition of these inflammatory mediators.
The surgical solutions also make use of "downstream" inhibitors to control the
pathophysiologic pathways. In vascular smooth muscle preparations that have
been
precontracted with a variety of neurotransmitters (e.g., serotonin, histamine,
endothelin,
and thromboxane) implicated in coronary vasospasm, ATP-sensitive potassium
channel
openers (KCOs) produce smooth muscle relaxation which is concentration
dependent
(Quast et al., 1994; Kashiwabara et al., 1994). The KCOs, therefore, provide a
significant
advantage to the surgical solutions in the settings of vasospasm and smooth
muscle spasm
by providing "dowxnstream" antispasmodic effects that are independent of the
physiologic
combination of agonists initiating the spasmodic event (see FIGURES 2 and 4).
Similarly, NO donors and voltage-gated calcium channel antagonists can limit
vasospasm
and smooth muscle spasm initiated by multiple mediators known to act earlier
in the
spasmodic pathway.
II. ANTI-INFLAMMATION/ANTI-PAIN AGENTS
The following is a description of suitable drugs falling in the aforementioned
classes of anti-inflammation/anti-pain agents, as well as suitable
concentrations for use in
solutions, of the present invention. While not wishing to be limited by
theory, the
justification behind the selection of the various classes of agents which is
believed to
render the agents operative is also set forth.
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A. SEROTONIN RECEPTOR ANTAGONISTS
Serotonin (5-HT) is thought to produce pain by stimulating serotonin2 (5-HT2)
and/or serotonin3 (5-HT3) receptors on nociceptive neurons in the periphery.
Most
researchers agree that 5-HT3 receptors on peripheral nociceptors mediate the
immediate
pain sensation produced by 5-HT (Richardson et al., 1985). In addition to
inhibiting
5-HT-induced pain, 5-HT3 receptor antagonists, by inhibiting nociceptor
activation, also
may inhibit neurogenic inflammation. Barnes P.J., et al., "Modulation of
Neurogenic
Inflammation; Novel Approaches to Inflammatory Disease," Ti°ends in
Pharmacological
Sciences 11:185-189 (1990). A study in rat ankle joints, however, claims the 5-
HT2
receptor is responsible for nociceptor activation by 5-HT. Grubb, B.D., et
al., "A Study
of 5-HT-Receptors Associated with Afferent Nerves Located in Normal and
Inflamed Rat
Ankle Joints," Agents Actions 25:216-18 (1988). Therefore, activation of 5-HT2
receptors also may play a role in peripheral pain and neurogenic inflammation.
One goal of the solution of the present invention is to block pain and a
multitude
of inflammatory processes. Thus, 5-HT~ and 5-HTg receptor antagonists are both
suitably used, either individually or together, in the solution of the present
invention, as
shall be described subsequently. Amitriptyline (ElavilTM) is a suitable 5-HT2
receptor
antagonist for use in the present invention. Amitriptyline has been used
clinically for
numerous years as an anti-depressant, and is found to have beneficial effects
in certain
chronic pain patients. Metoclopramide (ReglanTM) is used clinically as an anti-
emetic
drug, but displays moderate affinity for the 5-HTg receptor and can inhibit
the actions of
5-HT at this receptor, possibly inhibiting the pain due to 5-HT release from
platelets.
Thus, it also is suitable for use in the present invention.
Other suitable 5-HT2 receptor antagonists include imipramine, trazodone,
desipramine and lcetanserin. Ketanserin has been used clinically for its anti-
hypertensive
effects.. Hedner, T., et al., "Effects of a New Serotonin Antagonist,
Ketanserin, in
Experimental and Clinical Hypertension," Am. J. of Hypertension, pp.317s-23s
(Jul. 1988). Other suitable 5-HT3 receptor antagonists include cisapride and
ondansetron.
The cardiovascular and general vascular solution also may contain a
serotoninlB (also
lcnown as serotoninlDa) antagonist because serotonin has been shown to produce
significant vascular spasm via activation of the serotoninlB receptors in
humans.
Kaumann, A.J., et al., "Variable Participation of 5-HT1-Like Receptors and 5-
HT2
Receptors in Serotonin-Induced Contraction of Human Isolated Coronary
Arteries,"
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Ci~culatio~c 90:1141-53 (1994). Suitable serotonin~B receptor antagonists
include
yohimbine, N-[-methoxy-3-(4-methyl-1-piperanzinyl)phenyl]-2'-methyl-4'-(S-
methyl
1, 2, 4-oxadiazol-3-yl)[l, 1-biphenyl]-4-carboxamide ("GR127935") and
methiothepin.
Therapeutic and preferred concentrations for use of these drugs in the
solution of the
present invention are set forth in Table 5.
TABLE 5
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
PAIN/INFLAMMATION INHIBITORY AGENTS
Therapeutic Preferred
ConcentrationsConcentrations
Class of Agent (Nanomolar) (Nanomolar)
Serotonin2 Receptor Anta og
nists:
amitriptyline 0.1 - 1,000 50 - 500
imiprarnine 0.1 - 1,000 50 - 500
trazodone 0.1 - 2,000 50 - 500
desipramine 0.1 - 1,000 50 - 500
ketanserin 0.1 - 1,000 50 - 500
Serotonin3 Receptor Antagonists:
tropisetron 0.01 - 100 0.05 - 50
metoclopramide 10 - 10,000 200 - 2,000
cisapride 0.1 - 1,000 20 - 200
ondansetron 0.1 - 1,000 20 - 200
Serotoninl$~Human 1D~) Antagonists:
yohimbine 0.1 - 1,000 50 - 500
~
GR127935 0.1 - 1,000 10 - 500
methiothepin 0.1 - 500 1 - 100
B. SEROTON1N RECEPTOR AGONISTS
5-HTIp, 5-HT1B and 5-HT1D receptors are known to inhibit adenylate cyclase
activity. Thus including a low dose of these serotoninlp, serotoninlg and
serotoninlD
receptor agonists in the solution should inhibit neurons mediating pain and
inflammation.
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The same action is expected from serotoninlg and serotoninlF receptor agonists
because
these receptors also inhibit adenylate cyclase.
Buspirone is a suitable lA receptor agonist for use in the present invention.
Sumatriptan is a suitable 1 A, 1 B, 1 D and 1 F receptor agonist. A suitable 1
B and 1 D
receptor agonist is dihydroergotamine. A suitable iE receptor agonist is
ergonovine.
Therapeutic and preferred concentrations for these receptor agonists are
provided in
Table 6.
TABLE 6
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
PA1N/INFLAMMATION INHIBITORY AGENTS
Therapeutic Preferred
Concentrations Concentrations
Class of Agent (Nanomolar) (Nanomolar)
SerotoninlA A._gonists:
buspirone 1 - 1,000 10 - 200
surnatriptan 1 - 1,000 10 - 200
SerotoninlB Agonists:
dihydroergotamine 0.1 - 1,000 10 - 100
sumatriptan 1 - 1,000 10 - 200
SerotoninlD A~onists:
dihydroergotamine 0.1 - 1,000 10 - 100
surnatriptan 1 - 1,000 10 - 200
SerotoninlF.~,onists:
ergonovine 10 - 2,000 100 - 1,000
SerotoninlF A.gonists:
surnatriptan 1 - 1,000 10 - 200
C. HISTAMINE RECEPTOR ANTAGONISTS
Histamine receptors generally are divided into histamine) (Hl) and histamine2
(H2) subtypes. The classic inflammatory response to the peripheral
administration of
histamine is mediated via the H1 receptor. Douglas, 1985. Therefore, the
solution of the
present invention preferably includes a histamine H1 receptor antagonist.
Promethazine
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(PhenerganTM) is a commonly used anti-emetic drug which potently bloclcs H1
receptors,
and is suitable for use in the present invention. Interestingly, this drug
also has been
shown to possess local anesthetic effects but the concentrations necessary for
this effect
are several orders higher than that necessary to block H1 receptors, thus, the
effects are
believed to occur by different mechanisms. The histamine receptor antagonist
concentration in the solution is sufficient to inhibit H1 receptors involved
in nociceptor
activation, but not to achieve a "local anesthetic" effect, thereby
eliminating the concern
regarding systemic side effects.
Histamine receptors also are known to mediate vasomotor tone in the coronary
arteries. In vitro studies in the human heart have demonstrated that the
histamine)
receptor subtype mediates contraction of coronary smooth muscle. Ginsburg, R.,
et al.,
"Histamine Provocation of Clinical Coronary Artery Spasm: Implications
Concerning
Pathogenesis of Variant Angina Pectoris," Ame~icah Heat J. 102:819-822 (1980).
Some
studies suggest that histamine-induced hypercontractility in the human
coronary system is
most pronounced in the proximal arteries in the setting of atherosclerosis and
the
associated denudation of the arterial endothelium. Keitoku, M. et al.,
"Different
Histamine Actions in Proximal and Distal Human Coronary Arteries in Vitro,"
Cardiovascular Research 24:614-622 (1990). Therefore, histamine receptor
antagonists
may be included in the cardiovascular irrigation solution.
Other suitable H1 receptor antagonists include terfenadine, diphenhydramine,
amitriptyline, mepyramine and tripolidine. Because amitriptyline is also
effective as a
serotonin2 receptor antagonist, it has a dual function as used in the present
invention.
Suitable therapeutic and preferred concentrations for each of these H1
receptor
antagonists are set forth in Table 7.
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TABLE 7
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
PAIN/INFLAMMATION INHIBITORY AGENTS
Therapeutic Preferred
ConcentrationsConcentrations
Class of Agent (Nanomolar) (Nanomolar)
Histamine) Receptor Antagonists:
promethazine 0.1 - 1,000 50 - 200
diphenhydramine 0.1 - 1,000 50 - 200
amitriptyline 0.1 - 1,000 50 - 500
terfenadine 0.1 - 1,000 50 - 500
mepyramine (pyrilamine) 0.1 - 1,000 5 - 200
tripolidine 0.01 - 100 5 - 20
D. BRADYKIN1N RECEPTOR ANTAGONISTS
Bradykinin receptors generally are divided into bradykininl (B1) and
bradykinin~
(B2) subtypes. Studies have shown that acute peripheral pain and inflammation
produced
by bradykinin are mediated by the B2 subtype whereas bradykinin-induced pain
in the
setting of chronic inflammation is mediated via the B 1 subtype. Perkins,
M.N., et al.,
"Antinociceptive Activity of the Bradylcinin B l and B2 Receptor Antagonists,
des-Arg9,
[Leu8]-BK and HOE 140, in Two Models of Persistent Hyperalgesia in the Rat,"
Pain
53:191-97 (1993); Dray, A., et al., "Bradykinin and Inflammatory Pain," Trends
Neurosci. 16:99-104 (1993), each of which is hereby expressly incorporated by
reference.
At present, bradylcinin receptor antagonists are not used clinically. These
drugs
are peptides (small proteins), and thus they cannot be taken orally, because
they would be
digested. Antagonists to B2 receptors block bradykinin-induced acute pain and
inflammation. Dray et al., 1993. B 1 receptor antagonists inhibit pain in
chronic
inflammatory conditions. Perlcins et al., 1993; Dray et al., 1993. Therefore,
depending
on the application, the solution of the present invention preferably includes
either or both
bradylcinin B 1 and B~ receptor antagonists. For example, arthroscopy is
performed for
both acute and chronic conditions, and thus an irrigation solution for
arthroscopy could
include both B 1 and B~ receptor antagonists.
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Suitable bradykinin receptor antagonists for use in the present invention
include
the following bradylcininl receptor antagonists: the [des-Argl~] derivative of
D-Arg-(Hyp3-This-D-Tic7-Oic8)-BK ("the [des-Argl~] derivative of HOE 140",
available
from Hoechst Pharmaceuticals); and [Leu8] des-Arg9-BK. Suitable bradykinin2
receptor
antagonists include: [D-Phe7]-BK; D-Arg-(Hyp3-This~$-D-Phe7)-BK ("NPC 349");
D-Arg-(Hyp3--D-Phe7)-BK ("NPC 567"); and D-Arg-(Hyp3-This-D-Tic7-OicB)-BK
("HOE 140"). These compounds are more fully described in the previously
incorporated
Perkins et al. 1993 and Dray et al. 1993 references. Suitable therapeutic and
preferred
concentrations are provided in Table 8.
TABLE 8
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
PAIN/INFLAMMATION INHIBITORY AGENTS
Therapeutic Preferred
Concentrations Concentrations
Class of Agent (Nanomolar) (Nanomolax)
Bradykininl Receptor Anta og nists:
[Leug] des-Arg9-BK 1 - 1,000 50 - 500
[des-ArglO] derivative of HOE 140 1 - 1,000 50 - 500
[leu9 [des-Argl~] kalliden 0.1 - 500 10 - 200
Brad~kinin2 Receptor Antagonists:
[D-Phe7]-BK I00 - 10,000 200 - 5,000
NPC 349 1 - 1,000 50 - 500
NPC 567 1 - 1,000 50 - 500
HOE 140 1 - 1,000 50 - 500
E. KALLIKREIN INHIBITORS
The peptide bradylcinin is an important mediator of pain and inflammation, as
noted previously. Bradykinin is produced as a cleavage product by the action
of
lcallikrein on high molecular weight lcininogens in plasma. Therefore
kallikrein inhibitors
are believed to be therapeutic in inhibiting bradylcinin production and
resultant pain and
inflammation. A suitable lcallilcrein inhibitor for use in the present
invention is aprotinin.
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Suitable concentrations for use in the solutions of the present invention are
set forth
below in Table 9.
TABLE 9
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
PAIN/1NFLAMMATION INHIBITORY AGENTS
Therapeutic Preferred
Concentrations Concentrations
Class of Agent (Nanomolar) (Nanomolar)
Kallikrein Inhibitor:
Aprotinin 0.1 - 1,000 50 - 500
F. TACHYKININ RECEPTOR ANTAGONISTS
Tachykinins (TKs) are a family of structurally related peptides that include
substance P, neurokinin A (NKA) and neurokinin B (NKB). Neurons are the major
source of TKs in the periphery. An important general effect of TKs is neuronal
stimulation, but other effects include endothelium-dependent vasodilation,
plasma protein
extravasation, mast cell recruitment and degranulation and stimulation of
inflammatory
cells. Maggi, C.A., Gen. Phar~aacol. 22:1-24 (1991). Due to the above
combination of
physiological actions mediated by activation of TK receptors, targeting of TK
receptors is
a reasonable approach for the promotion of analgesia and the treatment of
neurogenic
inflammation.
1. NEUROK1N1N1 RECEPTOR SUBTYPE ANTAGONISTS
Substance P activates the neurokinin receptor subtype referred to as NKI.
Substance P is an undecapeptide that is present in sensory nerve terminals.
Substance P
is known to have multiple actions which produce inflammation and pain in the
periphery
after C-fiber activation, including vasodilation, plasma extravasation and
degranulation of
mast cells. Levine, J.D., et al., "Peptides and the Primary Afferent
Nociceptor,"
J. Neurosci. 13:2273 (1993). A suitable Substance P antagonist is ([D-
Pro9[spiro-
gamma-lactam]Leul~,Trpi 1]physalaemin-(1-11)) ("GR 82334"). Other suitable
antagonists for use in the present invention which act on the NK1 receptor
are: 1-imino-
2-(2-methoxy-phenyl)-ethyl)-7,7-diphenyl-4-perhydroisoindolone(3aR,7aR) ("RP
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67580"); and 2S,3S-cis-3-(2-methoxybenzylamino)-2-benzhydrylquinuclidine ("CP
96,345"). Suitable concentrations for these agents are set forth in Table 10.
TABLE 10
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
PAlN/INFLAMMATION INHIBITORY AGENTS
Therapeutic Preferred
Concentrations Concentrations
Class of Agent (Nanomolar) (Nanomolar)
Neurokininl Receptor Subty~,e Antagonists
GR 82334 1 - 1,000 10 - 500
CP 96,345 1-10,000 100-1,000
RP 67580 0.1-1,000 100-1,000
2. NEUROKININ2 RECEPTOR SUBTYPE ANTAGONISTS
Neurokinin A is a peptide which is colocalized in sensory neurons with
substance
P and which also promotes inflammation and pain. Neurokinin A activates the
specific
neurokinin receptor referred to as NK2. Edmonds-Alt, S., et al., "A Potent and
Selective
Non-Peptide Antagonist of the Neurokinin A (NK2) Receptor," Life Sci. SO:PL101
(1992). In the urinary tract, TKs are powerful spasmogens acting through only
the NK2
receptor in the human bladder, as well as the human urethra and ureter. Maggi,
C.A.,
Geh. Pha~macol. 22:1-24 (1991). Thus, the desired drugs for inclusion in a
surgical
solution for use in urological procedures would contain an antagonist to the
NK2 receptor
to reduce spasm. Examples of suitable NK2 antagonists include: ((S)-N-methyl-N-
[4-(4-
acetylamino-4-phenylpiperidino)-2- (3,4-dichlorophenyl)butyl]benzamide ("(~)-
SR
48968"); Met-Asp-Trp-Phe-Dap-Leu ("MEN 10,627"); and cyc(Gln-Trp-Phe-Gly-Leu-
Met) ("L 659,877"). Suitable concentrations of these agents are provided in
Table 11.
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TABLE 11
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
PAIN/INFLAMMATION INHIBITORY AGENTS
Therapeutic Preferred
Concentrations Concentrations
Class of Agent (Nanomolar) (Nanomolar)
Neurolcinin2 Receptor Subtype
Antagonists:
MEN 10,627 1-1,000 10-1,000
L 659,877 10-10,000 100-10,000
(~)-SR 48968 10-10,000 100-10,000
G. CGRP RECEPTOR ANTAGONISTS
Calcitonin gene-related peptide (CGRP) is a peptide which is also colocalized
in
sensory neurons with substance P, and which acts as a vasodilator and
potentiates the
actions of substance P. Brain, S.D., et al., "Inflammatory Oedema Induced by
Synergism
Between Calcitonin Gene-Related Peptide (CGRP) and Mediators of Increased
Vascular
Permeability," Bo. J. Pha~macol. 99:202 (1985). An example of a suitable CGRP
receptor antagonist is a-CGRP-(8-37), a truncated version of CGRP. This
polypeptide
inhibits the activation of CGRP receptors. Suitable concentrations for this
agent are
provided in Table 12.
TABLE 12
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
PAIN/1NFLAMMATION INHIBITORY AGENTS
Therapeutic Preferred
Concentrations Concentrations
Class of Agent (Nanomolar) (Nanomolar)
CGRP Receptor Antagonist:
a-CGRP-(8-37) 1-1,000 10-500
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H. INTERLEUKIN RECEPTOR ANTAGONIST
Interleukins axe a family of peptides, classified as cytokines, produced by
leukocytes and other cells in response to inflammatory mediators.
Interleulcins (IL) may
be potent hyperalgesic agents peripherally. Ferriera, S.H., et al.,
"Interleukin-1 ~3 as a
Potent Hyperalgesic Agent Antagonized by a Tripeptide Analogue,"
Natuf°e 334:698
(1988). An example of a suitable IL-1 ~3 receptor antagonist is Lys-D-Pro-Thr,
which is a
truncated version of IL-1 Vii. This tripeptide inhibits the activation of IL-1
(3 receptors.
Suitable concentrations for this agent are provided in Table I3.
TABLE 13
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
PAINlINFLAMMATION INHIBITORY AGENTS
Therapeutic Preferred
Concentrations Concentrations
Class of Agent (Nanomolar) (Nanomolar)
Interleukin Receptor Antagonist:
Lys-D-Pro-Thr 1-1,000 10-500
I. INHIBITORS OF ENZYMES ACTIVE IN THE SYNTHETIC
PATHWAY FOR AR.ACHIDONIC ACID METABOLITES
1. PHOSPHOLIPASE INHIBITORS
The production of arachidonic acid by phospholipase A2 (PLA2) results in a
cascade of reactions that produces numerous mediators of inflammation, know as
eicosanoids. There are a number of stages throughout this pathway that can be
inhibited,
thereby decreasing the production of these inflammatory mediators. Examples of
inhibition at these various stages are given below.
Inhibition of the enzyme PLA2 isoform inhibits the release of arachidonic acid
from cell membranes, and therefore inhibits the production of prostaglandins
and
Ieukotrienes resulting in decreased inflammation and pain. Glaser, K.B.,
"Regulation of
Phospholipase A2 Enzymes: Selective Inhibitors and Their Pharmacological
Potential,"
Adv. Pharmacol. 32:31 (1995). An example of a suitable PLA2 isoform inhibitor
is
manoalide. Suitable concentrations for this agent are included in Table 14.
Inhibition of
the phospholipase CY (PLCY) isoform also will result in decreased production
of
prostanoids and leukotrienes, and, therefore, will result in decreased pain
and
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inflammation. An example of a PLCY isoform inhibitor is 1-[6-((17(3-3-
methoxyestra-
1,3,5(10)-trim-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione.
TABLE 14
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
PA1N/INFLAMMATION INHIBITORY AGENTS
Therapeutic Preferred
Concentrations Concentrations
Class of Agent (Nanomolar) (Nanomolar)
PLA2 Isoform Inhibitor:
manoalide 100-100,000 500-10,000
2. CYCLOOXYGENASE INHIBITORS
Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used as
anti-inflammatory, anti-pyretic, anti-thrombotic and analgesic agents. Lewis,
R.A.,
"Prostaglandins and Leukotrienes," In: Textbook of Rheumatology, 3d ed.
(Kelley W.N.,
et al., eds.), p. 258 (1989). The molecular targets for these drugs are type I
and type II
cyclooxygenases (COX-1 and COX-2). These enzymes are also known as
Prostaglandin
H Synthase (PGHS)-1 (constitutive) and -2 (inducible), and catalyze the
conversion of
arachidonic acid to Prostaglandin H which is an intermediate in the
biosynthesis of
prostaglandins and thromboxanes. The COX-2 enzyme has been identified in
endothelial
cells, macrophages, and fibroblasts. This enzyme is induced by IL-1 and
endotoxin, and
its expression is upregulated at sites of inflammation. Constitutive activity
of COX-l and
induced activity of COX-2 both lead to synthesis of prostaglandins which
contribute to
pain and inflammation.
NSAIDs currently on the market (diclofenac, naproxen, indomethacin, ibuprofen,
etc.) are generally nonselective inhibitors of both isoforms of COX, but may
show greater
selectively for COX-1 over COX-2, although this ratio varies for the different
compounds. Use of COX-1 and 2 inhibitors to block formation of prostaglandins
represents a better therapeutic strategy than attempting to block interactions
of the natural
ligands with the seven described subtypes of prostanoid receptors. Reported
antagonists
of the eicosanoid receptors (EP-l, EP-2, EP-3) are quite rare and only
specific, high
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affinity antagonists of the thromboxane A2 receptor have been reported.
Wallace, J, and
Cirino, G., Ti~ev~ds in Pharna. Sci. 15:405-406 (1994).
The oral, intravenous or intramuscular use of cyclooxygenase inhibitors is
contraindicated in patients with ulcer disease, gastritis or renal impairment.
In the United
States, the only available injectable form of this class of drugs is ketorolac
(ToradolTM),
available from Syntex Pharmaceuticals, which is conventionally used
intramuscularly or
intravenously in postoperative patients but, again, is contraindicated for the
above-
mentioned categories of patients. The use of ketorolac, or any other
cyclooxygenase
inhibitor(s), in the solution in substantially lower dosages than currently
used
perioperatively may allow the use of this drug in otherwise contraindicated
patients. The
addition of a cyclooxygenase inhibitor to the solutions of the present
invention adds a
distinct mechanism for inhibiting the production of pain and inflammation
during
artlvoscopy or other therapeutic or diagnostic procedure.
Preferred cyclooxygenase inhibitors for use in the present invention are
keterolac
and indomethacin. Of these two agents, indomethacin is less preferred because
of the
relatively high dosages required. 'Therapeutic and preferred concentrations
for use in the
solution are provided in Table 15.
TABLE 15
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
PA1N/1NFLAMMATION INHIBITORY AGENTS
Therapeutic Preferred
Concentrations Concentrations
Class of Agent (Nanomolax) (Nanomolar)
C ~~clooxy~enase Inhibitors:
lcetorolac 100 - 10,000 500 - 5,000
indomethacin 1,000 - 500,000 10,000 - 200,000
' 3. LIPOOXYGENASE INHIBTTORS
Inhibition of the enzyme lipooxygenase inhibits the production of
leukotrienes,
such as leukotriene B~, which is lcnown to be an important mediator of
inflammation and
pain. Lewis, R.A., "Prostaglandins and Leukotrienes," In: Textbook of
Rheumatology,
3d ed. (Kelley W.N., et al., eds.), p. 258 (1989). An example of a 5-
lipooxygenase
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antagonist is 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-
benzoquinone ("AA
861 "), suitable concentrations for which are listed in Table 16.
TABLE 16
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
PAIN/INFLAMMATION INHIBITORY AGENTS
Therapeutic Preferred
Concentrations Concentrations
Class of Agent (Nanomolax) (Nanornolar)
Li~ooxyeenase Inhibitor:
AA 861 100-10,000 500-5,000
J. PROSTANOID RECEPTOR ANTAGONISTS
Specific prostanoids produced as metabolites of axachidonic acid mediate their
inflammatory effects through activation of prostanoid receptors. Examples of
classes of
specific prostanoid antagonists are the eicosanoid EP-1 and EP-4 receptor
subtype
antagonists and the thromboxane receptor subtype antagonists. A suitable
prostaglandin
EZ receptor antagonist is 8-chlorodibenz[b,fj[1,4]oxazepine-10(11H)-carboxylic
acid,
2-acetylhydrazide ("SC 19220"). A suitable thromboxane receptor subtype
antagonist is
[15-[Ia, 2(3(SZ), 3(3, 4a]-7-[3-[2-(phenylamino)-carbonyl] hydrazino] methyl]-
7-
oxobicyclo-[2,2,1]-hept-2-yl]-5-heptanoic acid ("SQ 29548"). Suitable
concentrations for
these agents are set forth in Table 17.
TABLE 17
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
PAIN/INFLAMMATION INHIBITORY AGENTS
Therapeutic Preferred
Concentrations Concentrations
Class of Agent (Nanomolax) (Nanomolar)
Eicosanoid EP-1 Antagonist:
SC 19220 100-10,000 500-5,000
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K. LEUKOTRIENE RECEPTOR ANTAGONISTS
The leul~otrienes (LTBø, LTC4, and LTD4) are products of the 5-lipooxygenase
pathway of arachidonic acid metabolism that are generated enzymatically and
have
important biological properties. Leukotrienes are implicated in a number of
pathological
conditions including inflammation. Specific antagonists axe currently being
sought by
many pharmaceutical companies for potential therapeutic intervention in these
pathologies. Halushka, P.V., et aL, Annu. Rev. Pharmacol. Toxicol. 29:213-239
(1989);
Ford-Hutchinson, A., C~it. Rev. Immunol. 10:1-12 (1990). The LTB4 receptor is
found in
certain immune cells including eosinophils and neutrophils. LTBø binding to
these
receptors results in chemotaxis and lysosomal enzyme release thereby
contributing to the
process of inflammation. The signal transduction process associated with
activation of
the LTB4 receptor involves G-protein-mediated stimulation of
phosphotidylinositol (PI)
metabolism and elevation of intracellular calcium (see FIGURE 2).
An example of a suitable leulcotriene Bq, receptor antagonist is SC (+)-(S)-7-
(3-(2-
(cyclopropylmethyl)-3-methoxy-4-[(methylamino)-carbonyl]phenoxy(propoxy)-3,4-
dihydro-8-propyl-2H-1-benzopyran-2-propanoic acid ("SC 53228"). Concentrations
for
this agent that axe suitable for the practice of the present invention are
provided in
Table 18. Other suitable leukotriene Bq. receptor antagonists include [3-[-2(7-
chloro-2-
quinolinyl)ethenyl]phenyl] [[3-(dimethylamino-3-oxopropyl)thio]
methyl]thiopropanoic
acid ("MK 0571 ") and the drugs LY 66,071 and ICI 20,3219. MK 0571 also acts
as a
LTD4 receptor subtype antagonist.
TABLE 18
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
PA1N/TNFLAMMATION INHIBITORY AGENTS
Therapeutic Preferred
Concentrations Concentrations
Class of Agent (Nanomolar) (Nanofnolar)
Leulcotriene Be Antagonist:
SC 53228 100-10;000 500-5,000
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f?
L. OPIOID RECEPTOR AGONISTS
Activation of opioid receptors results in anti-nociceptive effects and,
therefore,
agonists to these receptors are desirable. Opioid receptors include the ~,-, 8-
and K-opioid
receptor subtypes. The p,-receptors are located on sensory neuron terminals in
the
periphery and activation of these receptors inhibits sensory neuron activity.
Basbaum,
A.L, et al., "Opiate analgesia: How Central is a Peripheral Target?," N. E~gl.
J. Med.
325:1168 (199I). s- and x-receptors are located on sympathetic efferent
terminals and
inhibit the release of prostaglandins; thereby inhibiting pain and
inflammation. Taiwo,
Y.O., et al., "Kappa- and Delta-Opioids Block Sympathetically Dependent
Hyperalgesia,"
,l. Neu~osci. 11:928 (1991). The opioid receptor subtypes are members of the G-
protein
coupled receptor superfamily. Therefore, all opioid receptor agonists interact
and initiate
signaling through their cognate G-protein coupled receptor (see FIGURES 3 and
7).
Examples of suitable ~-opioid receptor agonists are fentanyl and Try-D-Ala-Gly-
[N-
MePhe]-NH(CH~)-OH ("DAMGO"). An example of a suitable 8-opioid receptor
agonist
is [D-Pen~,D-Pens]enkephalin ("DPDPE"). An example of a suitable K-opioid
receptor
agonist is (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyxrolidinyl)cyclohexyl]-
benzene
acetamide ("U50,488"). Suitable concentrations for each of these agents are
set forth in
Table 19.
TABLE 19
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
PAIN/1NFLAMMATION INHIBITORY AGENTS
Therapeutic Preferred
Concentrations Concentrations
Class of Agent (Nanomolar) (Nanomolax)
~,-Opioid A~onist:
DAMGO 0.1-100 0.5-20
sufentanyl 0.01-50 1-20
fentanyl O.I-500 10-200
PL 017 0.05-50 0.25-10
8-Opioid A~onist:
DPDPE 0.1-500 1.0-100
K-Opioid A~onist:
U50,488 0.1-500 1.0-100
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M. PUR1NOCEPTOR ANTAGONISTS AND AGONISTS
Extracellulax ATP acts as a signaling molecule through interactions with P2
purinoceptors. One major class of purinoceptors are the PBX purinoceptors
which are
ligand-gated ion channels possessing intrinsic ion channels permeable to Na+,
K~, and
Ca2+. P2X receptors described in sensory neurons are important for primary
afferent
neurotransmission and nociception. ATP is known to depolarize sensory neurons
and
plays a role in nociceptor activation since ATP released from damaged cells
stimulates
P2~ receptors leading to depolarization of nociceptive nerve-fiber terminals.
The P2X3
receptor has a highly restricted distribution (Chen, C.C., et al., Nature
377:428-431
(1995)) since it is selectively expressed in sensory C-fiber nerves that run
into the spinal
cord and many of these C-fibers are known to carry the receptors for painful
stimuli.
Thus, the highly restricted localization of expression for the P2X3 receptor
subunits make
these subtypes excellent targets for analgesic action (see FIGURES 3 and 7).
Suitable antagonists of P2x/ATP purinoceptors for use in the present invention
include, by way of example, suramin and pyridoxylphosphate-6-azophenyl-2,4-
disulfonic
acid ("PPADS"). Suitable concentrations for these agents are provided in Table
20.
Agonists of the P2y receptor, a G-protein coupled receptor, are known to
effect
smooth muscle relaxation through elevation of inositol triphosphate (IP3)
levels with a
subsequent increase in intracellular calcium. An example of a P2y receptor
agonist is
2-me-S-ATP.
TABLE 20
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
PAlN/INFLAMMATION INHIBITORY AGENTS
Therapeutic Preferred
Concentrations Concentrations
Class of Agent (Nanomolar) (Nanomolar)
Purinoceptor Anta o~ nists:
suramin 100-100,000 10,000-100,000
PPADS 100-100,000 10,000-100,000
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N. ADENOSINE TRIPHOSPHATE (ATP)-SENSITIVE POTASSIUM
CHANNEL OPENERS
ATP-sensitive potassium channels have been discovered in numerous tissues,
including vascular and non-vascular smooth muscle and brain, and binding
studies using
radiolabeled ligands have confirmed their existence. Opening of these channels
causes
potassium (K+) efflux and hyperpolarizes the cell membrane (see FIGURE 2).
This
hyperpolaxization induces a reduction in intracellular free calcium through
inhibition of
voltage-dependent calcium (Ca2+) channels and receptor operated Ca~~ channels.
These
combined actions drive the cell (e.g., smooth muscle cell) into a relaxed
state or one
which is more resistant to activation and, in the case of vascular smooth
muscle, results in
vasorelaxation. K+ channel openers (KCOs) have been characterized as having
potent
antihypertensive activity in vivo and vasorelaxant activity in vitro (see
FIGURE 4). K+
channel openers (KCOs) also have been shown to prevent stimulus coupled
secretion and
are considered to act on prejunctional neuronal receptors and thus will
inhibit effects due
to nerve stimulation and release of inflammatory mediators. Quast, U., et al.,
"Cellular
Pharmacology of Potassium Channel Openers in Vascular Smooth Muscle,"
Ca~diovasc.
Res. 28: 805-810 (1994).
Synergistic interactions between endothelin (ETA) antagonists and openers of
ATP-sensitive potassium channels (KCOs) are expected in achieving
vasorelaxation or
smooth muscle relaxation. A rationale for dual use is based upon the fact that
these drugs
have different molecular mechanisms of action in promoting relaxation of
smooth muscle
and prevention of vasospasm. .An initial intracellular calcium elevation in
smooth muscle
cells induced by the ETA receptor subsequently triggers activation of voltage-
dependent
channels and the entry of extracellular calcium which is required for
contraction.
Antagonists of the ETA receptor will specifically block this receptor mediated
effect but
not block increases in calcium triggered by activation of other G-protein
coupled
receptors on the muscle cell.
Potassium-channel opener drugs, such as pinacidil, will open these channels
causing K+ efflux and hyperpolaxization of the cell membrane. This
hyperpolarization
will act to reduce contraction mediated by other receptors by the following
mechanisms:
(1) it will induce a reduction in intracellular free calcium through
inhibition of
voltage-dependent Ca2+ channels by reducing the probability of opening L-type
or T-type
calcium channels, (2) it will restrain agonist induced (receptor operated
channels) Ca2+
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release from intracellular sources through inhibition of inositol triphosphate
(IPg)
formation, and (3) it will lower the efficiency of calcium as an activator of
contractile
proteins. Consequently, combined actions of these two classes of drugs will
clamp the
target cells into a relaxed state or one which is more resistant to
activation.
Suitable ATP-sensitive I~~ channel openers for the practice of the present
invention include: (-)pinacidil; cromakalim; nicorandil; minoxidil; N-cyano-N'-
[l,l-
dimethyl-[2,2,3,3 3H]propyl]-N"-(3-pyridinyl)guanidine ("P 1075"); and N-cyano-
N'-(2-
nitroxyethyl)-3-pyridinecarboximidamide monomethansulphonate ("KRN 2391 ").
Concentrations for these agents are set forth in Table 21.
TABLE 21
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
PA1N/1NFLAMMATION INHIBITORY AGENTS
Therapeutic Preferred
Concentrations Concentrations
Class of Agent (Nanomolar) (Nanomolar)
ATP-Sensitive I~+ Channel Opener:
cromakalim 10-10,000 100-10,000
nicorandil 10-10,000 I00-10,000
minoxidil 10-10,000 100-10,000
P 1075 0.1-1,000 IO-1,000
KRN 2391 1-10,000 100-1,000
(-)pinacidil 1-10,000 100-1,000
III. ANTI-SPASM AGENTS
A. MULTIFUNCTION AGENTS
Several of the anti-pain/anti-inflammatory agents described above also serve
to
inhibit vasoconstriction or smooth muscle spasm. As such, these agents also
perform the
function of anti-spasm agents, and thus are beneficially used in vascular and
urologic
applications. Anti-inflammatory/anti-pain agents that also serve as anti-spasm
agents
include: serotonin receptor antagonists, particularly, serotonin2 antagonists;
tachykinin
receptor antagonists and ATP-sensitive potassium channel openers.
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B. NITRIC OXIDE DONORS
Nitric oxide donors may be included in the solutions of the present invention
particularly for their anti-spasm activity. Nitric oxide (NO) plays a critical
role as a
molecular mediator of many physiological processes, including vasodilation and
regulation of normal vascular tone. Within endothelial cells, an enzyme known
as NO
synthase (NOS) catalyzes the conversion of L-arginine to NO which acts as a
diffusible
second messenger and mediates responses in adjacent smooth muscle cells (see
FIGURE 8). NO is continuously formed and released by the vascular endothelium
under
basal conditions which inhibits contractions and controls basal coronary tone
and is
produced in the endothelium in response to various agonists (such as
acetylcholine) and
other endothelium dependent vasodilators. Thus, regulation of NO synthase
activity and
the resultant levels of NO are key molecular targets controlling vascular tone
(see
FIGURE 8). Muramatsu, K., et al., Coron. Artery Dis. 5: 815-820 (1994).
Synergistic interactions between NO donors and openers of ATP-sensitive
potassium channels (KCOs) are expected to achieve vasorelaxation or smooth
muscle
relaxation. A rationale for dual use is based upon the fact that these drugs
have different
molecular mechanisms of action in promoting relaxation of smooth muscle and
prevention of vasospasm. There is evidence from cultured coronary arterial
smooth
muscle cells that the vasoconstrictors: vasopressin, angiotensin II and
endothelin, all
inhibit KATp currents through inhibition of protein kinase A. In addition, it
has been
reported that KATp current in bladder smooth muscle is inhibited by muscarinic
agonists.
The actions of NO in mediating smooth muscle relaxation occur via independent
molecular pathways (described above) involving protein kinase G (see FIGURE
8). This
suggests that the combination of the two classes of agents will be more
efficacious in
relaxing smooth muscle than employing a single class of agent alone.
Suitable nitric oxide donors for the practice of the present invention include
nitroglycerin, sodium nitroprusside, the drug FK 409, FR 144420,
3-morpholinosydnonimine , or linsidomine chlorohydrate, ("SIN-1"); and S-
nitroso-N-
acetylpenicillarnine ("SNAP"). Concentrations for these agents are set forth
in Table 22.
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TABLE 22
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
SPASM INHIBITORY AGENTS
Therapeutic Preferred
Concentrations Concentrations
Class of Agent (Nanomolar) (Nanomolar)
Nitric Oxide Donors:
Nitroglycerin 10-10,000 100-1,000
sodium nitroprusside 10-10,000 100-1,000
SIN-1 10-10,000 100-1,000
SNAP 10-10,000 100-1,000
FK 409 (NOR-3) 1-1,000 10-500
FR 144420 (NOR-4) 10 - 10,000 100 - 5,000
C. ENDOTHELIN RECEPTOR ANTAGONISTS
Endothelin is a 21 amino acid peptide that is one of the most potent
vasoconstrictors knoum. Three different human endothelin peptides, designated
ET-1,
ET-2 and ET-3 have been described which mediate their physiological effects
through at
least two receptor subtypes referred to as ETA and ETB receptors. The heart
and vascular
smooth muscle contain predominantly ETA receptors and this subtype is
responsible for
contraction in these tissues. Furthermore, ETA receptors have often been found
to
mediate contractile responses in isolated smooth muscle preparations.
Antagonists of
ETA receptors have been found to be potent antagonists of human coronary
artery
contractions. Thus, antagonists to the ETA receptor should be therapeutically
beneficial
I S in the perioperative inhibition of coronary vasospasm and may additionally
be useful in
inhibition of smooth muscle contraction in urological applications. Miller,
R.C., et al.,
Trends its Pharmacol. Sci. 14: 54-60 (1993).
Suitable endothelin receptor antagonists include: cyclo(D-Asp-Pro-D-Val-Leu-D-
Trp) ("BQ 123"); (N,N-hexamethylene)-carbamoyl-Leu-D-Trp-(CHO)-D-Trp-OH ("BQ
610"); (R)2-([R-2-[(s)-2-([1-hexahydro-1H-azepinyl]-carbonyl]amino-4-methyl-
pentanoyl) amino-3-(3[1-methyl-1H-indodyl])propionylamino-3(2-pyridyl)
propionic
acid ("FR 139317"); cyclo(D-Asp-Pro-D-Ile-Leu-D-Trp) ("JKC 301 "); cyclo(D-Ser-
Pro-
D-Val-Leu-D-Trp) ("JK 302"); 5-(dimethylamino)-N-(3,4-dimethyl-5-isoxazolyl)-1-
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naphthalenesulphonamide ("BMS 182874"); and N-[1-Formyl=N-[N-[(hexahydro-1H-
azepin-1-yl)carbonyl]-L-leucyl]-D-tryptophyl)-D-tryptophan ("BQ 610").
Concentrations
for a representative three of these agents is set forth in Table 23.
TABLE 23
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
SPASM INHIBITORY AGENTS
Therapeutic Preferred
Concentrations Concentrations
Class of Agent (Nanomolar) (Nanomolar)
Endothelin Receptor Anta o~'-nists:
BQ 123 0.01-1,000 10-1,000
FR 139317 1-100,000 100-10,000
BQ 610 0.01 to 10,000' 10 - 1,000
D. CA~+ CHANNEL ANTAGONISTS
Calcium channel antagonists are a distinct group of drugs that interfere with
the
transmembrane flux of calcium ions required for activation of cellular
responses
mediating neuroinflarnination. Calcium entry into platelets and white blood
cells is a key
event mediating activation of responses in these cells. Furthermore, the role
of
bradylcinin receptors and neurokinin receptors (NK1 and NK2) in mediating the
neuroinflammation signal transduction pathway includes increases in
intracellular
calcium, thus leading to activation of calcium channels on the plasma
membrane. In
many tissues, calcium channel antagonists, such as nifedipine, can reduce the
release of
arachidonic acid, prostaglandins, and leukotrienes that are evoked by various
stimuli.
Moncada, S., Flower, R. and Vane, J., in Goodman's and Gilmah's
Pharmacological
Basis of The~~apeutics (7th ed.), MacMillan Publ. Inc., pp. 660-5 (1995).
Calcium channel antagonists also interfere with the transmembrane flux of
calcium ions required by vascular smooth muscle for contractions. This effect
provides
the rationale for the use of calcium channel antagonists perioperatively
during procedures
in which the goal is to alleviate vasospasm and promote relaxation of smooth
muscle.
The dihydropyridines, including nisoldipine, act as specific inhibitors
(antagonists) of the
voltage-dependent gating of the L-type subtype of calcium channels. Systemic
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administration of the calcium channel antagonist nifedipine during cardiac
surgery
previously has been utilized to prevent or minimize coronary artery vasospasm.
Seitelberger, R., et al., Ci~~culatioh 83:460-468 (1991).
Calcium chamiel antagonists, which are among the anti-spasm agents useful in
the
present invention, exhibit synergistic effect when combined with other agents
of the
present invention. Calcium (Ca2+) channel antagonists and nitric oxide (NO)
donors
interact in achieving vasorelaxation or smooth muscle relaxation, i.e., in
inhibiting spasm
activity. A rationale for dual use is based upon the fact that these two
classes of drugs
have different molecular mechanisms of action, may not be completely effective
in
achieving relaxation used alone, and may have different time periods of
effectiveness. In
fact, there are numerous studies showing that calcium channel antagonists
alone cannot
achieve complete relaxation of vascular muscle that has been precontracted
with a
receptor agonist.
The effect of nisoldipine, used alone and in combination with nitroglycerin,
on
spasm of the internal mammary artery (IMA) showed that the combination of the
two
drugs produced a large positive synergistic effect in the prevention of
contraction
(Liu et al., 1994). These studies provide a scientific basis for combination
of a calcium
channel antagonist and nitric oxide (NO) donor for the efficacious prevention
of
vasospasm and relaxation of smooth muscle. Examples of systemic administration
of
nitroglycerin and nifedipine during cardiac surgery to prevent and treat
myocardial
ischemia or coronary artery vasospasm have been reported (Cohen et al., 1983;
Seitelberger et al., 1991).
Calcium channel antagonists also exhibit synergistic effect with endothelin
receptor subtype A (ETA) antagonists. Yanagisawa and coworkers observed that
dihydropyridine antagonists bloclced effects of ET-1, an endogenous agonist at
the ETA
receptor in coronary arterial smooth muscle, and hence speculated that ET-1 is
an
endogenous agonist of voltage-sensitive calcium channels. It has been found
that the
sustained phase of intracellular calcium elevation in smooth muscle cells
induced by ETA
receptor activation requires extracellular calcium and is at least partially
blocked by
nicardipine. Thus, the inclusion of a calcium channel antagonist would be
expected to
synergistically enhance the actions of an ETA antagonist when combined in a
surgical
solution.
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Calcium channel antagonists and ATP-sensitive potassium channel openers
likewise exhibit synergistic action. Potassium channels that are ATP-sensitive
(KA'rP)
couple the membrane potential of a cell to the cell's metabolic state via
sensitivity to .
adenosine nucleotides. KATp channels are inhibited by intracellular ATP but
are
stimulated by intracellular nucleotide diphosphates. The activity of these
channels is
controlled by the electrochemical driving force to potassium and intracellular
signals
(e.g., ATP or a G-protein), but are not gated by the membrane potential per
se. KATP
channels hyperpolaxize the membrane and thus allow them to control the resting
potential
of the cell. ATP-sensitive potassium currents have been discovered in skeletal
muscle,
brain, and vascular and nonvascular smooth muscle. Binding studies with
radiolabeled
ligands have confirmed the existence of ATP-sensitive potassium channels which
are the
receptor targets for the potassium-channel opener drugs such as pinacidil.
Opening of
these channels causes potassium efflux and hyperpolarizes the cell membrane.
This
hyperpolaxization (1) induces a reduction in intracellular free calcium
through inhibition
of voltage-dependent Ca2+ channels by reducing the probability of opening L-
type or
T-type calcium channels, (2) restrains agonist induced (at receptor operated
channels)
Ca2+ release from intracellular sources through inhibition of inositol
triphosphate (IP3)
formation, and (3) lowers the efficiency of calcium as an activator of
contractile proteins.
The combined actions of these two classes of drugs (ATP-sensitive potassium
channel
openers and calcium channel antagonists) will clamp the target cells into a
relaxed state
or one which is more resistant to activation.
Finally, calcium channel antagonists and tachykinin and bradykinin antagonists
exhibit synergistic effects in mediating neuroinflammation. The role of
neurokinin
receptors in mediating neuroinflammation has been established. The neurokinin,
(NKl)
and neurokinina (NK2) receptor (members of the G-protein coupled superfamily)
signal
transduction pathway includes increases in intracellular calcium, thus leading
to
activation of calcium channels on the plasma membrane. Similarly, activation
of
bradylcinin2 (BK~) receptors is coupled to increases in intracellular calcium.
Thus,
calcium channel antagonists interfere with a common mechanism involving
elevation of
intracellular calcium, part of which enters through L-type channels. This is
the basis for
synergistic interaction between calcium channel antagonists and antagonists to
neurolcinin
and bradylcinin2 receptors.
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Suitable calcium channel antagonists for the practice of the present invention
include nisoldipine, nifedipine, nimodipine, lacidipine, isradipine and
amlodipine.
Suitable concentrations for these agents are set forth in Table 24.
TABLE 24
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
SPASM INHIBITORY AGENTS
Therapeutic Preferred
Concentrations Concentrations
Class of Agent (Nanomolar) (Nanomolar)
Calcium Channel Antagonists:
nisoldipine 1-10,000 100-1,000
nifedipine 1-10,000 100-5,000
nimodipine 1-10,000 100-5,000
lacidipine 1-10,000 100-5,000
isradipine 1-10,000 100-5,000
amlodipine 1-10,000 100-5,000
IV. ANTI-RESTENOSIS AGENTS
Solutions of the present invention utilized for cardiovascular and general
vascular
procedures may optionally also include an anti-restenosis agent, particularly
for
angioplasty, rotational atherectomy and other interventional vascular uses.
The following
drugs are suitable for inclusion in the previously described cardiovascular
and general
vascular irrigation solutions when limitation of restenosis is indicated. The
following
anti-restenosis agents would preferably be combined with anti-spasm, and still
more
preferably also with anti-pain/anti-inflammation agents in the solutions of
the present
invention.
A. ANTIPLATELET AGENTS
At sites of arterial injury, platelets adhere to collagen and fibrinogen via
specific
cell surface receptors, and are then activated by several independent
mediators. A variety
of agonists are able to activate platelets, including collagen, ADP,
thromboxane A2,
epinephrine and thrombin. Collagen and thrombin serve as primary activators at
sites of
vascular injury, while ADP and thromboxane A2 act to recruit additional
platelets into a
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growing platelet plug. The activated platelets degranulate and release other
agents which
serve as chemoattractants and vasoconstrictors, thus promoting vasospasm and
platelet
accumulation. Thus, anti-platelet agents can be antagonists drawn from any of
the above
agonist-receptor targets.
Since platelets play such an important role in the coagulation cascade, oral
antiplatelet agents have been routinely administered to patients undergoing
vascular
procedures. Indeed, because of this multiplicity of activators and
observations that single
antiplatelet agents are not effective, some investigators have concluded that
a combined
treatment protocol is necessary for effectiveness. Recently, Willerson and
coworkers
reported the intravenous use of 3 combined agents, ridogrel (an antagonist of
thromboxane A2), ketanserin (a serotonin antagonist) and clopidogrel (an ADP
antagonist). They found that the combination of 3 antagonists inhibited
several relevant
platelet functions and reduced neointimal proliferation in a canine coronaxy
angioplasty
model (JACC Abstracts, Feb. 1995). It is still uncertain which approach to
treatment of
coronary thrombosis will be most successful. One possibility would be to
include an
antiplatelet agent and an antithrombotic agent in the cardiovascular and
general vascular
solutions of the present invention.
1. THROMBIN INHIBITORS AND RECEPTOR ANTAGONISTS
Thrombin plays a central role in vascular lesion formation and is considered
the
principal mediator of thrombogenesis. Thus, thrombus formation at vascular
lesion sites
during and after PTCA (percutaneous transluminal coronary angioplasty) or
other
vascular procedure is central to acute reocclusion and chronic restenosis.
This process
can be interrupted by application of direct anti-thrombins, including hirudin
and its
synthetic peptide analogs, as well as thrombin receptor antagonist peptides
(Harker, et al.,
Am. ,I. Ca~diol. 75:12B (1995)). Thrombin is also a potent growth factor which
initiates
smooth muscle cell proliferation at sites of vascular injury. In addition,
thrombin also
plays a role in modulating the effects of other growth factors such as PDGF
(platelet-derived growth factor), and it has been shown that thrombin
inhibitors reduce
expression of PDGF mRNA subsequent to vascular injury induced by balloon
angioplasty.
Hirudin is the prototypic direct antithrombin drug since it binds to the
catalytic
site and the substrate recognition site (exosite) of thrombin. Animal studies
using
baboons have shown that this proliferative response can be reduced 80% using
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recombinant hirudin (Ciba-Geigy). Hirulog (Biogen) is a dodecapeptide modeled
after
hirudin, and binds to the active site of thrombin via a Phe-Pro-Arg linker
molecule.
Large clinical trials of hirudin and hirulog are underway to test their
efficacy in reducing
vascular lesions after PTCA and Phase II data on these inhibitors to date is
positive, and
both drugs are believed to be suitable in the solutions of the present
invention.
Preliminary results of a 1,200 patient trial with repeat angiographic
assessment at
6 months to detect restenosis indicated superior short-term suppression of
ischemic
events with hirudin vs. heparin. An advantage of this approach is that no
significant
bleeding complications were reported. A sustained-release local hirulog
therapy was
found to decrease early thrombosis but not neointimal thickening after
arterial stenting in
pigs. Muller, D. et al., "Sustained-Release Local Hirulog Therapy Decreases
Early
Thrombosis but not Neointimal Thickening After Arterial Stenting," Am. Heart
J.
133(2):211-218 (1996). In this study, hirulog was released from an impregnated
polymer
placed around the artery.
Other active anti-thrombin agents being tested which are theorized to be
suitable
for the present invention are argatroban (Texas Biotechnology) and efegatran
(Lilly).
TABLE 25
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
RESTENOSIS INHIBITORY AGENTS
Therapeutic/Preferred
Concentrations More preferred
Class of Agent (Nanomolar) (Nanomolar)
Thrombin Inhibitors
and Receptor Antagonists:
hirudin 0.00003-3/0.0003-0.3 0.03
hirulog 0.2-20,000/2-2,000 200
2. ADP RECEPTOR ANTAGONISTS (PURINOCEPTOR
ANTAGONISTS)
Ticlopidine, an analog of ADP, inhibits both thromboxane and ADP-induced
platelet aggregation. It is likely that ticlopidine blocks interaction of ADP
with its
receptor, thereby inhibiting signal transduction by this G-protein coupled
receptor on the
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surface of platelet membranes. A preliminary study showed it to be more
effective than
aspirin in combination with dipyridamole. However, the clinical use of
ticlopidine has
been limited because it causes neutropenia. Clopidogrel, a ticlopidine analog,
is thought
to have fewer adverse side effects than ticlopidine and is currently being
studied for
prevention of ischemic events. It is theorized that these agents may be
suitable for use in
the solutions of the present invention.
3. THROMBOXANE INHIBITORS AND RECEPTOR
ANTAGONISTS
Agents currently utilized for conventional methods of treatment of thrombosis
rely upon aspirin, heparin and plasminogen activators. Aspirin irreversibly
acetylates
cycloxygenase and inhibits the synthesis of thromboxane A2 and prostacyclin.
While
data support a benefit of aspirin for PTCA, the underlying efficacy of aspirin
is
considered as only partial or modest. This is likely due to platelet
activation through
thromboxane A2 independent pathways that are not blocked by aspirin induced
acetylation of cyclooxygenase. Platelet aggregation and thrombosis may occur
despite
aspirin treatment. Aspirin in combination with dipyridamole has also been
shown to
reduce the incidence of acute complication during PTCA but not the incidence
of
restenosis.
Two thromboxane receptor antagonists appear to be more efficacious than
aspirin
and are believed suitable for use in the solutions and methods of the present
invention.
Ticlopidine inhibits both thromboxane and ADP-induced platelet aggregation.
Ridogrel
(R6~060) is a combined thromboxane B2 synthetase inhibitor and thromboxane-.
prostaglandin endoperoxide receptor blocker. It has been compared with
salicylate
therapy in an open-pilot study of patients undergoing PTCA administered in
combination
with heparin. Timmermans, C., et al., "Ridogrel in the Setting of Percutaneous
Transluminal Coronary Angioplasty," Am. J. Cardiol. 68:463-466 (1991).
Treatment
consisted of administering a slow intravenous injection of 300 mg just prior
to the start of
the PTCA procedure and continued orally after 12 hrs with a dose of 300
mg/twice daily.
From this study, ridogrel was found to be primarily successful since no early
acute
reocclusion occurred in 30 patients. Bleeding complications did occur in a
significant
number (34%) of patients, and this appears to be a complicating factor that
would require
special care. The study confirmed that ridogrel is a potent long-lasting
inhibitor of
thromboxane B2 synthetase.
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B. INHIBITORS OF CELL ADHESION MOLECULES
1. SELECTIN INHIBITORS
Selectin inhibitors block the interaction of a selectin with its cognate
ligand or
receptor. Representative examples of selectin targets at which these
inhibitors would act
include, but are not limited to, E-selectin and P-selectin receptors. Upjohn
Co. has
licensed rights to a monoclonal antibody developed by Cytel Corps that
inhibits the
activity of P-selectin. The product, CY 1748, is in preclinical development,
with a
potential indication being restenosis.
2. INTEGR1N INHIBITORS
The platelet glycoprotein IIb/IIIa complex is present on the surface of
resting as
well as activated platelets. It appears to undergo a transformation during
platelet
activation which enables it to serve as a binding site for fibrinogen and
other adhesive
proteins. Most promising new antiplatelet agents are directed at this integrin
cell surface
receptor which represents a final common pathway for platelet aggregation.
Several types of agents fit into the class of GPIIb/IIIa integrin antagonists.
A
monoclonal antibody, c7E3 (CentoRx; Centocor, Malvern PA), has been
intensively
studied to date in a 3,000 patient PTCA study. It is a chimeric human/murine
hybrid. A
0.25 mg/kg bolus of c7E3 followed by 10 ~,g/min intravenous infusion for 12
hrs
produced greater than 80% blockade of GPIIb/IIIa receptors for the duration of
the
infusion. This was correlated with a greater than 80% inhibition of platelet
aggregation.
The antibody was coadministered with heparin and an increased risk of bleeding
was
noted. Additional information was obtained from the EPIC trial which showed a
significant reduction in the primary end point, a composite of death rate,
incidence of
nonfatal myocardial infarction and need for coronary revascularization, and
suggested a
long term benefit. Tcheng, Am. Heat J. 130:673-679 (1995). ° A phase IV
study
(EPILOG) designed to address safety and efficacy issues with c7E3 Fab is
planned or in
progress. This monoclonal antibody can also be classified as a platelet
membrane
glycoprotein receptor antagonist directed against the glycoprotein IIb/IIIa
receptor.
The platelet glycoprotein IIb/IIIa receptor blocker, integrelin, is a cyclic
heptapeptide that is highly specific for this molecular target. In contrast to
the antibody,
it has a short biologic half life (about 10 minutes). The safety and efficacy
of integrelin
was first evaluated in the Phase II Impact trial. Either 4 or 12 hour
intravenous infusions
of 1.0 ~.g/lcg/min of integrelin were utilized (Topol, E., Am. J. Cardiol, 27B-
33B (1995)).
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It was provided in combination with other agents (heparin, aspirin) and was
shown to
exhibit potent anti-platelet aggregation properties (>80%). A phase III study,
the
IMPACT II trial, of 4000 patients showed that integrelin markedly reduced
ischemic
events in patients who had undergone Rotablator atherectomy (JACC Abstracts,
1996).
Suitable concentrations of the drugs c7E3 and integrelin for use in the
present invention
are set forth below.
In addition, two peptidomimetics, MK-383 (Merck) and RO 4483 (Hoffmann-
LaRoche), have been studied in Phase II clinicals. Since these are both small
molecules,
they have a short half life and high potency. However, these seem to also have
less
specificity, interacting with other closely related integrins. It is theorized
that these
peptidomimetics may also be suitable for use in the present invention.
TABLE 26
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
RESTENOSIS INHIBITORY AGENTS
Therapeutic/Preferred More preferred
Class of Agent Concentrations (Nanomolar) (Nanomolar)
Cell Adhesion Inhibitors:
c7E3 ' 0.5-50,000/5-5,000 500
Integrelin 0.1-10,000/1-1000 x Kd 100 x Kd
C. ANTI-CHEMOTACTIC AGENTS
Anti-chemotactic agents prevent the chemotaxis of inflammatory cells.
Representative examples of anti-chemotactic targets at which these agents
would act
include, but are not limited to, F-Met-Leu-Phe receptors, IL-8 receptors, MCP-
1
receptors, and MIP-1-a/R.ANTES receptors. Drugs within this class of agents
are early in
the development stage, but it is theorized that they may be suitable for use
in the present
invention.
D. 1NTERLEIJKIN RECEPTOR ANTAGONISTS
Interleulcin receptor antagonists are agents which block the interaction of an
interleukin with its cognate ligand or receptor. Specific receptor antagonists
for any of
the numerous interleulein receptors are early in the development process. The
exception
to this is the naturally occurring existence of a secreted form of the IL-1
receptor, referred
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to as IL-1 antagonist protein (IL-lAP). This antagonist binds IL-1 and has
been shown to
suppress the biological actions of IL-1, and is theorized to be suitable for
the practice of
the present invention.
E. INTRACELLULAR SIGNALING INHIBITORS
1. PROTEIN KINASE INHIBITORS
A. PROTEIN KINASE C (PKC) INHIBITORS
Protein kinase C (PKC) plays a crucial role in cell-surface signal
transduction for
a number of physiological processes. PKC isozymes can be activated as
downstream
targets resulting from initial activation of either G-protein coupled
receptors (e.g.,
serotonin, endothelin, etc.) or growth-factor receptors such as PDGF. Both of
these
receptor classes play important roles in mediating vascular spasm and
restenosis
subsequent to coronary balloon angioplasty procedures.
Molecular cloning analysis has revealed that PKC exists as a large family
consisting of at least 8 subspecies (isozymes). These isozymes differ
substantially in
structure and mechanism for linking receptor activation to changes in the
proliferative
response of specific cells. Expression of specific isozymes is found in a wide
variety of
cell types, including: platelets, neutrophils, myeloid cells, and smooth
muscle cells.
Inhibitors of PKC are therefore likely to effect signaling pathways in several
cell types
unless the inhibitor shows isozyme specificity. Thus, inhibitors of PKC can be
predicted
to be effective in blocking the proliferative response of smooth muscle cells
and may also
have an anti-inflammatory effect in blocking neutrophil activation and
subsequent
attachment. Several inhibitors have been described and initial reports
indicate an ICsp of
50 ~.M for calphostin C inhibitory activity. G-6203 (also known as Go 6976) is
a new,
potent PKC inhibitor with high selectivity for certain PKC isotypes with ICsp
values in
the 2-10 ~,M range. Concentrations of these and another drug, GF 109203X, also
known
as Go 6850 or bisindoylmaleimide I (available from Warner-Lambert), that are
believed
to be suitable for use in the present invention are set forth below.
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TABLE 27
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
RESTENOSIS INHIBITORY AGENTS
Therapeutic/Preferred Concentrations More preferred
Class of Agent (Nanomolar) (Nanomolar)
Protein Kinase C Inhibitors:
calphostin C 0.5-50,000/100-5,000 500
GF 109203X 0.1-10,000/1-1,000 100
G-6203 (Go 6976) 0.1-10,000/1-1,000 100
B. PROTEIN TYROSINE KINASE INHIBITORS
Although there is a tremendous diversity among the numerous members of the
receptors tyrosine-kinase (RTK) family, the signaling mechanisms used by these
receptors share many common features. Biochemical and molecular genetic
studies have
shown that binding of the ligand to the extracellular domain of the RTK
rapidly activates
the intrinsic tyrosine kinase catalytic activity of the intracellular domain
(see FIGURE 5).
The increased activity results in tyrosine-specific phosphorylation of a
number of
intracellular substrates which contain a common sequence motif. Consequently,
this
causes activation of numerous "downstream" signaling molecules and a cascade
of
intracellular pathways that regulate phospholipid metabolism, arachidonate
metabolism,
protein phosphorylation (involving mechanisms other than protein kinases),
calcium
mobilization and transcriptional activation (see FIGURE 2). Growth-factor-
dependent
tyrosine kinase activity of the RTK cytoplasmic domain is the primary
mechanism for
generation of intracellular signals that lead to cellular proliferation. Thus,
inhibitors have
the potential to block this signaling and thereby prevent the proliferative
response (see
FIGURE 5).
The platelet-derived growth factor (PDGF) receptor is of great interest as a
target
for inhibition in the cardiovascular field since it is believed to play a
significant role both
in atherosclerosis and restenosis. The release of PDGF by platelets at damaged
surfaces
of endothelium within blood vessels results in stimulation of PDGF receptors
on vascular
smooth muscle cells. As described above, this initiates a sequence of
intracellular events
leading to enhanced proliferation and neointimal thickening. An inhibitor of
PDGF
kinase activity would be expected to prevent proliferation and enhance the
probability of
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success following cardiovascular and general vascular procedures. Any of
several related
tyrphostin compounds have potential as specific inhibitors of PDGF-receptor
tyrosine
kinase activity (ICsps ih vitro in the 0.5-1.0 ~.M range), since they have
little effect on
other protein lcinases and other signal transduction systems. To date, only a
few of the
many tyrphostin compounds are commercially available, and suitable
concentrations for
these agents as used in the present invention axe set forth below. In
addition,
staurosporine has been reported to demonstrate potent inhibitory effects
against several
protein tyrosine kinases of the src subfamily and a suitable concentration for
this agent as
used in the present invention also is set forth below.
TABLE 28
THERAPEUTIC AND PREFERRED CONCENTRATIONS OF
RESTENOSIS INHIBITORY AGENTS
Therapeutic/Preferred More preferred
Class of Agent Concentrations (Nanomolar) (Nanomolar)
Protein I~inase
Inhibitors
lavendustin A 10-100,0001100-10,000 10,000
tyrphostin 10-100,000/100-20,000 10,000
AG1296
tyrphostin 10-100,000/100-20,000 ~ 10,000
AG1295
staurosporine 1-100,000/10-10,000 1,000
2. MODULATORS OF INTRACELLULAR
PROTEIN TYROSINE
PHOSPHATASES
Non-transmembrane protein tyrosine phosphatases (PTPases) containing
src-homology2 SH2 domains are known and nomenclature refers to them as SH-PTP1
and SH-PTP2. In addition, SH-PTP 1 is also known as PTP 1 C, HCP or SHP. SH-
PTP2 is
also known as PTP 1 D or PTP2C. Similarly, SH-PTP 1 is expressed at high
levels in
hematopoietic cells of all lineages and all stages of differentiation, and the
SH-PTP 1 gene
has been identified as responsible for. the motheaten (me) mouse phenotype and
this
provides a basis for predicting the effects of inhibitors that would block its
interaction
with its cellular substrates. Stimulation of neutrophils with chemotactic
peptides is
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known to result in the activation of tyrosine kinases that mediate neutrophil
responses
(Cui, et al., J. Immuuol. (1994)) and PTPase activity modulates agonist
induced activity
by reversing the effects of tyrosine kinases activated in the initial phases
of cell
stimulation. Agents that could stimulate PTPase activity could have potential
therapeutic
applications as anti-inflammatory mediators.
These same PTPases have also been shown to modulate the activity of certain
RTKs. They appear to counter-balance the effect of activated receptor kinases
and thus
may represent important drug targets. In vitro experiments show that injection
of PTPase
blocks insulin stimulated phosphorylation of tyrosyl residues on endogenous
proteins.
Thus, activators of PTPase activity could serve to reverse activation of PDGF-
receptor
action in restenosis, and are believed to be useful in the solutions of the
present invention.
In addition, receptor-linked PTPases also function as extracellular ligands,
similar to
those of cell adhesion molecules. The functional consequences of the binding
of a ligand
to the extracellular domain have not yet been defined but it is reasonable to
assume that
binding would serve to modulate phosphatase activity within cells (Fashena and
Zinn,
Cu~~ent Biology 5:1367-1369 (1995)). Such actions could block adhesion
mediated by
other cell surface adhesion molecules (NCAM) and provide an anti-inflammatory
effect.
No drugs have been developed yet for these applications.
3. INHIBITORS OF SH2 DOMAINS (SRC HOMOLOGY2
DOMAINS)
SH2 domains, originally identified in the src subfamily of protein tyrosine
kinases
(PTKs), are noncatalytic protein sequences and consist of about 100 amino
acids
conserved among a variety of signal transducing proteins (Cohen, et al.,
1995). SH2
domains function as phosphotyrosine-binding modules and thereby mediate
critical
protein-protein associations in signal transduction pathways within cells
(Pawson,
Nature, 573-580, 1995). In particular, the role of SH2 domains has been
clearly defined
as critical for receptor tyrosine kinase (RTK) mediated signaling such as in
the case of the
platelet-derived growth factor (PDGF) receptor. Phosphotyrosine-containing
sites on
autophosphorylated RTKs serve as binding sites for SH2-proteins and thereby
mediate
the activation of biochemical signaling pathways (see FIGURE 2) (Carpenter,
G.,
FASEB J. 6:3283-3289 (1992); Sierke, S. and Koland, J. Biochem. 32:10102-
10108,
(1993)). The SH2 domains are responsible for coupling the activated growth-
factor
receptors to cellular responses which include alterations in gene expression,
and
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ultimately cellular proliferation (see FIGURE 5). Thus, inhibitors that will
selectively
block the effects of activation of specific RTKs expressed on the surface of
vascular
smooth muscle cells are predicted 'to be effective in blocking proliferation
and the
restenosis process after PTCA or other vascular procedure. One RTK target of
current
interest is the PDGF receptor.
At least 20 cytosolic proteins have been identified that contain SH2 domains
and
function in intracellular signaling. The distribution of SH2 domains is not
restricted to a
particular protein family, but found in several classes of proteins, protein
kinases, lipid
kinases, protein phosphatases, phospholipases, Ras-controlling proteins and
some
transcription factors. Many of the SH2-containing proteins have known
enzymatic
activities while others (Grb2 and Crlc) function as "linkers" and "adapters"
between cell
surface receptors and "downstream" effector molecules (Marengere, L., et al.,
Nature
369:502-505 (1994)). Examples of proteins containing SH2 domains with
enzymatic
activities that are activated in signal transduction include, but are not
limited to, the src
subfamily of protein tyrosine kinases (src (pp60c-src)~ abl, lck, fyn, fgr and
others),
phospholipase Cy (PLCy), phosphatidylinositol 3-kinase (PI-3-kinase), p21-ras
GTPase
activating protein (GAP) and SH2 containing protein tyrosine phosphatases (SH-
PTPases) (Songyang, et al., Cell 72:767-778 (1993)). Due to the central role
these
various SH2-proteins occupy in transmitting signals from activated cell
surface receptors
into a cascade of additional molecular interactions that ultimately define
cellular
responses, inhibitors which block specific SH2 protein binding are desirable
as agents for
a variety of potential therapeutic applications.
In addition, the regulation of many immune/inflammatory responses is mediated
through receptors that transmit signals through non-receptor tyrosine kinases
containing
SH2 domains. T-cell activation via the antigen specific T-cell receptor (TCR)
initiates a
signal transduction cascade leading to lymphokine secretion and T-cell
proliferation. One
of the earliest biochemical responses following TCR activation is an increase
in tyrosine
kinase activity. In particular, neutrophil activation is in part controlled
through responses
of the cell surface immunoglobulin G receptors. Activation of these receptors
mediates
activation of unidentified tyrosine kinases which are known to possess SH2
domains.
Additional evidence indicates that several src-family kinases (lck, blk, fyn)
participate in
signal transduction pathways leading from cytokine and integrin receptors and
hence may
serve to integrate stimuli received from several independent receptor
structures. Thus,
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inhibitors of specific SH2 domains have the potential to block many neutrophil
functions
and serve as anti-inflammatory mediators.
Efforts to develop drugs targeted to SH2 domains currently are being conducted
at
the biochemical ih vitro and cellular level. Should such efforts be
successful, it is
theorized that the resulting drugs would be useful in the practice of the
present invention.
4. CALCIUM CHANNEL ANTAGONISTS
Calcium channel antagonists, previously described with relation to spasm
inhibitory function, also can be used as anti-restenotic agents in the
cardiovascular and
general vascular solutions of the present invention. Activation of growth
factor receptors,
such as PDGF, is known to result in an increase in intracellular calcium (see
FIGURE 2).
Studies at the cellular level have shown that actions of calcium channel
antagonists axe
effective at inhibiting mitogenesis of vascular smooth muscle cells.
F. SYNERGISTIC INTERACTIONS DERIVED FROM THERAPEUTIC
COMBINATIONS OF ANTI-RESTENOSIS AGENTS AND OTHER
AGENTS USED IN CARDIOVASCULAR AND GENERAL
VASCULAR SOLUTIONS
Given the complexity of the disease process associated with restenosis after
PTCA or other cardiovascular or general vascular therapeutic procedure and the
multiplicity of molecular targets involved, blockade or inhibition of a single
molecular
target is unlikely to provide adequate efficacy in preventing vasospasm and
restenosis
(see FIGURE 2). Indeed, a number of animal studies targeting different
individual
molecular receptors and or enzymes have not proven effective in animal models
or have
not yielded efficacy for both pathologies in clinical trials to date. (Freed,
M., et al., "An
Intensive Poly-pharmaceutical Approach to the Prevention of Restenosis: the
Mevacor,
Ace Inhibitor, Colchicine (BIG-MAC) Pilot Trial," J. AnZ. Coll. of Cardiol.
21:33A
(1993); Serruys, P., et al., "PARK: the Post Angioplasty Restenosis Ketanserin
Trial,"
J. Am. Coll. of Cardiol. 21:322A (1993)). Therefore, a therapeutic combination
of drugs
acting on distinct molecular targets and delivered locally appears necessary
for clinical
effectiveness in the therapeutic approach to vasospasm and restenosis. As
described
below, the rationale for this synergistic molecular targeted therapy is
derived from recent
advances in understanding fundamental biochemical mechanisms by which vascular
smooth muscle cells in the vessel wall transmit and integrate stimuli to which
they are
exposed during PTCA or other vascular interventional procedure.
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1. "CROSSTALK" AND CONVERGENCE IN MAJOR
SIGNALING PATHWAYS
The molecular switches responsible for cell signaling have been traditionally
divided into two major discrete signaling pathways, each comprising a distinct
set of
protein families that act as transducers for a particular set of extracellular
stimuli and
mediating distinct cell responses. One such pathway transducer signals from
neurotransmitters and hormones through G-protein coupled receptors (GPCRs) to
produce contractile responses using intracellular targets of trimeric G
proteins and Ca~+
(see FIGURE 2). These stimuli and their respective receptors mediate smooth
muscle
contraction and may induce vasospasm in the context of PTCA or other
cardiovascular or
general vascular therapeutic or diagnostic procedure. Examples of signaling
molecules
involved in mediating spasm through the GPCR pathway are 5-HT and endothelin
for
which antagonists have been included .acting via their respective G-protein
coupled
receptors.
A second major pathway transduces signals from growth factors, such as PDGF,
through tyrosine kinases, adaptor proteins and the Ras protein into regulation
of cell
proliferation and differentiation (see FIGURES 2 and 5). This pathway may also
be
activated during PTCA or other cardiovascular or general vascular procedure
leading to a
high incidence of vascular smooth muscle cell proliferation. An example of a
restenosis
drug target is the PDGF-receptor.
Signals transmitted from neurotransmitters and hormones stimulate either of
two
classes of receptors: G-protein-coupled receptors, composed of seven-helix
transmembrane regions, or ligand-gated ion channels. "Downstream" signals from
both
kinds of receptors converge on controlling the concentration of cytoplasmic
Ca2+ which
triggers contraction in smooth muscle cells (see FIGURE 2). Each GPCR
transmembrane
receptor activates a specific class of trimeric G proteins, including Gq, Gi
or many others.
Ga and/or GR,y subunits activate phospholipase Ca, resulting in activation of
protein
kinase C (PKC) and an increase in the levels of cytoplasmic calcium by release
of
calcium from intracellular stores.
Growth factor signaling, such as mediated by PDGF, converges on regulation of
cell growth. This pathway depends upon phosphorylation of tyrosine residues in
receptor
tyrosine kinases and "downstream" enzymes (phospholipase Cy, discussed above
with
regard to tyrosine kinases). Activation of the PDGF-receptor also leads to
stimulation of
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PKC and elevation of intracellular calcium, common steps shared by the GPCRs
(see
FIGURE 2). It is now recognized that ligand-independent "crosstalk" can
transactivate
tyrosine kinase receptor pathways in response to stimulation of GPCRs. Recent
work has
identified Shc, an adaptor protein in the tyrosine kinase/Ras pathway, as a
key
intermediary protein that relays messages from the GPCR pathway described
above to the
tyrosine kinase pathway (see FIGURE 2) (Lev et al., Nature 376:737 (1995)).
Activation
of Shc is calcium dependent. Thus, a combination of selective inhibitors which
blocks
transactivation of a common signaling pathway leading to vascular smooth
muscle cell
proliferation will act synergistically to prevent spasm and restenosis after
PTCA or other
cardiovascular or general vascular procedure. Specific examples are briefly
detailed
below.
2. SYNERGISTIC INTERACTIONS BETWEEN PKC
INHIBITORS AND CALCIUM CHANNEL ANTAGONISTS
In this case synergistic interactions among PKC inhibitors and calcium channel
antagonists in achieving vasorelaxation and inhibition of proliferation occur
due to
"crosstalk" between GPCR and tyrosine kinase signaling pathways (see FIGURE
2). A
rationale for dual use is based upon the fact that these drugs have different
molecular
mechanisms of action. As described above, GPCR stimulation results in
activation of
protein kinase C and an increase in the levels of cytoplasmic calcium by
release of
calcium from intracellular stores. Calcium-activated PKC is a central control
point in the
transmission of extracellular responses. "Crosstalk" from GPCR stimulated
pathways
through PKC can lead to mitogenesis of vascular smooth muscle cells and thus
calcium
channel antagonists will have the dual action of directly blocking spasm and
further
preventing activation of proliferation by inhibiting Shc activation.
Conversely, the PKC
inhibitor acts on part of the pathway leading to contraction.
3. SYNERGISTIC EFFECTS OF PKC INHIBITORS, 5-HT2
ANTAGONISTS AND ETp ANTAGONISTS
The 5-HT2 receptor family contains three members designated 5-HT2A, 5-HT2g,
and 5-HT2~, all of which share the common property of being coupled to
phosphotidylinositol turnover and increases in intracellular calcium (Hoyer et
al., 1988;
Hartig et al., 1989). The distribution of these receptors includes vascular
smooth muscle
and platelets and, due to their localization, these 5-HT receptors are
important in
mediating spasm, thrombosis and restenosis. It has been found that the
sustained phase of
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intracellular calcium elevation in smooth muscle cells induced by ETA receptor
activation
requires extracellular calcium and is at least partially blocked by
nicardipine. Since
activation of both 5-HT2 receptors and ETA receptors is mediated through
calcium, the
inclusion of a PKC inhibitor is expected to synergistically enhance the
actions of
antagonists to both of these receptors when combined in a surgical solution
(see
FIGURES 2 and 4).
4. SYNERGISTIC EFFECTS OF PROTEIN TYROSINE KINASE
INHIBITORS AND CALCIUM CHANNEL ANTAGONISTS
The mitogenic effect of PDGF (or basic fibroblast growth factor or insulin-
like-
growth-factor-1) is mediated through receptors that possess intrinsic protein
tyrosine
lcinase activity. The substrates for PDGF phosphorylation are many and lead to
activation
of mitogen-activated protein kinases (MAPK) and ultimately proliferation (see
FIGURE 5). The endothelin, 5-HT and thrombin receptors, which are members of
the
G-protein coupled superfamily, trigger a signal transduction pathway which
includes
increases in intracellular calcium, leading to activation of calcium channels
on the plasma
membrane. Thus, calcium channel antagonists interfere with a common mechanism
employed by these GPCRs. It has recently been shown that activation of certain
GPCRs,
including endothelin and bradykinin, leads to a rapid increase in tyrosine
phosphorylation
of a number of intracellular proteins. Some of the proteins phosphorylated
parallel those
known necessary for mitogenic stimulation. The rapidity of the process was
such that
changes were detectable in seconds and the targets acted upon likely play a
role in
mitogenesis. These tyrosine phosphorylation events were not blocked by a
selective PKC
inhibitor or apparently mediated by increased intracellular calcium. Thus,
since two
independent pathways, the GPCR and tyrosine phosphorylation pathways, can
drive the
vascular smooth muscle cells into a proliferative state, it is necessary to
block both
independent signaling arms. This is the basis for the synergistic interaction
between
calcium channel antagonists and tyrosine lcinase inhibitors in the surgical
solution.
Because the actions of the protein tyrosine kinase inhibitors in preventing
vascular
smooth muscle cell proliferation occur via independent molecular pathways
(described
above) from those involving calcium and protein kinase C, the combination of
the two
classes of drugs, calcium channel antagonists and protein tyrosine kinase
inhibitors, is
expected to be more efficacious in inhibiting spasm and restenosis than
employing either
single class of drug alone.
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5. SYNERGISTIC EFFECTS OF PROTEIN TYROSINE KINASE
INHIBITORS AND THROMBIN RECEPTOR ANTAGONISTS
Thrombin mediates its action via the thrombin receptor, another member of the
GPCR superfamily. Binding to the receptor stimulates platelet aggregation,
smooth
muscle cell contraction and mitogenesis. Signal transduction occurs through
multiple
pathways: activation of phospholipase (PLC) through G proteins and activation
of
tyrosine kinases. The activation of tyrosine lcinase activity is also
essential for
mitogenesis of the vascular smooth muscle cells. Experiments have shown that
inhibition
with a specific tyrosine kinase inhibitor was effective in blocking thrombin-
induced
mitosis, although there were no effects on the PLC pathway as monitored by
measurement of intracellular calcium (Weiss and Nucitelli, J. Biol. Chem.
267:5608-5613
(1992)). Because the actions of the protein tyrosine kinase inhibitors in
preventing
vascular smooth muscle cell proliferation occur via independent molecular
pathways
(described above) from those involving calcium and protein kinase C, the
combination of
protein tyrosine kinase inhibitors and thrombin receptor antagonists is
anticipated to be
more efficacious in inhibiting platelet aggregation, spasm and restenosis than
employing
either class of agent alone.
G. CYCLOOXYGENASE-2 (COX-2) INHIBITORS
Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used as anti-
inflammatory, anti-pyretic, anti-thrombotic and analgesic agents (Lewis, R.A.,
"Prostaglandins and Leukotrienes," In: Textbook of Rheumatology, 3d ed.
(Kelley W.,
et al., eds.), p. 258 (1989). The molecular target for these drugs is the
first enzyme in the
prostaglandin synthetic pathway, referred to as prostaglandin endoperoxide
synthase, or
fatty acid cyclooxygenase. It is now appreciated that there are two forms of
cyclooxygenase, termed cyclooxygenase-1 or type 1 (COX-1) and cyclooxygenase-2
or
type 2 (COX-2). These isozymes are also known as Prostaglandin H Synthase
(PGHS)-1
and PGHS-2, respectively. Both enzymes catalyze the conversion of arachidonic
acid to
unstable intermediates, PGG2 and PGH2, which are intermediates in the
biosynthesis of
prostaglandins and thromboxanes. COX-1 is present in platelets and endothelial
cells and
exhibits constitutive activity. COX-2 has been identified in endothelial
cells,
macrophages and fibroblasts, including synovial cells after treatment
(induction) with
cytolcines.
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The COX-2 isozyme is induced in settings of inflammation by cytokines and
inflammatory mediators, such as IL-1, TNF-a and endotoxin, and its expression
is
upregulated at sites of inflammation. The large increase in activity of COX-2
above basal
COX-1 activity concomitant with its upregulation, leads to synthesis of
prostaglandins
which contribute to pain and inflammation. Because COX-2 is usually expressed
only in
inflamed tissue or after exposure to mediators of inflammation, selective
inhibitors may
exhibit anti-inflammatory activity without simultaneous effects on
constitutively
expressed COX-1 activity present in platelets and other cell types which is
considered the
cause of undesirable side effects associated with use of nonselective NSAID
drugs (e.g.,
clotting time, bleeding, renal and gastrointestinal).
It has been established that the two COX isozymes are pharmacologically
distinct
and therefore it has been possible to develop isozyme-specific (selective)
cyclooxygenase
inhibitors that are useful for anti-inflammatory therapy. A variety of
biochemical,
cellular and animal assays have been developed to assess the relative
selectivity of
inhibitors for the COX-1 and COX-2 isoforms. These assays include measurements
of
prostaglandin E2 production in microsomes prepared from various cell types and
bioassay systems using intact human cells. For any given drug, despite
experimental
variation in the degree of selectivity noted among assay systems and between
biological
sources, compounds that are selective inhibitors for COX-2 have been
identified. In
general, a criteria for defining selectivity is the ratio of the COX-1/COX-2
inhibitory
constants (or COX-2/COX-1) obtained for a given biochemical or cellular assay
system.
The selectivity ratio accounts for different absolute ICSO values for
inhibition of
enzymatic activity that are obtained between microsomal and cellular assay
systems (e.g.,
platelets and macrophages, cell lines stably expressing recombinant human COX
isozymes).
Many of the conventional NSAIDs currently on the market (naproxen,
indomethacin, ibuprofen) are generally nonselective inhibitors of both
isoforms of COX,
but may show greater selectively for COX-1 over COX-2, although this ratio
varies for
the different compounds. The use of a COX-2 inhibitor to block formation of
prostaglandins represents a preferred therapeutic strategy rather than
attempting to block
interactions of the endogenous prostanoid ligands, such as PGE2, which are
produced by
COX-2 at the inflammatory site, with any of the eight described subtypes of
prostanoid
receptors. This approach is not currently feasible since selective and potent
antagonists
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for all of the of the prostanoid receptors (EP-1, EP-2, EP-3, EP-4, DP, FP, IP
and TP) do
not exist.
A study by Riendeau and coworkers compared the selectivity of more than
45 NSAIDs and selective COX-2 inhibitors using sensitive microsomal and
platelet
assays for the inhibition of human COX-1 based on the production of
prostaglandin E2 by
microsomes (Cah. J. Physiol. Pha~macol. 75:1088-95 (1997)). In this study,
among the
compounds that were reported to show selectivity for COX-2 vs. COX-l, the rank
order
of potency was DuP 697 > SC-58451, celecoxib > nimesulide = meloxicam =
piroxicam
= NS-398= RS-57067 >SC-57666 > SC-58125 > flosulide > etodolac > L-745,337 >
DFU-T-614, with ICSO values ranging from 7 ~M to 17 ~.M. A good correlation
was
obtained between the ICSo values for the inhibition of microsomal COX-1 and
both the
inhibition of TXB2 production by Ca2+ ionophore challenged platelets and the
inhibition
of prostaglandin E2 production by CHO cells stably expressing human COX-1. The
microsomal assay was more sensitive to inhibition than cell-based assays and
allowed the
detection of inhibitory effects on COX-1 for all NSAIDs and selective COX-2
inhibitors
examined with discrimination of their potency under conditions of limited
availability of
arachidonic acid.
From the molecular and cellular mechanism of action defined for selective COX-
2
inhibitors, such as celecoxib, as well as animal studies, these compounds are
expected to
exhibit anti-inflammatory action when applied intraoperatively in an
irrigation solution
directly to a tissue or a joint. In particular, it is expected to be an
effective drug delivered
by an irrigation solution during an arthroscopic, urologic, cardiovascular or
general
surgical procedure (periprocedurally). For example, a selective COX-2
inhibitor may
replace lcetorolac, a relatively non-selective cyclooxygenase inhibitor, in
Examples IV, V
& VI. The selective COX-2 inhibitor may be delivered alone, or in combination
with
other small molecule drugs, peptides, proteins, recombinant chimeric proteins,
antibodies,
or gene therapy vectors (viral and nonviral) to the spaces of the joint,
urogenital tract,
cardiovascular system or any cavity of the body. For example, the selective
COX-2
inhibitor can exert its actions on any cells associated with the fluid spaces
of the joint and
structures comprising the joint, and which are involved in the normal function
of the joint
or are present due to a pathological condition. These cells and structures
include, but are
not limited to: synovial cells including both Type A fibroblast and type B
macrophage
cells; the cartilaginous components of the joint such as chondrocytes; cells
associated
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with bone, including periosteal cells, osteoblasts, osteoclasts; the
immunological
components such as inflammatory cells including lymphocytes, mast cells,
monocytes,
eosinophils; and other cells like fibroblasts; and combinations of the above
cell types.
The selective COX-2 inhibitor may be delivered in a formulation useful for
introduction and administration of the drug into the targeted tissue or joint
that would
enhance the delivery, uptake, stability or pharmacokinetics of the inhibitor
drug. The
formulation could include, but is not limited to, administration using
microparticles,
microspheres or nanoparticles composed of lipids, proteins, carbohydrates,
synthetic
organic compounds, or inorganic compounds. Examples of formulation molecules
include, but are not limited to, lipids capable of forming liposomes or other
ordered lipid
structures, cationic lipids, hydrophilic polymers, polycations (e.g.,
protamine, spermidine,
polylysine), peptide or synthetic ligands and antibodies capable of targeting
materials to
specific cell types, gels, slow release matrices, soluble and insoluble
particles, as well as
formulation elements not listed.
The present invention describes the local delivery of a selective COX-2
inhibitor
drug using an irrigation solution containing the drug which is present at low
concentration and which enables the drug to be delivered directly to the
affected tissue or
joint. The drug-containing irrigation solution is employed perioperatively
during a
surgical procedure. Other conventional methods used for drug delivery have
required
systemic administration (e.g., intramuscular, intravenous, subcutaneous, oral)
which
necessitates higher concentrations of COX-2 drugs (and higher total dose) to
be
administered in order to achieve significant therapeutic concentrations in the
targeted
tissue or joint. Systemic administration also results in high concentrations
in tissues other
than the targeted tissue which is undesirable and, depending on the dose, may
result in
adverse side effects (e.g., bleeding, ulceration). These systemic methods of
delivery also
subject the drug to second pass metabolism and rapid degradation, thereby
limiting the
duration of the effective therapeutic concentration. The drug in the
irrigation solution is
administered directly to the desired tissue which provides a significant
advantage by
allowing the delivery of the selective COX-2 inhibitor drug in a
therapeutically effective
lower concentration and therapeutically effective lower total dose than by
other routes of
administration. Furthermore, the use of a COX-2 inhibitor in the solution in
substantially
lower dosages than currently used perioperatively may allow the use of this
drug in
otherwise contraindicated patients.
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Compounds suitable for the invention are listed in the Table below.
TABLE 29
CYCLOOXYGENASE-2 INHIBITORS
Compounds Therapeutic PreferredMost Preferred
Concentrations (q.M)Concentrations
(~M)
DuP 697 0.3-3,000 600
SC-58451 0.3-3,000 600
celecoxib 0.3-3,000 600
meloxicam 0.5-5,000 500
nimesulide 0.5-5,000 500
diclofenac 0.3-3,000 600
NS-398 0.3-3,000 600
L-745,337 0.2-10,000 1000
RS57067 0.2-10,000 1000
SC-58125 0.2-10,000 1000
SC-57666 0.2-10,000 1000
flosulide 0.2-10,000 1000
V. METHOD OF APPLICATION
The solution of the present invention has applications for a variety of
operative/interventional procedures, including surgical, diagnostic and
therapeutic
techniques. The irrigation solution is perioperatively applied during
arthroscopic surgery
of anatomic joints, urological procedures, cardiovascular and general vascular
diagnostic
and therapeutic procedures and for general surgery. As used herein, the term
"perioperative" encompasses application intraprocedurally, pre- and
intraprocedurally,
intra- and postprocedurally, and pre-, intra- and postprocedurally. Preferably
the solution
is applied preprocedurally and/or postprocedurally as well as
intraprocedurally. Such
procedures conventionally utilize physiologic irrigation fluids, such as
normal saline or
lactated Ringer's, applied to the surgical site by techniques well known to
those of
ordinary slcill in the art. The method of the present invention involves
substituting the
anti-pain/anti-inflammatory!anti-spasm/anti-restenosis irrigation solutions of
the present
invention for conventionally applied irrigation fluids. The irrigation
solution is applied to
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the wound or surgical site prior to the initiation of the procedure,
preferably before tissue
trauma, and continuously throughout the duration of the procedure, to
preemptively block
pain and inflammation, spasm and restenosis. As used herein throughout, the
term
"irrigation" is intended to mean the flushing of a wound or anatomic structure
with a
stream of liquid. The term "application" is intended to encompass irrigation
and other
methods of locally introducing the solution of the present invention, such as
introducing a
gellable version of the solution to the operative site, with the gelled
solution then
remaining at the site throughout the procedure. As used herein throughout, the
term
"continuously" is intended to also include situations in which there is
repeated and
frequent irrigation of wounds at a frequency sufficient to maintain a
predetermined
therapeutic local concentration of the applied agents, and applications in
which there may
be intermittent cessation of irrigation fluid flow necessitated by operating
technique.
The concentrations listed for each of the agents within the solutions of the
present
invention are the concentrations of the agents delivered locally, in the
absence of
metabolic transformation, to the operative site in order to achieve a
predetermined level
of effect at the operative site. It is understood that the drug concentrations
in a given
solution may need to be adjusted to account for local dilution upon delivery.
For
example, in the cardiovascular application, if one assumes an average human
coronary
artery blood flow rate of 80 cc per minute and an average delivery rate for
the solution of
5 cc per minute via a local delivery catheter (i.e., a blood flow-to-solution
delivery ratio
of 16 to 1), one would require that the drug concentrations within the
solution be
increased 16-fold over the desired ih vivo drug concentrations. Solution
concentrations
are not adjusted to account for metabolic transformations or dilution by total
body
distribution because these circumstances are avoided by local delivery, as
opposed to
oral, intravenous, subcutaneous or intramuscular application.
Arthroscopic techniques for which the present solution may be employed
include,
by way of non-limiting example, partial meniscectomies and ligament
reconstructions in
the knee, shoulder acromioplasties, rotator cuff debridements, elbow
synovectomies, and
wrist and ankle arthroscopies. The irrigation solution is continuously
supplied
intraoperatively to the joint at a flow rate sufficient to distend the joint
capsule, to remove
operative debris, and to enable unobstructed infra-articular visualization.
A suitable irrigation solution for control of pain and edema during such
arthroscopic techniques is provided in Example I herein below. For
arthroscopy, it is
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preferred that the solution include a combination, and preferably all, or any
of the
following: a serotonin2 receptor antagonist, a serotonin3 receptor antagonist,
a
histamine 1 receptor antagonist, a serotonin receptor agonist acting on the 1
A, 1 B, 1 D, 1 F
and/or lE receptors, a bradykininl receptor antagonist, a bradykinin2 receptor
antagonist,
and a cyclooxygenase inhibitor.
This solution utilizes extremely low doses of these pain and inflammation
inhibitors, due to the local application of the agents directly to the
operative site during
the procedure. For example, less than 0.05 mg of amitriptyline (a suitable
serotonin2 and
histamine) "dual" receptor antagonist) are needed per liter of irrigation
fluid to provide
the desired effective local tissue concentrations that would inhibit 5-HT2 and
H1
receptors. This dosage is extremely low relative to the 10-25 mg of oral
amitriptyline that
is the usual starting dose for this drug. This same rationale applies to the
anti-spasm and
anti-restenosis agents which are utilized in the solution of the present
invention to reduce
spasm associated with urologic, cardiovascular and general vascular procedures
and to
inhibit restenosis associated with cardiovascular and general vascular
procedures. For
example, less than 0.2 mg of nisoldipine (a suitable calcium channel
antagonist) is
required per liter of irrigation fluid to provide the desired effective local
tissue
concentrations that would inhibit the voltage-dependent gating of the L-
subtype of
calcium channels. This dose is extremely low compared to the single oral dose
of
nisoldipine which is 20 to 40 mg.
In each of the surgical solutions of the present invention, the agents are
included
in low concentrations and are delivered locally in low doses relative to
concentrations and
doses required with conventional methods of drug administration to achieve the
desired
therapeutic effect. It is impossible to obtain an equivalent therapeutic
effect by delivering
similarly dosed agents via other (i.e., intravenous, subcutaneous,
intramuscular or oral)
routes of drug administration since drugs given systemically are subject to
first- and
second-pass metabolism.
For example, using a rat model of arthroscopy, the inventors examined the
ability
of amitriptyline, a 5-HT2 antagonist, to inhibit 5-HT-induced plasma
extravasation in the
rat knee in accordance with the present invention. This study, described more
fully below
in Example XII, compared the therapeutic dosing of amitriptyline delivered
locally (i.e.,
intra-anticularly) at the knee and intravenously. The results demonstrated
that intra-
articular administration of amitriptyline required total dosing levels
approximately
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200-fold less than were required via the intravenous route to obtain the same
therapeutic
effect. Given that only a small fraction of the drug delivered intra-
articulaxly is absorbed
by the local synovial tissue, the difference in plasma drug levels between the
two routes
of administration is much greater than the difference in total amitriptyline
dosing levels.
Practice of the present invention should be distinguished from conventional
intra-
articular injections of opiates and/or local anesthetics at the completion of
arthroscopic or
"open" joint (e.g., knee, shoulder, etc.) procedures. The solution of the
present invention
is used for continuous infusion throughout the surgical procedure to provide
preemptive
inhibition of pain and inflammation. In contrast, the high concentrations
necessary to
achieve therapeutic efficacy with a constant infusion of local anesthetics,
such as
lidocaine (0.5-2% solutions), would result in profound systemic toxicity.
Upon completion of the procedure of the present invention, it may be desirable
to
inject or otherwise apply a higher concentration of the same pain acid
inflammation
inhibitors as used in the irrigation solution at the operative site, as an
alternative or
supplement to opiates.
The solution of the present invention also has application in cardiovascular
and
general vascular diagnostic and therapeutic procedures to potentially decrease
vessel wall
spasm, platelet aggregation, vascular smooth muscle cell proliferation and
nociceptor
activation produced by vessel manipulation. Reference herein to arterial
treatment is
intended to encompass the treatment of venous grafts harvested and placed in
the arterial
system. A suitable solution for such techniques is disclosed in Example II
herein below.
The cardiovascular and general vascular solution preferably includes any
combination,
and preferably all, of the following: a 5-HT2 receptor antagonist (Saxena, P.
R., et al.,
"Cardiovascular Effects of Serotonin Inhibitory Agonists and Antagonists,"
J. Ca~diovasc. Pharmacol. IS(Suppl. 7):517-S34 (1990); Douglas, 1985); a 5-HT3
receptor antagonist to block activation of these receptors on sympathetic
neurons and
C-fiber nociceptive neurons in the vessel walls, which has been shown to
produce brady-
and tachycardia (Saxena et al. 1990); a bradykininl receptor antagonist; and a
cyclooxygenase inhibitor to prevent production of prostaglandins at tissue
injury sites and
thereby decreasing pain and inflammation. In addition, the cardiovascular and
general
vascular solution also preferably will contain a serotoninlB (also known as
serotoninl~a)
antagonist because serotonin has been shown to produce significant vascular
spasm via
activation of the serotoninlg receptors in humans. Kaumann, A.J., et al.,
"Variable
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Participation of 5-HT1-Like Receptors and 5-HT2 Receptors in Serotonin-Induced
Contraction of Human Isolated Coronary Arteries," Ci~culatioh 90:1141-53
(1994). This
excitatory action of serotoninlg receptors in vessel walls, resulting in
vasoconstriction, is
in contrast to the previously-discussed inhibitory action of serotoninlg
receptors in
neurons. The cardiovascular and general vascular solution of the present
invention also
may suitably include one or more of the anti-restenosis agents disclosed
herein that
reduce the incidence and severity of post-procedural restenosis resulting
from, for
example, angioplasty or rotational atherectomy.
The solution of the present invention also has utility for reducing pain and
inflammation associated with urologic procedures, such as trans-urethra)
prostate
resection and similar urologic procedures. References herein to application of
solution to
the urinary tract or to the urological structures is intended to include
application to the
urinary tract per se, bladder and prostate and associated structures. Studies
have
demonstrated that serotonin, histamine and bradykinin produce inflammation in
lower
urinary tract tissues. Schwartz, M.M., et al., "Vascular Leakage in the Kidney
and Lower
Urinary Tract: Effects of Histamine, Serotonin and Bradylcinin," P~oc. Soc.
Exp. Biol.
Med. 140:535-539 (1972). A suitable irrigation solution for urologic
procedures is
disclosed in Example III herein below. The solution preferably includes a
combination,
and preferably all, of the following: a histamine) receptor antagonist to
inhibit histamine-
induced pain and inflammation; a 5-HT3 receptor antagonist to block activation
of these
receptors on peripheral C-fiber nociceptive neurons; a bradykininl antagonist;
a
bradykinin~ antagonist; and a cyclooxygenase inhibitor to decrease
pain/inflammation
produced by prostaglandins at the tissue injury sites. Preferably an anti-
spasm agent is
also included to prevent spasm in the urethra) canal and bladder wall.
Some of the solutions of the present invention may suitably also include a
gelling
agent to produce a dilute gel. This gellable solution may be applied, for
example, within
the urinary tract or an arterial vessel to deliver a continuous, dilute local
predetermined
concentration of agents.
The solution of the present invention may also be employed perioperatively for
the inhibition of pain and inflammation in surgical wounds, as well as to
reduce pain and
inflammation associated with burns. Burns result in the release of a
significant quantity
of biogenic amines, which not only produce pain and inflammation, but also
result in
profound plasma extravasation (fluid loss), often a life-threatening component
of severe
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burns. Holliman, C.J., et al., "The Effect of Ketanserin, a Specific Serotonin
Antagonist,
on Burn Shock Hemodynamic Parameters in a Porcine Burn Model," J. Ti~autna
23:867-
871 (1983). The solution disclosed in Example I for arthroscopy may also be
suitably
applied to a wound or burn for pain and inflammation control, and for surgical
procedures
such as arthroscopy. The agents of the solution of Example I may alternately
be included
in a paste or salve base, for application to the burn or wound.
VI. EXAMPLES
The following are several formulations in accordance with the present
invention
suitable for certain operative procedures followed by a summary of three
clinical studies
utilizing the agents of the present invention.
A. EXAMPLE I
IRRIGATION SOLUTION FOR ARTHROSCOPY
The following composition is suitable for use in anatomic joint irrigation
during
arthroscopic procedures. Each drug is solubilized in a carrier fluid
containing
physiologic electrolytes, such as normal saline or lactated Ringer's solution,
as are the
remaining solutions described in subsequent examples.
Concentration
(Nanomolar): Most
Class of Agent Drug TherapeuticPreferredPreferred
serotonin2 antagonistamitriptyline0.1-1,000 50-500 100
serotonin3 antagonistmetoclopramide10-10,000 200-2,0001,000
histamine) antagonistamitriptyline0.1-1,000 50-500 200
serotoninlA,1B,1D,1Fsumatriptan 1-1,000 10-200 50
agonist
bradykininl antagonist[des-Argl~] 1-1,000 50-500 200
derivative
of
HOE 140
bradylcinin2 antagonist HOE 140 1-1,000 50-500 200
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B. EXAMPLE II
IRRIGATION SOLUTION FOR CARDIOVASCULAR AND
GENERAL VASCULAR THERAPEUTIC AND DIAGNOSTIC
PROCEDURES
The following drugs and concentration ranges in solution in a physiologic
carrier
fluid are suitable for use in irrigating operative sites during cardiovascular
and general
vascular procedures.
Concentration
Class of Agent Drug (Nanomolar): Most
TherapeuticPreferredPreferred
serotonin~ antagonist trazodone 0.1-2,000 50-500 200
serotonin3 antagonist metoclopramide10-10,000 200-2,0001,000
serotoninlB antagonist yohimbine 0.1-1,000 50-500 200
bradykininl antagonist [des-Argl~]1-1,000 50-500 200
derivative
of
HOE 140
cyclooxygenase inhibitorketorolac 100-10,000 500-5,0003,000
C. EXAMPLE III
IRRIGATION SOLUTION FOR UROLOGIC PROCEDURES
The following drugs and concentration ranges in solution in a physiologic
carrier
fluid axe suitable for use in irrigating operative sites during urologic
procedures.
Concentration
Class of Agent Drug (Nanomolar):PreferredMost
Therapeutic Preferred
histamine) antagonistterfenadine 0.1-1,000 50-500 200
serotonin3 antagonistmetoclopramide10-10,000 200-2,0001,000
bradykininl antagonist[des-Argl~] 1-1,000 50-500 200
derivative
of
HOE 140
bradylcinin~ antagonistHOE 140 1-1,000 50-500 200
cyclooxygenase 100-10,000 500-5,0003,000
inhibitor
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D. EXAMPLE IV
IRRIGATION SOLUTION FOR ARTHROSCOPY, BURNS, GENERAL
SURGICAL WOUNDS AND ORAL/DENTAL APPLICATIONS
The following composition is preferred for use in anatomic irrigation during
arthroscopic and oral/dental procedures and the management of burns and
general
surgical wounds. While the solution set forth in Example I is suitable for use
with the
present invention, the following solution is even more preferred because of
expected
higher efficacy.
Concentration
Class of Agent Drug (Nanomolar): Most
TherapeuticPreferred Preferred
serotonin2 antagonistamitriptyline0.1 - 1,00050 - 500 200
serotonin3 antagonistmetoclopramide10 - 10,000200 - 2,0001,000
histamine) antagonistamitriptyline0.1 - 1,00050 - 500 200
serotonin lA, sumatriptan 1 - 1,000 10 - 200 100
ls,1D,
iF agonist
cyclooxygenase ketorolac 100 - 10,000500 - 5,0003,000
inhibitor
neurolcininl GR 82334 1 - 1,000 10 - 500 200
antagonist
neurokinin2 antagonist() SR 48968 1 - 1,000 10 - 500 200
purine2X antagonistPPADS 100 - 100,00010,000- 50,000
100,000
ATP-sensitive (-) pinacidil1 - 10,000 100 - 1,000500
K+
channel agonist
Ca2+ channel nifedipine 1 - 10,000 100 - 5,0001,000
antagonist
lcallilcrein aprotinin 0.1 - 1,00050 - 500 200
inhibitor
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E. EXAMPLE V
ALTERNATE IRRIGATION SOLUTION FOR CARDIOVASCULAR
AND GENERAL VASCULAR THERAPEUTIC AND DIAGNOSTIC
PROCEDURES
The following drugs and concentration ranges in solution in a physiologic
carrier
fluid are preferred for use in irrigating operative sites during
cardiovascular and general
vascular procedures. Again, this solution is preferred relative to the
solution set forth in
Example II above for higher efficacy.
Concentration
(Nanomolar) Most
:
Class of Agent Drug Therapeutic Preferred Preferred
serotonin2 trazodone 0.1 - 2,000 50 - 500 200
antagonist
cyclooxygenase ketorolac 100 - 10,000500 - 5,0003,000
inhibitor
endothelin BQ 123 0.01 - 1,00010 - 1,000 500
antagonist
ATP-sensitive (-) pinacidil1 - 10,000 100 - 1,000500
K+
channel agonist
Ca2+ channel nisoldipine1 - 10,000 100 - 1,000500
antagonist
nitric oxide donor SIN-1 10 - 10,000 100 - 1,000 500
F. EXAMPLE VI
ALTERNATE IRRIGATION SOLUTION FOR UROLOGIC
PROCEDURES
The following drugs and concentration ranges in solution in a physiologic
carrier
fluid are preferred for use in irrigating operative sites during urologic
procedures. The
solution is believed to have even higher efficacy than the solution set forth
in prior
Example III.
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Concentration
(Nanomolar): Most
Class of Agent Drug Therapeutic Preferred Preferred
Serotonin~ LY 53857 0.1 - 500 1 - 100 50
antagonist
Histamine) terfenadine 0.1 - 1,000 50 - 500 200
antagonist
cyclooxygenaseketorolac 100 - 10,000500 - 5,0003,000
inhibitor
neurokinin2 SR 48968 1 - 1,000 10 - 500 200
antagonist
purine2X antagonistppADS 100 - 100,00010,000-100,50,000
000
ATP-sensitive (-) pinacidil1 - 10,000 100 - 1,000500
K+
channel agonist
Ca2+ channel nifedipine1 - 10,000 100 - 5,0001,000
antagonist
kallikrein aprotinin 0.1 - 1,000 50 - 500 200
inhibitor
nitric oxide SIN-1 10 - 10,000 100 - 1,000500
donor
G. EXAMPLE VII
CARDIOVASCULAR AND GENERAL VASCULAR
ANTI-RESTENOSIS IRRIGATION SOLUTION
The following drugs and concentration ranges in solution in a physiologic
carrier
fluid are preferred for use in irrigation during cardiovascular and general
vascular
therapeutic and diagnostic procedures. The drugs in this preferred solution
may also be
added at the same concentration to the cardiovascular and general vascular
irrigation
solutions of Examples II and V described above or Example VIII described below
for
preferred anti-spasmodic, anti-restenosis, anti-pain/anti-inflammation
solutions.
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Concentration
(Nanomolar): Most
Class of Drug Therapeutic Preferred Preferred
Agent
thrombin hirulog 0.2-20,000 2-2,000 200
inhibitor
glycoproteinintegrelin 0.1-10,000 1-1000 x 100 x Kd
x Kd Kd
IIb/IIIa
receptor
bloclcer
PKC inhibitorGF 109203X* 0.1-10,000 1-1,000 200
protein tyrosinetyrphostin 10-100,000 100-20,000 10,000
lcinase inhibitorAG1296
* Also known as Go 6850 or Bisindoylmaleimide I (available from Warner-
Lambert)
H. EXAMPLE VIII
ALTERNATE IRRIGATION SOLUTION FOR CARDIOVASCULAR
AND GENERAL VASCULAR THERAPEUTIC AND DIAGNOSTIC
PROCEDURES
An additional preferred solution for use in cardiovascular and general
vascular
therapeutic and diagnostic procedures is formulated the same as the previously
described
formulation of Example V, except that the nitric oxide (NO donor) SIN-1 is
replaced by a
combination of two agents, FK 409 (NOR-3) and FR 144420 (NOR-4), at the
concentrations set forth below:
Conceht~atiou
(Nanomola~): ~ Most
Class ofAgent Drug Therapeutic Pt°efer~ed P~efe~t~ed
NO donor FK 409 (NOR-3) 1-1,000 10-500 250
NO donor FR 144420 (NOR-4) 10-10,000 100-5,000 1,000
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I. EXAMPLE IX
ALTERNATE IRRIGATION SOLUTION FOR ARTHROSCOPY,
GENERAL SURGICAL WOUNDS, BURNS AND ORAL/DENTAL
APPLICATIONS
An alternate preferred solution for use in irrigation of arthroscopic, general
surgical and oral/dental applications is formulated the same as in the
previously described
Example IV, ,with the following substitution, deletion and additions at the
concentrations
set forth below:
1) amitriptyline is replaced by mepyramine as the H1 antagonist;
2) the kallikrein inhibitor, aprotinin, is deleted;
3) a bradykininl antagonist, [leu9] [des-Argl~] kalliden, is added;
4) a bradykinin2 antagonist, HOE 140, is added; and
5) a ~.-opioid agonist, fentanyl, is added.
Concentration
(Nanomolar): Most
_Class of Drug Therapeutic PreferredPreferred
Agent
H1 antagonistmepyramine 0.1-1,000 5-200 100
bradykininl [leu9][des-Argl0.1-500 10-200 100
antagonist ~] lcalliden
bradylcinin2 HOE 140 1-1,000 50-500 200
antagonist
~.-opioid fentanyl 0.1-500 10-200 100
agonist
J. EXAMPLE X
ALTERNATE IRRIGATION SOLUTION FOR UROLOGIC
PROCEDURES
An alternate preferred solution for use in irrigation during urologic
procedures is
formulated the same as in the previously described Example VI with the
following
substitution, deletion and additions at the concentrations set forth below:
1) S1N-1 is replaced as the NO donor by a combination of two agents:
a) FK 409 (NOR-3); and
b) FR 144420 (NOR-4);
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2) the kallikrein inhibitor, aprotinin, is deleted;
3) a bradykininl antagonist, [leu9] [des-Argl~] kalliden, is added; and
4) a bradykinina antagonist, HOE 140, is added.
Concentration
(Nanomolar): Most
Class of Agent Drug Therapeutic Preferred Preferred
NO donor FK 409 1-1,000 10-500 250
(NOR-3)
NO donor FR 144420 10-10,000 100-5,000 1,000
(NOR-4)
bradylcininl [leu9][des- 0.1-500 10-200 100
antagonist Argl~] kalliden
bradylcinin2 HOE 140 1-1,000 50-500 200
antagonist
K. EXAMPLE XI
BALLOON DILATATION OF NORMAL ILIAC ARTERIES IN THE
NEW ZEALAND WHITE RABBIT AND THE INFLUENCE OF
HISTAM1NE/SEROTONIN RECEPTOR BLOCKADE ON THE
RESPONSE
The purpose of this study was twofold. First, a new in vivo model for the
study of
arterial tone was employed. The time course of arterial dimension changes
before and
after balloon angioplasty is described below. Second, the role of histamine
and serotonin
together in the control of arterial tone in this setting was then studied by
the selective
infusion of histamine and serotonin receptor blocking agents into arteries
before and after
the angioplasty injury.
1. DESIGN CONSIDERATIONS
This study was intended to describe the time course of change in arterial
lumen
dimensions in one group of arteries and to evaluate the effect of
histamine/serotonin
receptor blockade on these changes in a second group of similar arteries. To
facilitate the
comparison of the two different groups, both groups were treated in an
identical manner
with the exception of the contents of an infusion performed during the
experiment. In
control animals (arteries), the infusion was normal saline (the vehicle for
test solution).
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The histamine/serotonin receptor blockade treated arteries received saline
containing the
receptor antagonists at the same rate and at the same part of the protocol as
control
animals. Specifically, the test solution included: (a) the serotonin3
antagonist
metoclopramide at a concentration of 16.0 ~M; (b) the serotonin2 antagonist
trazodone at
a concentration of 1.6 ~.M; and (c) the histamine antagonist promethazine at
concentrations of 1.0 pM, all in normal saline. Drug concentrations within the
test
solution were 16-fold greater than the drug concentrations delivered at the
operative site
due to a 16 to 1 flow rate ratio between the iliac artery (80 cc per minute)
and the solution
delivery catheter (5 cc per minute). This study was performed in a
prospective,
randomized and blinded manner. Assignment to the specific groups was random
and
investigators were blinded to infusion solution contents (saline alone or
saline containing
the histamine/serotonin receptor antagonists) until the completion of the
angiographic
analysis.
2. ANIMAL PROTOCOL
This protocol was approved by the Seattle Veteran Affairs Medical Center
Committee on Animal Use and the facility is fully accredited by the American
Association for Accreditation of Laboratory Animal Care. The iliac arteries of
3-4 kg
male New Zealand white rabbits fed a regular rabbit chow were studied. The
animals
were sedated using intravenous xylazine (5 mg/kg) and ketamine (35 mg/kg)
dosed to
effect and a cutdown was performed in the ventral midline of the neck to
isolate a carotid
artery. The artery was ligated distally, an arteriotomy performed and a 5
French sheath
was introduced into the descending aorta. Baseline blood pressure and heart
rate were
recorded and then an angiogram of the distal aorta and bilateral iliac
arteries was recorded
on 35 mm cine film (frame rate 15 per second) using hand injection of
iopamidol 76%
(Squibb Diagnostics, Princeton, NJ) into the descending aorta. For each
angiogram, a
calibration object was placed in the radiographic field of view to allow for
correction for
magnification when diameter measurements were made. A 2.5 French infusion
catheter
(Advanced Cardiovascular Systems, Santa Clara, CA) was placed through the
carotid
sheath and positioned 1-2 cm above the aortic bifurcation. Infusion of the
test
solution -- either saline alone or saline containing the histamine/serotonin
receptor
antagonists -- was started at a rate of 5 cc per minute and continued for 15
minutes. At
5 minutes into the infusion, a second angiogram was performed using the
previously
described technique then a 2.5 mm balloon angioplasty catheter (the Lightning,
Cordis
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Corp., Miami, FL) was rapidly advanced under fluoroscopic guidance into the
left and
then the right iliac arteries. In each iliac the balloon catheter was
carefully positioned
between the proximal and distal deep femoral branches using bony landmarks and
the
balloon was inflated for 30 seconds to 12 ATM of pressure. The balloon
catheter was
inflated using a dilute solution of the radiographic contrast agent so that
the inflated
balloon diameter could be recorded on cine film. The angioplasty catheter was
rapidly
removed and another angiogram was recorded on cine film at a mean of 8 minutes
after
the infusion was begun. The infusion was continued until the 15 minute time
point and
another angiogram (the fourth) was performed. Then the infusion was stopped (a
total of
75 cc of solution had been infused) and the infusion catheter was removed. At
the
30 minute time point (15 minutes after the infusion was stopped), a final
angiogram was
recorded as before. Blood pressure and heart rate were recorded at the 15 and
30 minute
time points immediately before the angiograms. After the final angiogram, the
animal
was euthanized with an overdose of the anesthetic agents administered
intravenously and
the iliac arteries were retrieved and immersion fixed in formation for
histologic analysis.
3. ANGIOGRAPHIC ANALYSIS
The angiograms were recorded on 35 mm cine film at a frame rate of 15 per
second. For analysis, the angiograms were projected from a Vanguard projector
at a
distance of 5.5 feet. Iliac artery diameters at prespecified locations
relative to the balloon
angioplasty site were recorded based on hand held caliper measurement after
correction
for magnification by measurement of the calibration object. Measurements were
made at
baseline (before test solution infusion was begun), 5 minutes into the
infusion,
immediately post balloon angioplasty (a mean of 8 minutes after the test
solution was
begun), at 15 minutes (just before the infusion was stopped) and at 30 minutes
(15 minutes after the infusion was stopped). Diameter measurements were made
at three
sites in each iliac artery: proximal to the site of balloon dilatation, at the
site of balloon
dilatation and just distal to the site of balloon dilatation.
The diameter measurements were then converted to area measurements by the
formula:
Area = (Pi)(Diameter2)/4.
For calculation of vasoconstriction, baseline values were used to
represent the maximum area of the artery and percent
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vasoconstriction was calculated as: % Vasoconstriction =
f (Baseline area - Later time point area)Baseline area} x100.
4. STATISTICAL METHODS
All values are expressed as mean ~ 1 standard error of the mean. The time
course
of vasomotor response in control arteries was assessed using one way analysis
of variance
with correction for repeated measures. Post hoc comparison of data between
specific
time points was performed using the Scheffe test. Once the time points at
which
significant vasoconstriction occurred had been determined in control arteries,
the control
and histamine/serotonin receptor antagonist treated arteries were compared at
those time
points where significant vasoconstriction occurred in control arteries using
multiple
analysis of variance with treatment group identified as an independent
variable. To
compensate for the absence of a single a priori stated hypothesis, a p value
<0.01 was
considered significant. Statistics were performed using Statistica for
Windows,
version 4.5 (Statsoft, Tulsa, OIL).
5. RESULTS
The time course of arterial dimension changes before and after balloon
angioplasty in normal arteries receiving saline infusion was evaluated in 16
arteries from
8 animals (Table 30). Three segments of each artery were studied: the proximal
segment
immediately upstream from the balloon dilated segment, the balloon dilated
segment and
the distal segment immediately downstream from the balloon dilated segment.
The
proximal and distal segments demonstrated similar patterns of change in
arterial
dimensions: in each, there was significant change in arterial diameter when
all time
points were compared (proximal segment, p=0.0002 and distal segment, p<0.001,
ANOVA). Post hoc testing indicated that the diameters at the immediate post
angioplasty
time point were significantly less than the diameters at baseline or at the 30
minute time
point in each of these segments. On the other hand, the arterial diameters in
each
segment at the 5 minute, 15 minute and 30 minute time points were similar to
the baseline
diameters. The balloon dilated segment showed lesser changes in arterial
dimension than
the proximal and distal segments. The baseline diameter of this segment was
1.820.05 mm; the nominal inflated diameter of the balloon used for angioplasty
was
2.5 mm and the actual measured inflated diameter of the balloon was 2.20~0.03
mm
(p<0.0001 vs. baseline diameter of the balloon treated segment). Thus, the
inflated
balloon caused circumferential stretch of the balloon dilated segment, but
there was only
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slight increase in lumen diameter from baseline to the 30 minute time point
(1.820.05 mm to 1.940.07 mm, p--NS by post hoc testing).
TABLE 30
ANGIOGRAPHICALLY DETERMINED LUMEN DIAMETERS
AT THE SPECIFIED TIMES BEFORE AND AFTER
BALLOON DILATATION OF NORMAL ILIAC ARTERIES
Immediate
Segment Baseline 5 Minute Post PTA 15 Minute 30 Minute
Proximall 2.18~0.7 2.03~0.7 1.81~0.08* 2.00~.08 2.23~.08
Balloon2 1.82~.OS 1.77~.03 1.79~.OS 1.70~.04 1.94~.07
Distal3 1.76~.04 1.68~.04** 1.43~.04* 1.54~.03 1.69~.06
All measurements in mm. Means~SEM. PTA = percutaneous transluminal
angioplasty.
1 p=0.0002 (ANOVA within group comparison),
2 p=0.03 (ANOVA within group comparison),
3 p<0.0001 (ANOVA within group comparison). N=16 at all time points.
* p<0.01 versus baseline and 30 minute diameter measurements (Scheffe test for
post hoc
comparisons).
** p<0.01 versus immediate post PTA measurements (Scheffe test for post hoc
comparisons). All other post hoc comparisons were not significant using the
p<0.01
threshold.
Arterial lumen diameters were used to calculate lumen area then the area
measurements were used to calculate percent vasoconstriction by comparison of
the
5 minute, immediate post angioplasty, 15 and 30 minute data to the baseline
measurements. The proximal and distal segment data expressed as percent
vasoconstriction are shown in FIGURE 9; the changes in the amount of
vasoconstriction
over time axe significant (in the proximal segment, p=0.0008; in the distal
segment,
p=0.0001, ANOVA). Post hoc testing identifies the vasoconstriction at the
immediate
post angioplasty time point as significantly different from that present at
the 30 minute
time point (P<0.001 in both segments). In the distal segment, the immediate
post
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angioplasty vasoconstriction was also significantly less than that at 5
minutes (p<0.01);
no other differences in intra-time point comparisons were significant by post
hoc testing.
The luminal changes in control arteries can be summarized as follows:
1) Vasoconstriction with loss of approximately 30% of baseline luminal area
occurs in
the segments of artery proximal and distal to the balloon dilated segment
immediately
after balloon dilatation. There are trends to smaller amounts of
vasoconstriction in the
proximal and distal segments before dilatation and at the 15 minute time point
(approximately 7 minutes after dilatation) also but, by the 30 minute time
point
(approximately 22 minutes after dilatation), a trend towards vasodilatation
has replaced
the previous vasoconstriction; 2) In the balloon dilated segment, only minor
changes in
lumen dimensions are present, and, despite the use of a balloon with a
significantly larger
inflated diameter than was present in this segment at baseline, there was no
significant
increase in lumen diameter of the dilated segment. These findings lead to a
conclusion
that any effects of the putative histamine/serotonin treatment would only be
detectable in
the proximal and distal segments at the time points where vasoconstriction was
present.
The histamine/serotonin receptor blockade solution was infused into 16
arteries
(~ animals); angiographic data was available at all time points in 12
arteries. Heart rate
and systolic blood pressure measurements were available in a subset of animals
(Table 31). There were no differences in heart rate or systolic blood pressure
when the
two animal groups were compared within specific time points.
Histamine/serotonin
treated animals showed trends toward a decrease in the systolic blood pressure
from
baseline to 30 minutes (-145 mm Hg, p=0.04) and a lower heart rate (-2610,
p=0.05).
Within the control animals, there was no change in heart rate or systolic
blood pressure
over the duration of the experiment.
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TABLE 31
SYSTOLIC BLOOD PRESSURE
AND HEART RATE
MEASUREMENTS 1N
CONTROL AND HISTAM1NE/SEROTON1N
TREATED ANIMALS
Group Baseline 5 Minute 15 Minute30 Minute
Systolic Blood Pressure
Control 834 (8) 844 (8) 826 (8) 804 (8)
Histamine/Serotonin935 (6) 879 (4) 829 (6) 808 (6)*
Heart Rate
Control 22118 (5) 23418 (4) 21723 22722 (5)
(5)
Histamine/Serotonin2328 (5) 2328 (5) 20914 20612 (5)**
(5)
Systolic blood pressure in mm Hg and heart rate in beats per minute. Mean~SEM.
* p=0.04 for decrease in systolic blood pressure from baseline to 30 minutes
and
**p=0.05 for decrease in heart rate from baseline to 30 minutes within the
histamine/serotonin treated animals.
The proximal acid distal segments of histamine/serotonin treated arteries were
compared to control arteries using the percent vasoconstriction measurement.
FIGURE l0A shows the effects of the histamine/serotonin infusion on proximal
segment
vasoconstriction relative to the vasoconstriction present in the control
arteries. When the
findings in the two treatment groups were compared at the baseline, immediate
post
angioplasty and 15 minute time points, histamine/serotonin infusion resulted
in
significantly less vasoconstriction compared to the control saline infusion
(p=0.003,
2-way ANOVA). Comparison of the two treatment groups in the distal segment is
illustrated in FIGURE lOB. Despite observed differences in mean diameter
measurements in the distal segment, solution treated vessels exhibited less
vasoconstriction than saline treated control vessels at baseline, immediate
post-
angioplasty and 15 minute time points, this pattern did not achieve
statistical significance
(p=0.32, 2-way ANOVA). Laclc of statistical significance may be attributed to
smaller
than expected vasoconstriction values in the control vessels.
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L. EXAMPLE XII
AMITRIPTYLINE INHIBITION OF 5-HYDROXYTRYPTAM1NE-
INDUCED KNEE JOINT PLASMA EXTRAVASATION -
COMPARISON OF 1NTRA-ARTICULAR VERSUS INTRAVENOUS
ROUTES OF ADMINISTRATION
The following study was undertaken in order to compare two routes of
administration of the 5-HT2 receptor antagonist, amitriptyline: 1) continuous
intra-
articular infusion; versus 2) intravenous injection, in a rat knee synovial
model of
inflammation. The ability of amitriptyline to inhibit 5-HT-induced joint
plasma
extravasation by comparing both the efficacy and total drug dose of
amitriptyline
delivered via each route was determined.
1. ANIMALS
Approval from the Institutional Animal Care Committee at the University of
California, San Francisco was obtained for these studies. Male Sprague-Dawley
rats
(Bantin and Kingman, Fremont, CA) weighing 300-450 g were used in these
studies.
Rats were housed under controlled lighting conditions (lights on 6 A.M. to 6
P.M.), with
food and water available ad libitum.
2. PLASMA EXTRAVASATION
Rats were anesthetized with sodium pentobarbital (65 mg/kg) and then given a
tail
vein injection of Evans Blue dye (50 mg/kg in a volume of 2.5 ml/kg), which is
used as a
marker for plasma protein extravasation. The knee joint capsule was exposed by
excising
the overlying skin, and a 30-gauge needle was inserted into the joint and used
for the
infusion of fluid. The infusion rate (250 ~1/min) was controlled by a Sage
Instruments
Syringe pump (Model 341B, Orion Research Inc., Boston, MA). A 25-gauge needle
was
also inserted into the joint space and perfusate fluid was extracted at 250
~,1/min,
controlled by a Sage Instruments Syringe pump (Model 351).
The rats were randomly assigned to three groups: 1) those receiving only intra-
articular (IA) 5-HT (1 ~.M), 2) those receiving amitriptyline intravenously
(IV) (doses
ranging from 0.01 to 1.0 mg/kg) followed by IA 5-HT (1 mM), and 3) those
receiving
amitriptyline intra-articularly (IA) (concentrations ranging from 1 to 100 ~M)
followed
by IA 5-HT (1 ~,M) plus IA amitriptyline. In all groups, baseline plasma
extravasation
levels were obtained at the beginning of each experiment by perfusing 0.9%
saline
intra-articularly and collecting three perfusate samples over a 15 min period
(one every
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min). The first group was then administered 5-HT IA for a total of 25 min.
Perfusate
samples were collected every 5 min for a total of 25 min. Samples were then
analyzed
for Evans Blue dye concentration by spectrophotometric measurement of
absorbance at
620 nm, which is linearly related to its concentration (Carr and Wilhelm,
1964). The
5 IV amitriptyline group was administered the drug during the tail vein
injection of the
Evans Blue dye. The knee joints were then perfused for 15 min with saline
(baseline),
followed by 25 min perfusion with 5-HT (1 ~M). Perfusate samples were
collected every
5 min for a total of 25 min. Samples were then analyzed using
spectrophotometry. In the
IA amitriptyline group, amitriptyline was perfused infra-articularly for 10
min after the
15 min saline perfusion, then amitriptyline was perfused in combination with 5-
HT for an
additional 25 min. Perfusate samples were collected every 5 min and analyzed
as above.
Some rat knees were excluded from the study due to physical damage of knee
joint or inflow and outflow mismatch (detectable by presence of blood in
perfusate and
high baseline plasma extravasation levels or knee joint swelling due to
improper needle
placement).
A. 5-HT-INDUCED PLASMA EXTRAVASATION
Baseline plasma extravasation was measured in all knee joints tested (total
n=22).
Baseline plasma extravasation levels were low, averaging 0.022 ~ 0.003
absorbance units
at 620 nm (average ~ standard error of the mean). This baseline extravasation
level is
shown in FIGURES 11 and 12 as a dashed line.
5-HT (1 ~.M) perfused into the rat knee joint produces a time-dependent
increase
in plasma extravasation above baseline levels. During the 25 min perfusion of
5-HT
infra-articularly, maximum levels of plasma extravasation were achieved by 15
min and
continued until the perfusion was terminated at 25 min (data not shown).
Therefore,
5-HT-induced plasma extravasation levels reported are the average of the 15,
20 and 25
min time points during each experiment. 5-HT-induced plasma extravasation
averaged
0.192 ~ 0.011, approximately an 8-fold stimulation above baseline. This data
is graphed
in FIGURES 11 and 12, corresponding to the "0" dose of IV amitriptyline and
the "0"
concentration of IA amitriptyline, respectively.
B. EFFECT OF INTRAVENOUS AMITRIPTYLINE ON
5-HT-INDUCED PLASMA EXTRAVASATION
Amitriptyline administered via tail vein injection produced a dose-dependent
decrease in 5-HT-induced plasma extravasation as shown in FIGURE 11. The ICSO
for
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IV amitriptyline inhibition of 5-HT-induced plasma extravasation is
approximately
0.025 mg/kg. 5-HT-induced plasma extravasation is completely inhibited by an
IV amitriptyline dose of 1 mg/kg, the plasma extravasation averaging 0.034 ~
0.010.
C. EFFECT OF INTRA-ARTICULAR AMITRIPTYLINE
ON 5-HT-INDUCED PLASMA EXTRAVASATION
Amitriptyline administered alone in increasing concentrations intra-
articularly did
not affect plasma extravasation levels relative to baseline, with the plasma
extravasation
averaging 0.018 ~ 0.002 (data not shown). Amitriptyline co-perfused in
increasing
concentrations with 5-HT produced a concentration-dependent decrease in 5-HT-
induced
plasma extravasation as shown in FIGURE 12. 5-HT-induced plasma extravasation
in
the presence of 3 ~.M IA amitriptyline was not significantly different from
that produced
by 5-HT alone, however, 30 ~M amitriptyline co-perfused with 5-HT produced a
greater
than 50% inhibition, while 100 ~M amitriptyline produced complete inhibition
of
5-HT-induced plasma extravasation. The ICSO for IA amitriptyline inhibition of
5-HT-
induced plasma extravasation is approximately 20 ~,M.
The major finding of the present study is that 5-HT (1 ~,M) perfused intra-
articularly in the rat knee joint produces a stimulation of plasma
extravasation that is
approximately 8-fold above baseline levels and that either intravenous or
intra-articular
administration of the 5-HT2 receptor antagonist, amitriptyline, can inhibit 5-
HT-induced
plasma extravasation. The total dosage of administered amitriptyline, however,
differs
dramatically between the two methods of drug delivery. The ICSO for IV
amitriptyline
inhibition of 5-HT-induced plasma extravasation is 0.025 mg/kg, or 7.5 x 10-3
mg in a
300 g adult rat. The ICSO for IA amitriptyline inhibition of 5-HT-induced
plasma
extravasation is approximately 20 ~M. Since 1 ml of this solution was
delivered every
five minutes for a total of 35 min during the experiment, the total dosage
perfused into
the lcnee was 7 ml, for a total dosage of 4.4 x 10'5 mg perfused into the
knee. This IA
amitriptyline dose is approximately 200-fold less than the IV amitriptyline
dose.
Furthermore, it is likely that only a small fraction of the IA perfused drug
is systemically
absorbed, resulting in an even greater difference in the total delivered dose
of drug.
Since 5-HT may play an important role in surgical pain and inflammation, as
discussed earlier, 5-HT antagonists such as amitriptyline may be beneficial if
used during
the peri-operative period. A recent study attempted to determine the effects
of oral
amitriptyline on post-operative orthopedic pain (Derrick et al., 1993). An
oral dose as
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low as 50 mg produced undesirable central nervous system side-effects, such as
a
"decreased feeling of well-being". Their study, in addition, also showed that
oral
amitriptyline produced higher pain scale scores than placebo (P<0.05) in the
post-
operative patients. Whether this was due to the overall unpleasantness
produced by oral
amitriptyline is not known. In contrast, an intra-articular route of
administration allows
an extremely low concentration of drug to be delivered locally to the site of
inflammation, possibly resulting in maximal benefit with minimal side-effects.
M. EXAMPLE XIII
EFFECTS OF CARDIOVASCULAR AND GENERAL VASCULAR
SOLUTION ON ROTATIONAL ATHERECTOMY-INDUCED
VASOSPASM 1N RABBIT ARTERIES
1. SOLUTION TESTED
This study utilized an irrigation solution consisting of the agents set forth
in
Example V above, with the following exceptions. Nitroprusside replaced SIN-1
as the
nitric oxide donor and nicardipine replaced nisoldipine as the Ca2+ channel
antagonist.
The concentration of nitroprusside was selected based on its previously-
defined
pharmacological activity (ECSp). The concentrations of the other agents in
this test
solution were determined based on the binding constants of the agents with
their cognate
receptors. Furthermore, all concentrations were adjusted based on a blood flow
rate of
80 cc per minute in the distal aorta of the rabbit and a flow rate of 5 cc per
minute in the
solution delivery catheter. Three components were mixed in one cc or less
DMSO, and
then these components and the remaining three components were mixed to their
final
concentrations in normal saline. A control solution consisting of normal
saline was
utilized. The test solution or the control solution was infused at a rate of 5
cc per minute
for 20 minutes. A brief pause in the infusion was necessary at the times blood
pressure
measurements were made, so each animal received about 95 cc of the solution in
the
20 minute treatment period.
2. ANIMAL PROTOCOL
This protocol was approved by the Seattle Veteran Affairs Medical Center
Committee on Animal Use, which is accredited by the American .Association for
Accreditation of Laboratory Animal Care. The iliac arteries of 3-4 lcg male
New Zealand
white rabbits fed a 2% cholesterol rabbit chow for 3-4 weeks were studied. The
animals
were sedated using intravenous xylazine (5 mg/kg) and ketamine (35 mglkg)
dosed to
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effect and a cutdown was performed in the ventral midline of the neck to
isolate a carotid
artery. The artery was ligated distally, an arteriotomy performed and a 5
French sheath
was introduced into the descending aorta and positioned at the level of the
renal arteries.
Baseline blood pressure and heart rate were recorded. An angiogram of the
distal aorta
and bilateral iliac arteries was recorded on 35 mm cine film (frame rate 15
per second)
using hand injection of iopamido176% (Squibb Diagnostics, Princeton, NJ) into
the
descending aorta.
For each angiogram, a calibration object was placed in the radiographic field
of
view to allow for correction for magnification when diameter measurements were
made.
Infusion of either the above described test solution or a saline control
solution was started
through the side arm of the 5 French sheath (and delivered to the distal
aorta) at a rate of
5 cc per minute and continued for 20 minutes. At 5 minutes into the infusion,
a second
angiogram was performed using the previously described technique. Then a 1.25
mm or
a 1.50 mm rotational atherectomy burr (Heart Technology/Boston Scientific
Inc.) was
advanced to the iliac arteries. The rotational.atherectomy burr was advanced
three times
over a guide wire in each of the iliac arteries at a rotation rate of 150,000
to 200,000
RPM. In each iliac, the rotational atherectomy burr was advanced from the
distal aorta to
the mid portion of the iliac artery between the first and second deep femoral
branches.
The rotational atherectomy burr was rapidly removed and another angiogram was
recorded on cine film at a mean of 8 minutes after the infusion was begun.
The infusion was continued until the 20 minute time point, and another
angiogram
(the fourth) was performed. Then the infusion was stopped. A total of about 95
cc of the
control or test solution had been infused. At the 30 minute time point (15
minutes after
the infusion was stopped), a final angiogram was recorded as before. Blood
pressure and
heart rate were recorded at the 15 and 30 minute time points immediately
before the
angiograms. After the final angiogram, the animal was euthanized with an
overdose of
the anesthetic agents administered intravenously.
3. ANGIOGR.APHIC ANALYSIS
The angiograms were recorded on 35 mm cine film at a frame rate of 15 per
second. Angiograms were reviewed in random order without knowledge of
treatment
assignment. For analysis, the angiograms were projected from a Vanguaxd
projector at a
distance of 5.5 feet. The entire angiogram for each animal was reviewed to
identify the
anatomy of the iliac arteries and to identify the sites of greatest spasm in
the iliac arteries.
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A map of the iliac anatomy was prepared to assist in consistently identifying
sites for
measurement. Measurements were made on the 15 minute post rotational
atherectomy
angiogram first, then in random order on the remaining angiograms from that
animal.
Measurements were made using an electronic hand-held caliper (Brown & Sharpe,
Inc.,
N. Kingston, RI). Iliac artery diameters were measured at three locations:
proximal to
the first deep femoral branch of the iliac artery; at the site of most severe
spasm (this
occurred between the first and second deep femoral artery branches in all
cases); and at a
distal site (near or distal to the origin of the second deep femoral artery
branch of the iliac
artery). Measurements were made at baseline (before test solution infusion was
begun),
5 minutes into the infusion, immediately post rotational atherectomy (a mean
of
8 minutes after the test solution was begun), at 20 minutes just after the
infusion was
stopped (this was 15 minutes after the rotational atherectomy was begun) and
at
minutes after the infusion was stopped (30 minutes after the rotational
atherectomy
was begun). The calibration object was measured in each angiogram.
15 The diameter measurements were then converted to area measurements by the
formula:
Area = (Pi)(Diameter~)/4.
For calculation of vasoconstriction, baseline values were used to represent
the
maximum area of the artery and percent vasoconstriction was calculated as:
% Vasoconstriction = f (Baseline area - Later time point area)/Baseline areas
x100.
4. STATISTICAL METHODS
All values are expressed as mean ~1 standard error of the mean. The time
course
of vasomotor response in control arteries was assessed using one way analysis
of variance
with correction for repeated measures. Post hoc comparison of data between
specific
time points was performed using the Scheffe test. Test solution treated
arteries were
compared to saline treated arteries at specified locations in the iliac
arteries and at
specified time points using multiple analysis of variance (MANOVA). To
compensate
for the absence of a single a priori hypothesis, a p value < 0.01 was
considered
significant. Statistics were performed using Statistica for Windows, version
4.5 (Statsoft,
Tulsa, OK).
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5. RESULTS
Eight arteries in 4 animals received saline solution and 13 arteries in seven
animals received test solution. In each artery, regardless of the solution
used, rotational
atherectomy was performed with the rotating burr passing from the distal aorta
to the
mid-portion of the iliac artery. Thus, the proximal iliac artery segment and
the segment
designated as the site of maximal vasoconstriction were subjected to the
rotating burr.
The guide, wire for the rotational atherectomy catheter passed through the
distal segment,
but the rotating burr of the rotational atherectomy catheter itself did not
enter the distal
segment.
Iliac artery diameters in saline treated arteries at the three specified
segments are
summarized in Table 32. In the proximal segment, there was no significant
change in the
diameter of the artery over the time course of the experiment (p=0.88, ANOVA).
In the
mid-iliac artery at the site of maximal vasoconstriction, there was a
significant reduction
in diameter with the largest reduction occurring at the 15 minute post-
rotational
atherectomy time point (p<0.0001, ANOVA comparing measurements at all 5 time
points). The distal segment diameter did not significantly change over the
time course of
the experiment (p=0.19, ANOVA comparing all time points) although there was a
trend
towards a smaller diameter at the immediate post- and 15 minute post-
rotational
atherectomy time points.
TABLE 32
ILIAC ARTERY LUMEN DIAMETERS AT SPECIFIED TIME POINTS 1
1N SALINE TREATED ARTERIES
5 Minutes Immediate 15 Minute 30 Minutes
Baseline into Infusion Post R.A after RA after RA
Segment N=8 N=8 N=8 N=8 N=8
Proximall 2.40.18 2.32.14 2.320.13 2.38.13 2.34~.07*
Mid2 2.01.08 1.84.09 1.57.15 1.24.13 1.87.06**
Distal3 2.01.10 1.86.08 1.79.08 1.81.09 1.96.06***
RA=rotational atherectomy
1 Proximal iliac artery measurement site, proximal to the first deep femoral
branch
~ Mid iliac artery at the site of maximal vasospasm
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3 Distal iliac artery measurement site, near or distal to the second deep
femoral branch
* p=0.88 by ANOVA comparing diameters in the proximal segment at the five time
points.
***p=0.000007 by ANOVA comparing diameters at site of maximal vasospasm at the
five time points.
*** p=0.19 by ANOVA comparing diameters in the distal segment at the five time
points.
The diameters of iliac arteries treated with the test solution are shown in
Table 33.
Angiograms were not recorded in three of these arteries at the 5 minute post-
initiation of
the infusion time point and angiographic data were excluded from two arteries
(one
animal) at the 30 minute post-rotational atherectomy time point because the
animal
received an air embolus at the 15 minute angiogram that resulted in
hemodynamic
instability. Because there is a variable number of observations at the five
time points, no
ANOVA statistic was applied to this data. Still it is apparent that the
magnitude of
change in the diameter measurements within segments in the test solution
treated arteries
over the time course of the experiment is less than was seen in the saline
treated arteries.
TABLE 33
ILIAC ARTERY LUMEN DIAMETERS AT SPECIFIED TIME POINTS IN TEST
SOLUTION TREATED ARTERIES
5 Minutes Immediate 15 Minute 30 Minutes
Baseline into InfusionPost RA after RA after RA
Segment N=13 N=10 N=13 N=13 N=11
Proximate2.28.06 2.07.07 2.22.05 2.42.06 2.39.08
Mid2 1.97.06 1.79.06 1.74.04 1.95.07 1.93.08
Distal3 2.00.06 1.92.04 1.90.04 ~ 2.00.06 2.01.07
RA=rotational atherectomy
1 Proximal iliac autery measurement site, proximal to the first deep femoral
branch
2 Mid iliac artery at the site of maximal vasospasm
3 Distal iliac artery measurement site, near or distal to the second deep
femoral branch
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Because of the different number of observations at the various time points,
ANOVA was
not performed to determine the statistical similarity/difference in diameters
within
specific segments.
The primary endpoint for this study was the comparison of the amounts of
vasoconstriction in saline treated and test solution treated arteries.
Vasoconstriction was
based on arterial lumen areas derived from artery diameter measurements. Area
values at
the 5 minute, immediate post-rotational atherectomy and later time points were
compared
to the baseline area values to calculate the relative change in area. The
results were
termed "vasoconstriction" if the lumen area was smaller at the later time
point than at
baseline, and "vasodilatation" if the lumen area was larger at the later time
point
compared to the baseline area (Tables 27 and 28). To facilitate statistical
analysis with
the largest number of observations possible in both treatment groups, the test
solution and
saline treated artery data were compared at the immediate post- and at the 15
minute
postrotational atherectomy time points.
In the proximal segment (FIGURE 13), there was essentially no change in lumen
area with either treatment at the immediate post-rotational atherectomy time
point, but
there was some vasodilatation in this segment by the 15 minute post-rotational
atherectomy time point. Test solution did not alter the results of rotational
atherectomy
compared to saline treatment in this segment. In the mid-vessel (FIGURE 14) at
the site
of maximal vasoconstriction however, test solution significantly blunted the
vasoconstriction, caused by rotational atherectomy in the saline treated
arteries
(p=0.0004, MANOVA corrected for repeated measures). In the distal segment
(FIGURE 15), there was little vasoconstriction in the saline treated arteries
and test
solution did not significantly alter the response to rotational atherectomy.
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TABLE 34
AMOUNT OF VASOCONSTRICTION (NEGATIVE VALUES) OR
VASODILATATION (POSITIVE VALUES) AT SPECIFIED
TIME POINTS IN
SALINE TREATED ARTERIES
Minutes Immediate 15 Minute 30 Minutes
into Infusion Post RA after RA after RA
Segment N=8 N=8 N=8 N=8
Proximall -3%~.8% -1%~10% 3%~8% 3%~13%
Mid2 -14%~7% -35%~10% -58%~7% -11%~.9%
Distal3 -9%x.10% -14%x.14% -14%~10% 2%x.12%
5 1 Proximal iliac artery measurement site, proximal to the first deep femoral
branch
2 Mid iliac artery at the site of maximal vasospasm
3 Distal iliac artery measurement site, near or distal to the second deep
femoral branch
TABLE 35
AMOUNT OF VASOCONSTRICTION (NEGATIVE VALUES) OR
VASODILATATION (POSITIVE VALUES)
AT SPECIFIED TIME POINTS IN TEST SOLUTION TREATED
ARTERIES
5 Minutes Immediate 15 Minute 30 Minutes
into Infusion Post RA after RA after RA
Segment N=10 N=13 N=13 N=11
Proximall -17%~.5% -4%~3% 14%~6% 7%~9%
Mid2 -14%~5% -20%~5% 0.3%~7% -5%~.5%
Distal3 -8%~.4% -9%~.4% 1%~4% 3%~.6%
1 Proximal iliac artery measurement site, proximal to the first deep femoral
branch
2 Mid iliac artery at the site of maximal vasospasm
3 Distal iliac artery measurement site, near or distal to the second deep
femoral branch
The hemodynamic response in the saline and test solution treated arteries is
summarized in Table 36. Compared to saline treated animals, test solution
treated
animals sustained substantial hypotension and significant tachycardia during
the solution
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infusion. By 15 minutes after completion of the infusion (or at the 30 minute
postrotational atherectomy time point), test solution treated animals showed
some partial,
but not complete, return of blood pressure towards baseline.
TABLE 36
BLOOD PRESSURE AND HEART RATES DURING
THE PROTOCOL
Group Baseline 5 Minute 15 Minute 30 Minute
Systolic Blood Pressure
Saline 839 (4) 936 (3) 9211 (4) 8310 (4)*
Test Solution 925 (7) 355 (7) 355 (7) 465 (7)**
Heart Rate
Saline 20216 (3) 2043 (3) 19822 (3) 19329 (3)*
Test Solution 187111 24611 (7) 2405 (7) 24716 (7)**
(7)
* There was no significant change in systolic blood pressure or heart rate in
this group
(p=0.37 for systolic blood pressure and p=0.94 for heart rate, ANOVA).
* * There was a highly significant change in systolic blood pressure and heart
rate in this
group (p<0.0001 for systolic blood pressure and p=0.002 for heart rate,
ANOVA).
6. SUMMARY OF STUDY
1. Rotational atherectomy in hypercholesterolemic New Zealand white
rabbits results in prominent vasospasm in the mid-portion of iliac arteries
subjected to the
rotating burr. The vasospasm is most apparent 15 minutes after rotational
atherectomy
treatment and has almost completely resolved without pharmacologic
intervention by
30 minutes after rotational atherectomy.
2. Under the conditions of rotational atherectomy treatment studied in this
protocol, test solution treatment in accordance with the present invention
almost
completely abolishes the vasospasm seen after the mid-iliac artery is
subjected to the
rotating burr.
3. Treatment with test solution of the present invention given the
concentration of components used in this protocol results in profound
hypotension during
the infusion of the solution. The attenuation of vasospasm after rotational
atherectomy by
test solution occurred in the presence of severe hypotension.
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While the preferred embodiment of the invention has been illustrated and
described, it will be appreciated that various changes to the disclosed
solutions and
methods can be made therein without departing from the spirit and scope of the
invention.
For example, alternate pain inhibitors and anti-inflammation and anti-spasm
and anti-
s restenosis agents may be discovered that may augment or replace the
disclosed agents in
accordance with the disclosure contained herein. It is therefore intended that
the scope of
letters patent granted hereon be limited only by the definitions of the
appended claims.
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