U.S. patent application number 10/288997 was filed with the patent office on 2003-10-16 for irrigation solution and methods for inhibition of tumor cell adhesion, pain and inflammation.
This patent application is currently assigned to Omeros Corporation. Invention is credited to Demopulos, Gregory A., Herz, Jeffrey M., Pierce-Palmer, Pamela, Tanelian, Darrell L..
Application Number | 20030195461 10/288997 |
Document ID | / |
Family ID | 27491010 |
Filed Date | 2003-10-16 |
United States Patent
Application |
20030195461 |
Kind Code |
A1 |
Demopulos, Gregory A. ; et
al. |
October 16, 2003 |
Irrigation solution and methods for inhibition of tumor cell
adhesion, pain and inflammation
Abstract
A method and solution for perioperatively inhibiting tumor cell
adhesion and a variety of pain and inflammation processes at wounds
from general surgical procedures including oral/dental procedures.
The solution preferably includes at least one anti-tumor cell
adhesion agent and multiple pain and inflammation inhibitory agents
at dilute concentration in a physiologic carrier, such as saline or
lactated Ringer's solution. The solution is applied by continuous
irrigation of a wound during a surgical procedure for preemptive
inhibition of pain and while avoiding undesirable side effects
associated with oral, intramuscular, subcutaneous or intravenous
application of larger doses of the agents. One preferred solution
to inhibit tumor cell adhesion, pain and inflammation includes at
least one anti-tumor cell adhesion agent, a serotonin.sub.2
antagonist, a serotonin.sub.3 antagonist, a histamine antagonist, a
serotonin agonist, a cyclooxygenase inhibitor, a neurokinin.sub.1
antagonist, a neurokinin.sub.2 antagonist, a purinoceptor
antagonist, an ATP-sensitive potassium channel opener, a calcium
channel antagonist, a bradykinin.sub.1 antagonist, a
bradykinin.sub.2 antagonist and a .mu.-opioid agonist.
Inventors: |
Demopulos, Gregory A.;
(Mercer Island, WA) ; Pierce-Palmer, Pamela; (San
Francisco, CA) ; Herz, Jeffrey M.; (Mill Creek,
WA) ; Tanelian, Darrell L.; (Dallas, TX) |
Correspondence
Address: |
OMEROS MEDICAL SYSTEMS, INC.
1420 FIFTH AVENUE
SUITE 2675
SEATTLE
WA
98101
US
|
Assignee: |
Omeros Corporation
|
Family ID: |
27491010 |
Appl. No.: |
10/288997 |
Filed: |
November 6, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10288997 |
Nov 6, 2002 |
|
|
|
09658815 |
Sep 11, 2000 |
|
|
|
6492332 |
|
|
|
|
09658815 |
Sep 11, 2000 |
|
|
|
09072913 |
May 4, 1998 |
|
|
|
6261279 |
|
|
|
|
09072913 |
May 4, 1998 |
|
|
|
08670699 |
Jun 26, 1996 |
|
|
|
5820583 |
|
|
|
|
08670699 |
Jun 26, 1996 |
|
|
|
PCT/US95/16028 |
Dec 12, 1995 |
|
|
|
PCT/US95/16028 |
Dec 12, 1995 |
|
|
|
08353775 |
Dec 12, 1994 |
|
|
|
60162416 |
Oct 28, 1999 |
|
|
|
Current U.S.
Class: |
604/59 ;
514/1 |
Current CPC
Class: |
A61K 31/4439 20130101;
A61K 31/4045 20130101; A61K 31/4174 20130101; A61K 31/4164
20130101; A61K 31/352 20130101; A61K 45/06 20130101; A61K 31/444
20130101; A61K 31/538 20130101; A61K 31/4168 20130101; A61K 31/4406
20130101; A61K 31/498 20130101; A61K 38/00 20130101; A61K 31/506
20130101; A61K 31/439 20130101; A61K 31/48 20130101; A61K 31/4427
20130101; A61K 31/00 20130101 |
Class at
Publication: |
604/59 ;
514/1 |
International
Class: |
A61K 031/00; A61M
031/00 |
Claims
The embodiments of the invention in which an exclusive property or
privilege is claimed are defined as follows:
1. A solution for use in inhibiting tumor cell adhesion, pain and
inflammation at a wound during a surgical procedure, comprising:
therapeutically effective amounts of at least one tumor-cell
anti-adhesion agent and at least one pain/inflammation inhibitory
agent in a dilute solution within a liquid irrigation carrier, the
agents being selected to act on differing molecular targets, the at
least one tumor-cell anti-adhesion agent being selected to be
deliverable by irrigation of the wound to provide without metabolic
transformation a therapeutic anti-adhesion effect.
2. The solution of claim 1, wherein said anti-adhesion agent is
selected from the group consisting of CD44 receptor antagonists,
integrin receptor antagonists and selectin receptor
antagonists.
3. The solution of claim 1, wherein said anti-adhesion agent
comprises an inhibitor of matrix metalloproteinases.
4. The solution of claim 1, wherein the pain/inflammation
inhibitory agents are selected from the group consisting of:
serotonin receptor antagonists; serotonin receptor agonists;
histamine receptor antagonists; bradykinin receptor antagonists;
kallikrein inhibitors; tachykinin receptor antagonists including
neurokinin.sub.1 receptor subtype antagonists and neurokinin.sub.2
receptor subtype antagonists; calcitonin gene-related peptide
receptor antagonists; interleukin receptor antagonists;
phospholipase inhibitors including PLA.sub.2 isoform inhibitors and
PLC.sub..gamma. isoform inhibitors; cyclooxygenase inhibitors;
lipooxygenase inhibitors; prostanoid receptor antagonists including
eicosanoid EP-1 receptor subtype antagonists and eicosanoid EP-4
receptor subtype antagonists and thromboxane receptor subtype
antagonists; leukotriene receptor antagonists including leukotriene
B.sub.4 receptor subtype antagonists and leukotriene D.sub.4
receptor subtype antagonists; opioid receptor agonists including
.mu.-opioid receptor subtype agonists, .delta.-opioid receptor
subtype agonists, and .kappa.-opioid receptor subtype agonists;
purinoceptor agonists and antagonists including P.sub.2Y receptor
agonists and P.sub.2X receptor antagonists; and ATP-sensitive
potassium channel openers.
5. The solution of claim 1, wherein the pain/inflammation
inhibitory agents are selected from the group consisting of:
serotonin receptor antagonists; serotonin receptor agonists;
histamine receptor antagonists; bradykinin receptor antagonists;
kallikrein inhibitors; tachykinin receptor antagonists including
neurokinin.sub.1 receptor subtype antagonists and neurokinin.sub.2
receptor subtype antagonists; calcitonin gene-related peptide
receptor antagonists; phospholipase inhibitors including PLA.sub.2
isoform inhibitors and PLC.sub..gamma. isoform inhibitors;
lipooxygenase inhibitors; prostanoid receptor antagonists including
eicosanoid EP-1 receptor subtype antagonists and eicosanoid EP-4
receptor subtype antagonists and thromboxane receptor subtype
antagonists; leukotriene receptor antagonists including leukotriene
B.sub.4 receptor subtype antagonists and leukotriene D.sub.4
receptor subtype antagonists; opioid receptor agonists including
.mu.-opioid receptor subtype agonists, .delta.-opioid receptor
subtype agonists, and .kappa.-opioid receptor subtype agonists;
purinoceptor agonists and antagonists including P.sub.2Y receptor
agonists and P.sub.2X receptor antagonists; and ATP-sensitive
potassium channel openers.
6. The solution of claim 1, further comprising a plurality of
pain/inflammation inhibitory agents.
7. The solution of claim 1, wherein each of the agents in the
solution applied is included at a concentration or dosage that is
sufficient to provide a level of inhibitory effect at the wound
when locally applied, and that results in a plasma concentration
that is less than a plasma concentration which would be required to
provide the same level of inhibitory effect at the wound if applied
systemically.
8. The solution of claim 1, wherein each of the plurality of agents
in the solution is delivered locally at a concentration of no
greater than 100,000 nanomolar.
9. The solution of claim 8, wherein each of the plurality of agents
in the solution is delivered locally at a concentration of no
greater than 10,000 nanomolar.
10. A solution for use in the inhibition of tumor cell attachment
and implantation during a surgical procedure within a body cavity
of a patient in need of such treatment, comprising: therapeutically
effective amounts of at least one tumor-cell anti-adhesion agent
and at least one pain/inflammation inhibitory agent in a dilute
solution within a liquid irrigation carrier, the agents being
selected to act on differing molecular targets, the at least one
tumor-cell anti-adhesion agent being selected to be deliverable by
irrigation in the body cavity to provide without metabolic
transformation a therapeutic anti-tumor cell attachment and
anti-tumor cell implantation effect.
11. A solution for use in inhibiting tumor cell adhesion, pain and
inflammation at a wound during a surgical procedure, comprising:
therapeutically effective amounts of at least one tumor-cell
anti-adhesion agent and at least one pain/inflammation inhibitory
agent in a dilute solution within a liquid irrigation carrier, the
agents being selected to act on differing molecular targets, the at
least one tumor-cell anti-adhesion agent being selected from the
group consisting of CD44 receptor antagonists, integrin receptor
antagonists, selectin receptor antagonists and inhibitors of matrix
metalloproteinases and being selected to be deliverable by
irrigation of the wound to provide without metabolic transformation
a therapeutic anti-adhesion effect.
12. A solution for use in inhibiting tumor cell adhesion, pain and
inflammation at a wound during a surgical procedure, comprising:
therapeutically effective amounts of at least one tumor-cell
anti-adhesion agent and at least one pain/inflammation inhibitory
agent in a dilute solution within a liquid carrier, the agents
being selected to act on a plurality of differing molecular
targets, the at least one tumor-cell anti-adhesion agent being
selected from the group consisting of CD44 receptor antagonists,
integrin receptor antagonists and selectin receptor antagonists,
and being selected to be deliverable to the wound to provide
without metabolic transformation a therapeutic anti-adhesion
effect.
13. A solution for use in inhibiting tumor cell adhesion at a wound
during a surgical procedure, comprising: therapeutically effective
amounts of a first tumor-cell anti-adhesion agent and a second
tumor-cell anti-adhesion agent in a dilute solution within a liquid
irrigation carrier, the agents being selected to act on differing
molecular targets, at least one of the agents being selected to be
deliverable by irrigation of the wound to provide without metabolic
transformation a therapeutic anti-adhesion effect.
14. The solution of claim 13, wherein each of the agents in the
solution applied is included at a concentration or dosage that is
sufficient to provide a level of inhibitory effect at the wound
when locally applied, and that results in a plasma concentration
that is less than a plasma concentration which would be required to
provide the same level of inhibitory effect at the wound if applied
systemically.
15. The solution of claim 13, wherein each of the plurality of
agents in the solution is delivered locally at a concentration of
no greater than 100,000 nanomolar.
16. The solution of claim 13, wherein each of the plurality of
agents in the solution is delivered locally at a concentration of
no greater than 10,000 nanomolar.
17. The solution of claim 13, further comprising at least one
pain/inflammation inhibitory agent.
18. The solution of claim 17, wherein each of the agents in the
solution applied is included at a concentration or dosage that is
sufficient to provide a level of inhibitory effect at the wound
when locally applied, and that results in a plasma concentration
that is less than a plasma concentration which would be required to
provide the same level of inhibitory effect at the wound if applied
systemically.
19. The solution of claim 17, wherein each of the plurality of
agents in the solution is delivered locally at a concentration of
no greater than 100,000 nanomolar.
20. The solution of claim 17, wherein each of the plurality of
agents in the solution is delivered locally at a concentration of
no greater than 10,000 nanomolar.
21. The solution of claim 13, wherein at least one of the agents is
selected from the group consisting of CD44 receptor antagonists,
integrin receptor antagonists, selectin receptor antagonists and
inhibitors of matrix metalloproteinases.
22. A solution for use in inhibiting tumor cell adhesion, pain and
inflammation at a wound during a surgical procedure, comprising:
therapeutically effective amounts of at least one tumor-cell
anti-adhesion agent and at least one pain/inflammation inhibitory
agent in a dilute solution within a sustained release carrier, the
agents being selected to act on differing molecular targets, the at
least one tumor-cell anti-adhesion agent being selected to provide
without metabolic transformation a therapeutic anti-adhesion effect
at the wound.
23. The solution of claim 22, wherein the sustained release carrier
comprises a gel.
24. The solution of claim 22, wherein said anti-adhesion agent is
selected from the group consisting of CD44 receptor antagonists,
integrin receptor antagonists and selectin receptor
antagonists.
25. A solution for use in inhibiting tumor cell adhesion at a wound
during a surgical procedure, comprising: therapeutically effective
amounts of a first tumor-cell anti-adhesion agent and a second
tumor-cell anti-adhesion agent in a dilute solution within a
sustained release carrier, the agents being selected to act on
differing molecular targets, at least one of the agents being
selected to provide without metabolic transformation a therapeutic
anti-adhesion effect.
26. The solution of claim 25, wherein the sustained release carrier
comprises a gel.
27. The solution of claim 25, wherein said anti-adhesion agent is
selected from the group consisting of CD44 receptor antagonists,
integrin receptor antagonists and selectin receptor
antagonists.
28. A solution for use in inhibition of tumor cell attachment and
implantation during a surgical procedure within a body cavity of a
patient in need of such treatment, comprising: therapeutically
effective amounts of at least one tumor-cell anti-adhesion agent
and at least one pain/inflammation inhibitory agent in a dilute
solution within a sustained release carrier, the agents being
selected to act on differing molecular targets, the at least one
tumor-cell anti-adhesion agent being selected to provide in the
body cavity without metabolic transformation a therapeutic
anti-tumor cell attachment and anti-tumor cell implantation
effect.
29. A solution for use in inhibition of tumor cell attachment and
implantation during a surgical procedure within a body cavity of a
patient in need of such treatment, comprising: therapeutically
effective amounts of a first tumor-cell anti-adhesion agent and a
second tumor-cell anti-adhesion agent in a dilute solution within a
sustained release carrier, the agents being selected to act on
differing molecular targets, at least one of the agents being
selected to provide in the body cavity without metabolic
transformation a therapeutic anti-tumor cell attachment and
anti-tumor cell implantation effect.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of co-pending U.S.
application Ser. No. 09/658,815 filed Sep. 11, 2000, which is a
continuation-in-part of U.S. application Ser. No. 09/072,913 filed
May 4, 1998, which is a continuation of U.S. patent application
Ser. No. 08/670,699 filed Jun. 26, 1996, (U.S. Pat. No. 5,820,583),
which is a continuation-in-part of International Patent Application
PCT/US95/16,028, filed Dec. 12, 1995, which designates the United
States and which is a continuation-in-part of U.S. patent
application Ser. No. 08/353,775, filed Dec. 12, 1994 (abandoned),
and claims the benefit of U.S. Provisional Application Serial No.
60/162,416 filed Oct. 28, 1999, priority of the filing date of each
application which are hereby claimed under 35 U.S.C. .sctn.120 or
.sctn.119(e).
FIELD OF THE INVENTION
[0002] 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
[0003] 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 skin
and body wall. Through similar portal incisions, surgical
instruments may be placed in a body cavity, their use guided by
arthroscopic visualization. As endoscopists' skills 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.
[0004] 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.
Pat. No. 4,504,493 to Marshall discloses an isomolar solution of
glycerol in water for a non-conductive and optically clear
irrigation solution for arthroscopy.
[0005] Irrigation is also used in other procedures, such as
cardiovascular and general vascular diagnostic and therapeutic
procedures, urologic procedures and the treatment of burns and any
operative wounds. In 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/or invasion and/or local tumor cell metastasis, nor do they
provide analgesic, anti-inflammatory, anti-spasm and
anti-restenotic effects.
[0006] 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 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).
[0007] 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 spread of cancer enables development of pharmaceutical
compositions that block the attachment of tumor cells to
extracellular matrix proteins and/or inhibit tumor cell invasion
during surgical procedures.
[0008] 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 intramuscular) 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.
[0009] 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-Bradykinin
Potentiation in the Pain Receptors in Man, Life Sci. 4, pp. 309-316
(1965); Rosenthal, S. R., Histamine as the Chemical Mediator for
Cutaneous Pain, J. Invest. Dermat. 69, pp. 98-105 (1977);
Richardson, B. P., et al., Identification of Serotonin M-Receptor
Subtypes and their Specific Blockade by a New Class of Drugs,
Nature 316, pp. 126-131 (1985); Whalley, E. T., et al., The Effect
of Kinin Agonists and Antagonists, Naunyn-Schmiedeb Arch.
Pharmacol. 36, pp. 652-57 (1987); Lang, E., et al.,
Chemo-Sensitivity of Fine Afferents from Rat Skin In Vitro, J.
Neurophysiol. 63, pp. 887-901 (1990).
[0010] For example, 5-HT applied to a human blister base (denuded
skin) has been demonstrated to cause pain that can be inhibited by
5-HT.sub.3 receptor antagonists. Richardson et al., (1985).
Similarly, peripherally applied bradykinin produces pain that can
be blocked by bradykinin receptor antagonists. Sicuteri et al.,
1965; Whalley et al., 1987; Dray, A., et al., Bradykinin and
Inflammatory Pain, Trends Neurosci. 16, pp. 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) and their Antagonists", in Goodman, L. S., et al., ed.,
The Pharmacological Basis of Therapeutics, MacMillan Publishing
Company, New York, pp. 605-638 (1985); Rumore, M. M., et al.,
Analgesic Effects of Antihistaminics, Life Sci 36, pp. 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, pp. 467-476
(1992).
[0011] In the body, 5-HT is located in platelets and in central
neurons, histamine is found in mast cells, and 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, pp. 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. Ringkamp, M., et al., Activated Human
Platelets in Plasma Excite Nociceptors in Rat Skin, In Vitro,
Neurosci. Lett. 170, pp. 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, pp. 635-647 (1973); Douglas, 1985; Dray et al. (1993).
[0012] 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 Publishing
Company, New York, pp. 674-715 (1985). Cyclooxygenase 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.
[0013] As discussed, 5-HT, histamine, bradykinin 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 are
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.
[0014] 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 are 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, Pain 27, pp. 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, pp. 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.
[0015] Amitriptyline, in addition to blocking 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 are 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 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.
[0016] 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.sub.1 (H.sub.1) receptor antagonists to
act as local anesthetics for surgical procedures. This anesthetic
effect is not believed to be mediated via H.sub.1 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
[0017] 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 local metastasis during surgical
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, 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. 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 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.
[0018] 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.
[0019] 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.
[0020] 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 adhesion or anti-invasion or anti-local metastasis agent that
directly reduces or prevents the attachment of tumor cells to
either the extracellular matrix or to other cells (both normal and
tumor). Suitable 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,
selecting, 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.
[0021] 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 or 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.
[0022] 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 kinase C, and mitogen-activated protein kinases (MAPK).
Such enzyme inhibitors may interact with a particular member 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.
[0023] 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
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
pharmaceutical 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.,
laparoscopic 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.
[0024] 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 ant-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.
[0025] 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.
[0026] 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 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.
[0027] 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-pain/anti-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) kallikrein inhibitors; (6)
tachykinin receptor antagonists, including neurokinin, and
neurokinin.sub.2 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 PLA.sub.2 isoform inhibitors
and PLC.sub..gamma. 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 including leukotriene B.sub.4
receptor subtype antagonists and leukotriene D.sub.4 receptor
subtype antagonists; (12) opioid receptor agonists, including
.mu.-opioid, .delta.-opioid, and .kappa.-opioid receptor subtype
agonists; (13) purinoceptor agonists and antagonists including
P.sub.2X receptor antagonists and P.sub.2Y receptor agonists; and
(14) adenosine triphosphate (ATP)-sensitive potassium channel
openers. Each of the above agents functions either as an
anti-inflammatory agent and/or 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.
[0028] Several preferred embodiments of the solution of the present
invention also include anti-spasm agents for particular
applications. For example, anti-spasm agents may be included alone
or in combination with anti-pain/anti-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-inflammatory/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).
[0029] 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
purinoceptor.sub.1 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 kinase C (PKC) inhibitors and protein tyrosine kinase
inhibitors, (b) modulators of intracellular protein tyrosine
phosphatases, (c) inhibitors of src homology.sub.2 (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.
[0030] The present invention also provides a method for
manufacturing a medicament compounded as a dilute irrigation
solution for use in continuously irrigating an operative 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-inflammato- ry 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.
[0031] 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.
[0032] 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.
[0033] The advantages of local administration of the agents via
luminal irrigation or other fluid application are the following:
(1) local administration guarantees a known 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 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.
[0034] 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 more substantial than if given after the damage has been
initiated.
[0035] 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 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 antagonists 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.
[0036] 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.
[0037] 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
[0038] The present invention will now be described in greater
detail, by way of example, with reference to the accompanying
drawings in which:
[0039] FIG. 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 are 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.
[0040] FIG. 2 provides a detailed diagram of the signaling pathways
illustrating "crosstalk" between G-protein coupled receptor (GPCR)
pathways and receptor tyrosine kinase (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 (FIGS. 3 and 7) leading to pain
transmission in other cell types (e.g., neurons).
[0041] FIG. 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.
[0042] FIG. 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 FIG. 5).
[0043] FIG. 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 FIG. 4).
[0044] FIG. 6 provides a diagram of the G-Protein Coupled Receptor
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
urologic solution are identified.
[0045] FIG. 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.
[0046] FIG. 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
(PKG) 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.
[0047] FIGS. 9, 10A and 10B 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
arteries, 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.
[0048] FIGS. 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.
[0049] FIGS. 13, 14 and 15 provide charts of mean vasoconstriction
(negative values) or vasodilation (positive values), .+-.1 standard
error of the mean for the proximal (FIG. 13), mid (FIG. 14) and
distal (FIG. 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.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0050] 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
[0051] A. Metalloproteinase Antagonists and Tissue Inhibitors of
Metalloproteinases (TIMP)
[0052] Matrix metalloproteinases (MMPs) are a family of
zinc-dependent neutral endopeptidases whose enzymatic activity is
capable of controlled degradation of the extracellular 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.
[0053] 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 52 to 58
amino acids.
[0054] Cells do not generally constitutively express MMPs, but
rather their expression is rapidly induced in response to exogenous
signals, cytokines 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.
[0055] Expression of MMPs is primarily regulated at the level of
transcription, and their proteolytic activity is regulated by
zymogen activation and inhibition of activity. Extracellular
signals such as cytokines (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), 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.
[0056] 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, L-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.
[0057] 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.
[0058] 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 deficient
mice show reduced angiogenesis and tumor progression. Infiltration
of inflammatory cells is also a 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.
[0059] 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.
[0060] Based upon in vitro, in vivo, in 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.
[0061] 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 (ERK1,2,SAPK/JNK 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.
[0062] 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 vitro
and in vivo. An MMP-9 antisense-ribozyme expression construct has
been shown to inhibit expression of MMP-9 and the invasion of rat
osteocarcinoma cells.
[0063] 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 specifically 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-1 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 in vitro and in vivo.
[0064] 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 and/or 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.
[0065] One skilled 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 administered for any
particular treatment protocol and that these amounts can be readily
determined.
[0066] MMP inhibitors suitable for use in the present invention are
listed in the Table below.
1TABLE 1 Metalloproteinase Antagonists and TIMPs Therapeutic
Preferred Most Preferred Agent 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 Bay12-9566 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 BB16 10-1,000,000 10,000 XS309
100-10,000,000 100,000 Antisense RNA 0.5-5,000 5,000 TIMPS 1
0.1-10,000 1,000
[0067] B. CD44 Receptor Antagonists and Inhibitors of CD44
Signaling
[0068] 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 (v1-y10) 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 (v1-y10) inserted into the
extracellular domain are restricted in their expression to
proliferating epithelial cells, activated lymphocytes and malignant
cells.
[0069] 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
glycosaminoglycan, CD44 has also been termed the hyaluronate
receptor. It can also bind some other extracellular matrix ligands
(chondroitin sulphate, heparin sulphate, and fibronectin) with
lower affinity.
[0070] 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.
[0071] The CD44 molecule is an 85 kD glycosylated molecule with
N-terminal sequence homology to cartilage link proteins. Isoforms
of CD44 of varying 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 85 kD 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 proinflammatory molecule
involved in immune cell activation as well as metastasis of certain
tumor cell types.
[0072] 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.
[0073] 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, are among several that are required by invasive
and metastatic tumor cells. In 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.
[0074] 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.
[0075] 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 isoforms
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.
[0076] At the cellular level, recent studies have characterized
cytoplasmic signal transduction pathways for CD44 in activated
endothelial cells that involve tyrosine 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-receptor 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 PKC
activation triggered the cytoplasmic kinase cascade involving Raf-1
kinase, MAP kinase (MEK-1), and extracellular signal-regulated
kinase (ERK-1). ERK is a dual-specificity kinase that controls
expression of proteins relevant to tumorigenesis, as well as tumor
cell proliferation, and motility.
[0077] 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, PKC 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.
[0078] 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 in vivo was demonstrated
by local administration of a mutant, nonhyaluronate binding,
CD44-Ig fusion protein which had no effect on subcutaneous melanoma
growth in mice whereas the infusion of wild-type CD44-Ig blocked
tumor development.
[0079] 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-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.
[0080] 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. Pat. 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.
[0081] 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 and 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 Fe antibody (termed Fe 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.
[0082] 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.
[0083] 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.
[0084] CD44 receptor 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.
2TABLE 2 CD44 Receptor Antagonists Therapeutic Preferred Most
Preferred Agent Concentrations (.mu.g/ml) Concentrations (.mu.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 oligomers 0.1-1000 1000
solubleCD44receptor 1.0-10000 1000
[0085] C. Integrin Receptor Antagonists and Inhibitors of Integrin
Signaling
[0086] 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.
[0087] The integrins are a family of heterodimeric, transmembrane
.alpha..beta. receptors that are expressed on a wide variety of
cells. The receptor family includes at least 14 known
.alpha.-subunits and 8 .beta.-subunits that associate with each
other to form numerous subtype combinations that demonstrate
differing ligand specificities. The .alpha.subunit appears to be a
critical determinant of ligand specificity and integrins containing
.alpha.v demonstrate specificity for vitronectin, .alpha.5 for
fibronectin, and .alpha.3 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, .alpha.4.beta.1 and .alpha.4.beta.7.
Mucosal addressin cell adhesion molecule 1 (MadCAM-1) is also
recognized as a ligand for .alpha.4.beta.7 integrin.
[0088] 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 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 .alpha.5.beta.1 fibronectin-specific integrin binds
to the central RGD/PHSRN site. The .alpha.4.beta.1 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.
[0089] 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
kinase (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.
[0090] A peptide sequence motif responsible for the cell attachment
in the fibronectin ligand for the integrin receptor
(arginine-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 in vitro.
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 in vitro have been examined.
[0091] 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-glycyl-seryl-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
.alpha.2.beta.1 and .alpha.3.beta.1 integrin expression was
markedly increased on OCUM-2MD3 cells and the ability of these
cells to bind to the extracellular 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, laminin or peptides
containing the RGDS motif.
[0092] 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. Pat. 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 present invention provides for the use of peptides
and peptide analogs which are specific for fibronectin-binding
integrins, in particular the .alpha..sub.5 .beta..sub.1 integrin.
In addition, specific agents for inhibition of binding to
.alpha..sub.2, .alpha..sub.6 and .alpha..sub.v integrin subunits as
part of .beta..sub.1 integrin heterodimers are also provided.
[0093] 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 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 cell 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 .alpha.V.beta.3 and not to .alpha.5.beta.1 or other
abundant integrins. The related disintegrin echistatin specifically
inhibited .sup.125I-labeled kistrin binding to .alpha.V.beta.3,
while a structurally distinct disintegrin, decorsin had 1000-fold
lower affinity.
[0094] Two recently isolated disintegrins from the venom of Chinese
viper (Agkistrodon ussuriensis), 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 IC.sub.50s were about ten
times higher. US-1 also dose-dependently inhibited the adhesion of
human melanoma cells to fibrinogen and fibronectin with an
IC.sub.50=17-33 nM, while US-2 did not inhibit the cell adhesion to
fibronectin.
[0095] An additional heterodimeric disintegrin, EC3 (Mr=14,762),
isolated from Echis carinatus venom is a potent antagonist of
.alpha.4 integrins. Each subunit contains 67 residues and displays
a high degree of homology to other disintegrins. EC3 inhibited
adhesion of cells expressing .alpha.4.beta.1 and .alpha.4.beta.7
integrins to natural ligands such as vascular cell adhesion
molecule 1 (VCAM-1) and mucosal addressin cell adhesion molecule 1
(MadCAM-1) with IC.sub.50=6-30 nM, adhesion of K562 cells
(.alpha.5.beta.1) to fibronectin with IC.sub.50=150 nM, and the
adhesion of .alpha.IIb.beta.3 Chinese hamster ovary cells to
fibrinogen with IC.sub.50=500 nM.
[0096] 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.
3TABLE 3 Therapeutic and Preferred Concentrations of Integrin
Receptor Antagonists Therapeutic Preferred Most Preferred Compounds
Concentrations (.mu.M) Concentrations (.mu.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).sub.3
0.1-1000 10 US-1 0.03-3000 3 kistrin 0.1-1000 10 EC-3 0.1-1000
1
[0097] D. Selectin Receptor Antagonists and Inhibitors of Selectin
Signaling
[0098] 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.sup.a) and sialyl Lewis X (SLe.sup.x), 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
cells to vascular endothelium and thus contribute to metastasis of
cancer. The selectins are inducible endothelial-expressed adhesion
molecules involved in leukocyte recruitment. The initial adhesion
mediated by these molecules triggers activation of integrin
molecules through the action of several cytokines. The degree of
expression of the carbohydrate ligands at the surface of cancer
cells is well correlated with the frequency of metastasis.
Monoclonal antibodies directed to Sle.sup.a or Sle.sup.x 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.
[0099] P selectin, also known as GMP-140 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.
[0100] 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 neuramimidase. KM12-L4 cells interact with P-selectin through
a PSGL-1-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.
[0101] Recent studies evaluated the role of E-selectin-sialyl Lewis
x (Sle.sup.x)/sialyl Lewis a (Sle.sup.a) interaction in mediating
in vitro adhesion of two human colon cancer cell lines, HT-29 and
COLO 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.sup.x and antibodies
directed against related Lewis epitopes, Le.sup.x and Le.sup.a, had
no significant effect on adhesion. Three antibodies directed
against Sle.sup.a differed in their capacity to inhibit the
adhesion of HT-29 and COLO 201 cells. Thus, the Sle.sup.a epitope
is important for adhesion of colon cancer cells mediated
predominantly by E-selectin.
[0102] Additional studies have demonstrated that pretreatment of
HUVEC with tumor necrosis factor-.alpha. (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 BB11, an
anti-E-selectin mAb, prior to the addition of COLO 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 Lewis.sup.x-containing
oligosaccharide (Sle.sup.x-OS) are effective in inhibiting cell
adhesion.
[0103] Similar in vitro and in 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 Sle.sup.a and SLe.sup.x antigens, CD44H, and
.beta.1 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
Sle.sup.a and against .beta.1 integrin. In animal studies,
treatments with the antibodies against Sle.sup.a and .beta.1
integrin each inhibited the development of liver metastasis in nude
mice with SW1990 cells and prolonged their survival.
[0104] 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 kinase 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.
[0105] 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 pp125 FAK 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.
[0106] 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 Lewis.sup.a (Sle.sup.a) mAb in combination with either
one or more anti-inflammatory and/or anti-pain and/or anti-spasm
and/or anti-restenosis agents.
[0107] 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.
[0108] 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.
4TABLE 4 Therapeutic and Preferred Concentrations of Selectin
Receptor Antagonists Therapeutic Preferred Most Preferred Agent
Concentrations (.mu.g/ml) Concentrations (.mu.g/ml) anti-SLe.sup.x
mAb PB1.3 0.1-1000 100 anti-SLe.sup.a mAb 0.1-1000 100 DREG-200
Slex-OS 0.1-1000 100 anti-E-selectin mAb 0.1-10000 1000 BB11
anti-P-selectin IgG 0.1-10000 1000 anti-E-selectin IgG 1.0-10000
1000 enoxaparin 0.1-1000 100
[0109] 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 and/or 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,
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. 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).
[0110] 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.
[0111] The present invention also provides for formulations of the
active therapeutic agent(s) which may be delivered in a formulation
useful for introduction and administration to the operative site
that would enhance the delivery, uptake, stability or
pharmacokinetics 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 ligands 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 skilled in the
art.
[0112] 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 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.
[0113] The present invention provides for the local delivery of a
combination of tumor cell anti-adhesion drug(s) 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.
[0114] 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.
[0115] 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
which may include physiologic electrolytes, such as normal saline
or lactated Ringer's solution.
[0116] 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.sub.1 and
neurokinin.sub.2 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 PLA.sub.2 isoform inhibitors
and PLC.sub..gamma. 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 including leukotriene B.sub.4
receptor subtype antagonists and leukotriene D.sub.4 receptor
subtype antagonists; (12) opioid receptor agonists, including
.mu.-opioid, .delta.-opioid, and .epsilon.-opioid receptor subtype
agonists; (13) purinoceptor agonists and antagonists including
P.sub.2X receptor antagonists and P.sub.2Y receptor agonists; and
(14) adenosine triphosphate (ATP)-sensitive potassium channel
openers.
[0117] 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).
[0118] 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
purinoceptor.sub.1 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 kinase C (PKC) inhibitors and protein tyrosine
phosphatases, (b) modulators of intracellular protein tyrosine
kinase inhibitors, (c) inhibitors of src homology.sub.2 (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.
[0119] 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, K.sub.d. 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 K.sub.d 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 K.sub.d
nanomolar and most preferably at approximately 100 times K.sub.d
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.
[0120] A solution in accordance with the present invention can
include just a single or multiple inhibitor(s) 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.
[0121] 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
specific drugs that are 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 ET.sub.A in hypertension, these drug companies
specifically are targeting the development of selective antagonists
to the ET.sub.A 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 FIGS. 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 ET.sub.A, is likely to be
ineffective.
[0122] 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 FIG. 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".
[0123] An example of "upstream" inhibition is the cyclooxygenase
antagonists in the setting of pain and inflammation. The
cyclooxygenase enzymes (COX.sub.1 and COX.sub.2) 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 thromboxanes.
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 bradykinins, 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.
[0124] 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 "downstream"
antispasmodic effects that are independent of the physiologic
combination of agonists initiating the spasmodic event (see FIGS. 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
[0125] 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.
[0126] A. Serotonin Receptor Antagonists
[0127] Serotonin (5-HT) is thought to produce pain by stimulating
serotonin.sub.2 (5-HT.sub.2) and/or serotonin.sub.3 (5-HT.sub.3)
receptors on nociceptive neurons in the periphery. Most researchers
agree that 5-HT.sub.3 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-HT.sub.3
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, Trends in Pharmacological Sciences 11, pp. 185-189 (1990).
A study in rat ankle joints, however, claims the 5-HT.sub.2
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, pp. 216-18 (1988). Therefore, activation of 5-HT.sub.2
receptors also may play a role in peripheral pain and neurogenic
inflammation.
[0128] One goal of the solution of the present invention is to
block pain and a multitude of inflammatory processes. Thus,
5-HT.sub.2 and 5-HT.sub.3 receptor antagonists are both suitably
used, either individually or together, in the solution of the
present invention, as shall be described subsequently.
Amitriptyline (Elavil.TM.) is a suitable 5-HT.sub.2 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 (Reglan.TM.) is used clinically as an anti-emetic
drug, but displays moderate affinity for the 5-HT.sub.3 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.
[0129] Other suitable 5-HT.sub.2 receptor antagonists include
imipramine, trazodone, desipramine and ketanserin. 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 (July 1988). Other suitable 5-HT.sub.3 receptor
antagonists include cisapride and ondansetron. The cardiovascular
and general vascular solution also may contain a serotonin.sub.1B
(also known as serotonin.sub.1D.beta.) antagonist because serotonin
has been shown to produce significant vascular spasm via activation
of the serotonin.sub.1B receptors in humans. Kaumann, A. J., et
al., Variable Participation of 5-HT1-Like Receptors and 5-H2
Receptors in Serotonin-Induced Contraction of Human Isolated
Coronary Arteries, Circulation 90, pp. 1141-53 (1994). Suitable
serotonin.sub.1B receptor antagonists include yohimbine,
N-[-methoxy-3-(4-methyl-1-piperanzinyl)phenyl]-2'-methyl-4'-(5-methyl-1,2-
,4-oxadiazol-3-yl) [1,1-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.
5TABLE 5 Therapeutic and Preferred Concentrations of
Pain/Inflammation Inhibitory Agents Therapeutic Preferred
Concentrations Concentrations Class of Agent (Nanomolar)
(Nanomolar) Serotonin.sub.2 Receptor Antagonists: amitriptyline
0.1-1,000 50-500 imipramine 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
Serotonin.sub.3 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 Serotonin.sub.1B (Human 1D.sub..beta.)
Antagonists: yohimbine 0.1-1,000 50-500 GR127935 0.1-1,000 10-500
methiothepin 0.1-500 1-100
[0130] B. Serotonin Receptor Agonists
[0131] 5-HT.sub.1A, 5-HT.sub.1B and 5-HT.sub.1D receptors are known
to inhibit adenylate cyclase activity. Thus including a low dose of
these serotonin.sub.1A, serotonin.sub.1B and serotonin.sub.1D
receptor agonists in the solution should inhibit neurons mediating
pain and inflammation. The same action is expected from
serotonin.sub.1E and serotonin.sub.1F receptor agonists because
these receptors also inhibit adenylate cyclase.
[0132] Buspirone is a suitable 1A receptor agonist for use in the
present invention. Sumatriptan is a suitable 1A, 1B, 1D and 1F
receptor agonist. A suitable 1B and 1D receptor agonist is
dihydroergotamine. A suitable 1E receptor agonist is ergonovine.
Therapeutic and preferred concentrations for these receptor
agonists are provided in Table 6.
6TABLE 6 Therapeutic and Preferred Concentrations of
Pain/Inflammation Inhibitory Agents Therapeutic Preferred
Concentrations Concentrations Class of Agent (Nanomolar)
(Nanomolar) Serotonin.sub.1A Agonists: buspirone 1-1,000 10-200
sumatriptan 1-1,000 10-200 Serotonin.sub.1B Agonists:
dihydroergotamine 0.1-1,000 10-100 sumatriptan 1-1,000 10-200
Serotonin.sub.1D Agonists: dihydroergotamine 0.1-1,000 10-100
sumatriptan 1-1,000 10-200 Serotonin.sub.1E Agonists: ergonovine
10-2,000 100-1,000 Serotonin.sub.1F Agonists: sumatriptan 1-1,000
10-200
[0133] C. Histamine Receptor Antagonists
[0134] Histamine receptors generally are divided into
histamine.sub.1 (H.sub.1) and histamine.sub.2 (H.sub.2) subtypes.
The classic inflammatory response to the peripheral administration
of histamine is mediated via the H.sub.1 receptor. Douglas, 1985.
Therefore, the solution of the present invention preferably
includes a histamine H.sub.1 receptor antagonist. Promethazine
(Phenergan.TM.) is a commonly used anti-emetic drug which potently
blocks H.sub.1 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
H.sub.1 receptors, thus, the effects are believed to occur by
different mechanisms. The histamine receptor antagonist
concentration in the solution is sufficient to inhibit H.sub.1
receptors involved in nociceptor activation, but not to achieve a
"local anesthetic" effect, thereby eliminating the concern
regarding systemic side effects.
[0135] 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.sub.1 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,
American Heart J., Vol. 102, pp. 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, pp. 614-622, (1990). Therefore,
histamine receptor antagonists may be included in the
cardiovascular irrigation solution.
[0136] Other suitable H.sub.1 receptor antagonists include
terfenadine, diphenhydramine, amitriptyline, mepyramine and
tripolidine. Because amitriptyline is also effective as a
serotonin.sub.2 receptor antagonist, it has a dual function as used
in the present invention. Suitable therapeutic and preferred
concentrations for each of these H.sub.1 receptor antagonists are
set forth in Table 7.
7TABLE 7 Therapeutic and Preferred Concentrations of
Pain/Inflammation Inhibitory Agents Therapeutic Preferred
Concentrations Concentrations Class of Agent (Nanomolar)
(Nanomolar) Histamine.sub.1 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
[0137] D. Bradykinin Receptor Antagonists
[0138] Bradykinin receptors generally are divided into
bradykinin.sub.1 (B.sub.1) and bradykinin.sub.2 (B.sub.2) subtypes.
Studies have shown that acute peripheral pain and inflammation
produced by bradykinin are mediated by the B.sub.2 subtype whereas
bradykinin-induced pain in the setting of chronic inflammation is
mediated via the B.sub.1 subtype. Perkins, M. N., et al.,
Antinociceptive Activity of the Bradykinin B1 and B2 Receptor
Antagonists, des-Arg.sup.9, [Leu.sup.8]-BK and HOE 140, in Two
Models of Persistent Hyperalgesia in the Rat, Pain 53, pp. 191-97
(1993); Dray, A., et al., Bradykinin and Inflammatory Pain, Trends
Neurosci 16, pp. 99-104 (1993), each of which references is hereby
expressly incorporated by reference.
[0139] At present, bradykinin 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 B.sub.2 receptors block bradykinin-induced acute
pain and inflammation. Dray et al., 1993. B.sub.1 receptor
antagonists inhibit pain in chronic inflammatory conditions.
Perkins et al., 1993; Dray et al., 1993. Therefore, depending on
the application, the solution of the present invention preferably
includes either or both bradykinin B.sub.1 and B.sub.2 receptor
antagonists. For example, arthroscopy is performed for both acute
and chronic conditions, and thus an irrigation solution for
arthroscopy could include both B.sub.1 and B.sub.2 receptor
antagonists.
[0140] Suitable bradykinin receptor antagonists for use in the
present invention include the following bradykinin.sub.1 receptor
antagonists: the [des-Arg.sup.10] derivative of
D-Arg-(Hyp.sup.3-Thi.sup.5-D-Tic.sup.7- -Oic.sup.8)-BK ("the
[des-Arg.sup.10] derivative of HOE 140", available from Hoechst
Pharmaceuticals); and [Leu.sup.8] des-Arg.sup.9-BK. Suitable
bradykinin.sub.2 receptor antagonists include: [D-Phe.sup.7]-BK;
D-Arg-(Hyp.sup.3-Thi.sup.5,8-D-Phe.sup.7)-BK ("NPC 349");
D-Arg-(Hyp.sup.3--D-Phe.sup.7)-BK ("NPC 567"); and
D-Arg-(Hyp.sup.3-Thi.sup.5-D-Tic.sup.7-Oic.sup.8)-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.
8TABLE 8 Therapeutic and Preferred Concentrations of
Pain/Inflammation Inhibitory Agents Therapeutic Preferred
Concentrations Concentrations Class of Agent (Nanomolar)
(Nanomolar) Bradykinin.sub.1 Receptor Antagonists: [Leu.sup.8]
des-Arg.sup.9-BK 1-1,000 50-500 [des-Arg.sup.10] derivative of HOE
140 1-1,000 50-500 [leu.sup.9] [des-Arg.sup.10] kalliden 0.1-500
10-200 Bradykinin.sub.2 Receptor Antagonists: [D-Phe.sup.7]-BK
100-10,000 200-5,000 NPC 349 1-1,000 50-500 NPC 567 1-1,000 50-500
HOE 140 1-1,000 50-500
[0141] E. Kallikrein Inhibitors
[0142] The peptide bradykinin is an important mediator of pain and
inflammation, as noted previously. Bradykinin is produced as a
cleavage product by the action of kallikrein on high molecular
weight kininogens in plasma. Therefore kallikrein inhibitors are
believed to be therapeutic in inhibiting bradykinin production and
resultant pain and inflammation. A suitable kallikrein inhibitor
for use in the present invention is aprotinin. Suitable
concentrations for use in the solutions of the present invention
are set forth below in Table 9.
9TABLE 9 Therapeutic and Preferred Concentrations of
Pain/Inflammation Inhibitory Agents Therapeutic Preferred
Concentrations Concentrations Class of Agent (Nanomolar)
(Nanomolar) Kallikrein Inhibitor: Aprotinin 0.1-1,000 50-500
[0143] F. Tachykinin Receptor Antagonists
[0144] 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. Pharmacol., Vol. 22, pp. 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. Neurokinin.sub.1 Receptor Subtype Antagonists
[0145] Substance P activates the neurokinin receptor subtype
referred to as NK.sub.1. 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,
p. 2273 (1993). A suitable Substance P antagonist is
([D-Pro.sup.9[spiro-gamma-lactam]Leu.sup.10,Trp.sup.11]phys-
alaemin-(1-11)) ("GR 82334"). Other suitable antagonists for use in
the present invention which act on the NK.sub.1 receptor are:
1-imino-2-(2-methoxy-phenyl)-ethyl)-7,7-diphenyl-4-perhydroisoindolone(3a-
R,7aR) ("RP 67580"); and
2S,3S-cis-3-(2-methoxybenzylamino)-2-benzhydrylqu- inuclidine ("CP
96,345"). Suitable concentrations for these agents are set forth in
Table 10.
10TABLE 10 Therapeutic and Preferred Concentrations of
Pain/Inflammation Inhibitory Agents Therapeutic Preferred
Concentrations Concentrations Class of Agent (Nanomolar)
(Nanomolar) Neurokinin.sub.1 Receptor Subtype 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. Neurokinin.sub.2 Receptor Subtle Antagonists
[0146] 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 NK.sub.2. Edmonds-Alt, S., et al., A Potent and
Selective Non-Peptide Antagonist of the Neurokinin A (NK.sub.2)
Receptor, Life Sci. 50:PL111 (1992). In the urinary tract, TKs are
powerful spasmogens acting through only the NK.sub.2 receptor in
the human bladder, as well as the human urethra and ureter. Maggi,
C. A., Gen. Pharmacol., Vol. 22, pp. 1-24 (1991). Thus, the desired
drugs for inclusion in a surgical solution for use in urological
procedures would contain an antagonist to the NK.sub.2 receptor to
reduce spasm. Examples of suitable NK.sub.2 antagonists include:
((S)-N-methyl-N-[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dic-
hlorophenyl)butyl]benzamide ("(1)-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.
11TABLE 11 Therapeutic and Preferred Concentrations of
Pain/Inflammation Inhibitory Agents Therapeutic Preferred
Concentrations Concentrations Class of Agent (Nanomolar)
(Nanomolar) Neurokinin.sub.2 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
[0147] G. CGRP Receptor Antagonists
[0148] 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, Br. J. Pharmacol. 99, p. 202
(1985). An example of a suitable CGRP receptor antagonist is
I-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.
12TABLE 12 Therapeutic and Preferred Concentrations of
Pain/Inflammation Inhibitory Agents Therapeutic Preferred
Concentrations Concentrations Class of Agent (Nanomolar)
(Nanomolar) CGRP Receptor Antagonist: I-CGRP-(8-37) 1-1,000
10-500
[0149] H. Interleukin Receptor Antagonist
[0150] Interleukins are a family of peptides, classified as
cytokines, produced by leukocytes and other cells in response to
inflammatory mediators. Interleukins (1L) may be potent
hyperalgesic agents peripherally. Ferriera, S. H., et al.,
Interleukin-1.theta. as a Potent Hyperalgesic Agent Antagonized by
a Tripeptide Analogue, Nature 334, p. 698 (1988). An example of a
suitable IL-1.theta. receptor antagonist is Lys-D-Pro-Thr, which is
a truncated version of IL-1.theta.. This tripeptide inhibits the
activation of IL-1.theta. receptors. Suitable concentrations for
this agent are provided in Table 13.
13TABLE 13 Therapeutic and Preferred Concentrations of
Pain/Inflammation Inhibitory Agents Therapeutic Preferred
Concentrations Concentrations Class of Agent (Nanomolar)
(Nanomolar) Interleukin Receptor Antagonist: Lys-D-Pro-Thr 1-1,000
10-500
[0151] I. Inhibitors of Enzymes Active in the Synthetic Pathway for
Arachidonic Acid Metabolites
1. Phospholipase Inhibitors
[0152] The production of arachidonic acid by phospholipase A.sub.2
(PLA.sub.2) 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.
[0153] Inhibition of the enzyme PLA.sub.2 isoform inhibits the
release of arachidonic acid from cell membranes, and therefore
inhibits the production of prostaglandins and leukotrienes
resulting in decreased inflammation and pain. Glaser, K. B.,
Regulation of Phospholipase A2 Enzymes: Selective Inhibitors and
Their Pharmacological Potential, Adv. Pharmacol. 32, p. 31 (1995).
An example of a suitable PLA.sub.2 isoform inhibitor is manoalide.
Suitable concentrations for this agent are included in Table 14.
Inhibition of the phospholipase C.sub..gamma. (PLC.sub..gamma.)
isoform also will result in decreased production of prostanoids and
leukotrienes, and, therefore, will result in decreased pain and
inflammation. An example of a PLC.sub..gamma. isoform inhibitor is
1-[6-((17.beta.-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-p-
yrrole-2,5-dione.
14TABLE 14 Therapeutic and Preferred Concentrations of
Pain/Inflammation Inhibitory Agents Therapeutic Preferred
Concentrations Concentrations Class of Agent (Nanomolar)
(Nanomolar) PLA.sub.2 Isoform Inhibitor: manoalide 100-100,000
500-10,000
2. Cyclooxygenase Inhibitors
[0154] 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-1 and induced activity of COX-2 both lead to synthesis of
prostaglandins which contribute to pain and inflammation.
[0155] 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-1, EP-2, EP-3)
are quite rare and only specific, high affinity antagonists of the
thromboxane A2 receptor have been reported. Wallace, J. and Cirino,
G. Trends in Pharm. Sci., Vol. 15 pp. 405-406 (1994).
[0156] 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
(Toradol.TM.), 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 arthroscopy or other
therapeutic or diagnostic procedure.
[0157] 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.
15TABLE 15 Therapeutic and Preferred Concentrations of
Pain/Inflammation Inhibitory Agents Therapeutic Preferred
Concentrations Concentrations Class of Agent (Nanomolar)
(Nanomolar) Cyclooxygenase Inhibitors: ketorolac 100-10,000
500-5,000 indomethacin 1,000-500,000 10,000-200,000
3. Lipooxygenase Inhibitors
[0158] Inhibition of the enzyme lipooxygenase inhibits the
production of leukotrienes, such as leukotriene B4, which is known
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 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.
16TABLE 16 Therapeutic and Preferred Concentrations of
Pain/Inflammation Inhibitory Agents Therapeutic Preferred
Concentrations Concentrations Class of Agent (Nanomolar)
(Nanomolar) Lipooxygenase Inhibitor: AA 861 100-10,000
500-5,000
[0159] J. Prostanoid Receptor Antagonists
[0160] Specific prostanoids produced as metabolites of arachidonic
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 E.sub.2 receptor antagonist is
8-chlorodibenz[b,f][1,4]oxazepine-10(11H)-carboxylic acid,
2-acetylhydrazide ("SC 19220"). A suitable thromboxane receptor
subtype antagonist is [15-[1.alpha., 2.beta.(5Z), 3.beta.,
4.alpha.]-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.
17TABLE 17 Therapeutic and Preferred Concentrations of
Pain/Inflammation Inhibitory Agents Therapeutic Preferred
Concentrations Concentrations Class of Agent (Nanomolar)
(Nanomolar) Eicosanoid EP-1 Antagonist: SC 19220 100-10,000
500-5,000
K. Leukotriene Receptor Antagonists
[0161] The leukotrienes (LTB.sub.4, LTC.sub.4, and LTD.sub.4) 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 are 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. Crit. Rev.
Immunol. 10: 1-12 (1990). The LTB.sub.4 receptor is found in
certain immune cells including eosinophils and neutrophils.
LTB.sub.4 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 LTB.sub.4 receptor involves G-protein-mediated
stimulation of phosphotidylinositol (PI) metabolism and elevation
of intracellular calcium (see FIG. 2).
[0162] An example of a suitable leukotriene B.sub.4 receptor
antagonist is SC
(+)-(S)-7-(3-(2-(cyclopropylmethyl)-3-methoxy-4-[(methylamino)-carbony-
l]phenoxy(propoxy)-3,4-dihydro-8-propyl-2H-1-benzopyran-2-propanoic
acid ("SC 53228"). Concentrations for this agent that are suitable
for the practice of the present invention are provided in Table 18.
Other suitable leukotriene B.sub.4 receptor antagonists include
[3-[-2(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino-3-oxopropy-
l)thio] methyl]thiopropanoic acid ("MK 0571") and the drugs LY
66,071 and ICI 20,3219. MK 0571 also acts as a LTD.sub.4 receptor
subtype antagonist.
18TABLE 18 Therapeutic and Preferred Concentrations of
Pain/Inflammation Inhibitory Agents Therapeutic Preferred
Concentrations Concentrations Class of Agent (Nanomolar)
(Nanomolar) Leukotriene B.sub.4 Antagonist: SC 53228 100-10,000
500-5,000
[0163] L. Opioid Receptor Agonists
[0164] Activation of opioid receptors results in anti-nociceptive
effects and, therefore, agonists to these receptors are desirable.
Opioid receptors include the .mu.-, .delta.- and .kappa.-opioid
receptor subtypes. The .mu.-receptors are located on sensory neuron
terminals in the periphery and activation of these receptors
inhibits sensory neuron activity. Basbaum, A. I., et al., Opiate
analgesia: How Central is a Peripheral Target?, N. Engl. J. Med.,
325:1168 (1991). .delta.- and .kappa.-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, J. Neurosci., Vol. 11, page 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 FIGS. 3 and 7). Examples of suitable
.mu.-opioid receptor agonists are fentanyl and
Try-D-Ala-Gly-[N-MePhe]-NH(CH.sub.2)-OH ("DAMGO"). An example of a
suitable 6-opioid receptor agonist is
[D-Pen.sup.2,D-Pen.sup.5]enkephalin ("DPDPE"). An example of a
suitable .kappa.-opioid receptor agonist is
(trans)-3,4-dichloro-N-methyl-N-[2-(1--
pyrrolidnyl)cyclohexyl]-benzene acetamide ("U50,488"). Suitable
concentrations for each of these agents are set forth in Table
19.
19TABLE 19 Therapeutic and Preferred Concentrations of
Pain/Inflammation Inhibitory Agents Therapeutic Preferred
Concentrations Concentrations Class of Agent (Nanomolar)
(Nanomolar) .mu.-Opioid Agonist: DAMGO 0.1-100 0.5-20 sufentanyl
0.01-50 1-20 fentanyl 0.1-500 10-200 PL 017 0.05-50 0.25-10
.delta.-Opioid Agonist: DPDPE 0.1-500 1.0-100 .kappa.-Opioid
Agonist: U50,488 0.1-500 1.0-100
[0165] M. Purinoceptor Antagonists and Agonists
[0166] Extracellular ATP acts as a signaling molecule through
interactions with P.sub.2 purinoceptors. One major class of
purinoceptors are the P.sub.2x purinoceptors which are ligand-gated
ion channels possessing intrinsic ion channels permeable to
Na.sup.+, K.sup.+, and Ca.sup.2+. P.sub.2X 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 P.sub.2X receptors leading
to depolarization of nociceptive nerve-fiber terminals. The
P2X.sub.3 receptor has a highly restricted distribution (Chen, C.
C., et al., Nature, Vol. 377, pp. 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 P2X.sub.3 receptor subunits make
these subtypes excellent targets for analgesic action (see FIGS. 3
and 7).
[0167] Suitable antagonists of P.sub.2X/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.
[0168] Agonists of the P.sub.2Y receptor, a G-protein coupled
receptor, are known to effect smooth muscle relaxation through
elevation of inositol triphosphate (IP.sub.3) levels with a
subsequent increase in intracellular calcium. An example of a
P.sub.2Y receptor agonist is 2-me-S-ATP.
20TABLE 20 Therapeutic and Preferred Concentrations of
Pain/Inflammation Inhibitory Agents Therapeutic Preferred
Concentrations Concentrations Class of Agent (Nanomolar)
(Nanomolar) Purinoceptor Antagonists: suramin 100-100,000
10,000-100,000 PPADS 100-100,000 10,000-100,000
[0169] N. Adenosine Triphosphate (ATP)-Sensitive Potassium Channel
Openers
[0170] 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.sup.+) efflux and hyperpolarizes the cell membrane
(see FIG. 2). This hyperpolarization induces a reduction in
intracellular free calcium through inhibition of voltage-dependent
calcium (Ca.sup.2+) channels and receptor operated Ca.sup.2+
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.sup.+ channel openers (KCOs) have been
characterized as having potent antihypertensive activity in vivo
and vasorelaxant activity in vitro (see FIG. 4). K.sup.+ 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, Cardiovasc. Res., Vol. 28, pp. 805-810 (1994).
[0171] 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 ET.sub.A receptor subsequently triggers activation
of voltage-dependent channels and the entry of extracellular
calcium which is required for contraction. Antagonists of the
ET.sub.A 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.
[0172] Potassium-channel opener drugs, such as pinacidil, will open
these channels causing K.sup.+ efflux and hyperpolarization 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 Ca.sup.2+ channels
by reducing the probability of opening L-type or T-type calcium
channels, (2) it will restrain agonist induced (receptor operated
channels) Ca.sup.2+ release from intracellular sources through
inhibition of inositol triphosphate (IP.sub.3) 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.
[0173] Suitable ATP-sensitive K.sup.+ channel openers for the
practice of the present invention include: (-)pinacidil;
cromakalim; nicorandil; minoxidil;
N-cyano-N'-[1,1-dimethyl-[2,2,3,3-.sup.3H]propyl]-N"-(3-pyridi-
nyl)guanidine ("P 1075"); and
N-cyano-N'-(2-nitroxyethyl)-3-pyridinecarbox- imidamide
monomethansulphonate ("KRN 2391"). Concentrations for these agents
are set forth in Table 21.
21TABLE 21 Therapeutic and Preferred Concentrations of
Pain/Inflammation Inhibitory Agents Therapeutic Preferred
Concentrations Concentrations Class of Agent (Nanomolar)
(Nanomolar) ATP-Sensitive K.sup.+ Channel Opener: cromakalim
10-10,000 100-10,000 nicorandil 10-10,000 100-10,000 minoxidil
10-10,000 100-10,000 P 1075 0.1-1,000 10-1,000 KRN 2391 1-10,000
100-1,000 (-)pinacidil 1-10,000 100-1,000
III. Anti-Spasm Agents
[0174] A. Multifunction Agents
[0175] 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, serotonin.sub.2 antagonists; tachykinin receptor
antagonists and ATP-sensitive potassium channel openers.
[0176] B. Nitric Oxide Donors
[0177] 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 FIG. 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 FIG. 8). Muramatsu, K., et al., Coron. Artery
Dis., Vol. 5, pp. 815-820 (1994).
[0178] 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, angotensin II and endothelin, all inhibit K.sub.ATP
currents through inhibition of protein kinase A. In addition, it
has been reported that K.sub.ATP 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 FIG. 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.
[0179] 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-acetylpenicillamine
("SNAP"). Concentrations for these agents are set forth in Table
22.
22TABLE 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
[0180] C. Endothelin Receptor Antagonists
[0181] Endothelin is a 21 amino acid peptide that is one of the
most potent vasoconstrictors known. 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 ET.sub.A and ET.sub.B
receptors. The heart and vascular smooth muscle contain
predominantly ET.sub.A receptors and this subtype is responsible
for contraction in these tissues. Furthermore, ET.sub.A receptors
have often been found to mediate contractile responses in isolated
smooth muscle preparations. Antagonists of ET.sub.A receptors have
been found to be potent antagonists of human coronary artery
contractions. Thus, antagonists to the ET.sub.A receptor should be
therapeutically beneficial 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 in Pharmacol. Sci., Vol. 14, pp. 54-60
(1993).
[0182] Suitable endothelin receptor antagonists include:
cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) ("BQ 123");
(N,N-hexamethylene)-carbamoy- l-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-1
H-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-naphth-
alenesulphonamide ("BMS 182874"); and
N-[1-Formyl-N-[N-[(hexahydro-1H-azep-
in-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.
23TABLE 23 Therapeutic and Preferred Concentrations of Spasm
Inhibitory Agents Therapeutic Preferred Concentrations
Concentrations Class of Agent (Nanomolar) (Nanomolar) Endothelin
Receptor Antagonists: 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
[0183] D. Ca.sup.2+ Channel Antagonists
[0184] 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 neuroinflammation.
Calcium entry into platelets and white blood cells is a key event
mediating activation of responses in these cells. Furthermore, the
role of bradykinin receptors and neurokinin receptors (NK.sub.1 and
NK.sub.2) 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 Gilman's Pharmacological
Basis of Therapeutics, (7th ed.), MacMillan Publ. Inc., pp. 660-5
(1995).
[0185] 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 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., Circulation, Vol. 83, pp. 460-468 (1991).
[0186] Calcium channel 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
(Ca.sup.2+) 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.
[0187] 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).
[0188] Calcium channel antagonists also exhibit synergistic effect
with endothelin receptor subtype A (ET.sub.A) antagonists.
Yanagisawa and coworkers observed that dihydropyridine antagonists
blocked effects of ET-1, an endogenous agonist at the ET.sub.A
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
ET.sub.A 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 ET.sub.A antagonist when combined in a
surgical solution.
[0189] Calcium channel antagonists and ATP-sensitive potassium
channel openers likewise exhibit synergistic action. Potassium
channels that are ATP-sensitive (K.sub.ATP) couple the membrane
potential of a cell to the cell's metabolic state via sensitivity
to adenosine nucleotides. K.sub.ATP 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. K.sub.ATP channels hyperpolarize 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 hyperpolarization (1)
induces a reduction in intracellular free calcium through
inhibition of voltage-dependent Ca.sup.2+ channels by reducing the
probability of opening L-type or T-type calcium channels, (2)
restrains agonist induced (at receptor operated channels) Ca.sup.2+
release from intracellular sources through inhibition of inositol
triphosphate (IP.sub.3) 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.
[0190] 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.sub.1
(NK.sub.1) and neurokinin.sub.2 (NK.sub.2) 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
bradykinin.sub.2 (BK.sub.2) 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 neurokinin and bradykinin.sub.2
receptors.
[0191] 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.
24TABLE 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
[0192] 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.
[0193] A. Antiplatelet Agents
[0194] 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
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.
[0195] 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 coronary angioplasty
model (JACC Abstracts, February 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
[0196] 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., 1995, Am. J. Cardiol 75, 12B). 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.
[0197] 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 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, No. 2, pp.
211-218, (1996). In this study, hirulog was released from an
impregnated polymer placed around the artery.
[0198] Other active anti-thrombin agents being tested which are
theorized to be suitable for the present invention are argatroban
(Texas Biotechnology) and efegatran (Lilly).
25TABLE 25 Therapeutic and Preferred Concentrations of Restenosis
Inhibitory Agents Therapeutic/Preferred Concentrations More
preferred Class of Agent (Nanomolar) (Nanomolar) Thrombin
Inhibitors and Receptor Angtagonists: 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)
[0199] 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
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
[0200] 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.
[0201] 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 (R68060) 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, pp. 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.
[0202] B. Inhibitors of Cell Adhesion Molecules
1. Selectin Inhibitors
[0203] 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. Integrin inhibitoIrs
[0204] 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.
[0205] 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 .mu.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,
(1995) Am. Heart J. 130, 673-679. 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.
[0206] 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 .mu.g/kg/min of
integrelin were utilized (Topol, E., 1995 Am. J. Cardiol, 27B-33B).
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.
[0207] 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.
26TABLE 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 .times. K.sub.d 100 .times. K.sub.d
[0208] C. Anti-Chemotactic Agents
[0209] 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-1-1/RANTES 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.
[0210] D. Interleukin Receptor Antagonists
[0211] Interleukin 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 interleukin
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 to as IL-1 antagonist protein (IL-1AP).
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.
[0212] E. Intracellular Signaling Inhibitors
1. Protein Kinase Inhibitors
a. Protein Kinase C (PKC) Inhibitors
[0213] 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.
[0214] 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 IC.sub.50 of 50 .mu.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 IC.sub.50 values in the 2-10 .mu.M range. Concentrations of
these and another drug, GF 109203.times., 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.
27TABLE 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
[0215] 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 FIG. 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 FIG. 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 FIG. 5).
[0216] 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 success following
cardiovascular and general vascular procedures. Any of several
related tyrphostin compounds have potential as specific inhibitors
of PDGF-receptor tyrosine kinase activity (IC.sub.50s in vitro in
the 0.5-1.0 .mu.M range), since they have little effect on other
protein kinases 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 are 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.
28TABLE 28 Therapeutic and Preferred Concentrations of Restenosis
Inhibitory Agents Therapeutic/Preferred More preferred Class of
Agent Concentrations (Nanomolar) (Nanomolar) Protein Kinase
Inhibitors lavendustin A 10-100,000/100-10,000 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
[0217] Non-transmembrane protein tyrosine phosphatases (PTPases)
containing src-homology.sub.2 SH2 domains are known and
nomenclature refers to them as SH-PTP1 and SH-PTP2. In addition,
SH-PTP1 is also known as PTP1C, HCP or SHP. SH-PTP2 is also known
as PTP1D or PTP2C. Similarly, SH-PTP1 is expressed at high levels
in hematopoietic cells of all lineages and all stages of
differentiation, and the SH-PTP1 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 known to
result in the activation of tyrosine kinases that mediate
neutrophil responses (Cui, et al., 1994 J. Immunol.) 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.
[0218] 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, 1995, Current Biology, 5,
1367-1369). 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 Homology.sub.2 Domains)
[0219] 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 FIG. 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
ultimately cellular proliferation (see FIG. 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.
[0220] 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 Crk) 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
(pp60.sup.c-src), abl, Ick, fyn, fgr and others),
phospholipaseC.gamma. (PLC.gamma.), 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.
[0221] 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, inhibitors of specific SH2 domains have the
potential to block many neutrophil functions and serve as
anti-inflammatory mediators.
[0222] Efforts to develop drugs targeted to SH2 domains currently
are being conducted at the biochemical in 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
[0223] 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 FIG. 2). Studies at the cellular level
have shown that actions of calcium channel antagonists are
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
[0224] 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 FIG. 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. Am. Coll. of Cardiol. 21, p. 33A, (1993).
Serruys, P., et al., PARK: the Post Angioplasty Restenosis
Ketanserin Trial, J. Am. Coll. of Cardiol. 21, p. 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.
1. "Crosstalk" and Convergence in Major Signaling Pathways
[0225] 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 transduces
signals from neurotransmitters and hormones through G-protein
coupled receptors (GPCRs) to produce contractile responses using
intracellular targets of trimeric G proteins and Ca.sup.2+ (see
FIG. 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.
[0226] 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 FIGS. 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.
[0227] 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
Ca.sup.2+ which triggers contraction in smooth muscle cells (see
FIG. 2). Each GPCR transmembrane receptor activates a specific
class of trimeric G proteins, including G.sub.q, G.sub.i or many
others. G.sub.I and/or G.sub..theta.K subunits activate
phospholipase C.sub..theta., resulting in activation of protein
kinase C (PKC) and an increase in the levels of cytoplasmic calcium
by release of calcium from intracellular stores.
[0228] 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 C.sub.K, discussed above
with regard to tyrosine kinases). Activation of the PDGF-receptor
also leads to stimulation of PKC and elevation of intracellular
calcium, common steps shared by the GPCRs (see FIG. 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 She, 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 FIG. 2) (Lev et al., 1995, Nature
376:737). 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
[0229] 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 FIG. 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-HT.sub.2 Antagonists
and ET.sub.A Antagonists
[0230] The 5-HT.sub.2 receptor family contains three members
designated 5-HT.sub.2A, 5-HT.sub.2B, and 5-HT.sub.2C, 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 intracellular calcium elevation in smooth muscle
cells induced by ET.sub.A receptor activation requires
extracellular calcium and is at least partially blocked by
nicardipine. Since activation of both 5-HT.sub.2 receptors and
ET.sub.A 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 FIGS. 2 and 4).
4. Synergistic Effects of Protein Tyrosine Kinase Inhibitors and
Calcium Channel Antagonists
[0231] 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 kinase activity.
The substrates for PDGF phosphorylation are many and lead to
activation of mitogen-activated protein kinases (MAPK) and
ultimately proliferation (see FIG. 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 kinase 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.
5. Synergistic Effects of Protein Tyrosine Kinase Inhibitors and
Thrombin Receptor Antagonists
[0232] 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 phospholipse (PLC) through G proteins and activation
of tyrosine kinases. The activation of tyrosine kinase 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, 1992, J. Biol. Chem. 267:5608-5613). 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.
[0233] G. Cyclooxygenase-2 (COX-2) Inhibitors
[0234] 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,
PGG.sub.2 and PGH.sub.2, 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 cytokines.
[0235] The COX-2 isozyme is induced in settings of inflammation by
cytokines and inflammatory mediators, such as IL-1, TNF-.alpha. 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).
[0236] 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 IC.sub.50 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).
[0237] 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 for all of the of
the prostanoid receptors (EP-1, EP-2, EP-3, EP-4, DP, FP, IP and
TP) do not exist.
[0238] 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 (Can J. Physiol. Pharmacol (1997) 75:1088-95). In this
study, among the compounds that were reported to show selectivity
for COX-2 vs. COX1, the rank order of potency was DuP
697>SC-58451,
celecoxib>nimesulide=meloxicam=piroxicam=NS-398=RS-57-
067>SC-57666>SC-58125>flosulide>etodolac>L-745,337>DFU-T-
-614, with IC.sub.50 values ranging from 7 .mu.M to 17 .mu.M. A
good correlation was obtained between the IC.sub.50 values for the
inhibition of microsomal COX-1 and both the inhibition of TXB.sub.2
production by Ca.sup.2+ 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.
[0239] 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 ketorolac, 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 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.
[0240] 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.
[0241] 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.
[0242] Compounds suitable for the invention are listed in the Table
below.
29TABLE 29 Cyclooxygenase-2 Inhibitors Therapeutic Preferred Most
Preferred Compounds Concentrations (.mu.M) Concentrations (.mu.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
[0243] 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 skill in the art. The method of the present
invention involves substituting the anti-pain/anti-inflammatory/a-
nti-spasm/anti-restenosis irrigation solutions of the present
invention for conventionally applied irrigation fluids. The
irrigation solution is applied to 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.
[0244] 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 in 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.
[0245] 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 intra-articular
visualization.
[0246] 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 preferred that the solution include a
combination, and preferably all, or any of the following: a
serotonin.sub.2 receptor antagonist, a serotonin.sub.3 receptor
antagonist, a histamine.sub.1 receptor antagonist, a serotonin
receptor agonist acting on the 1A, 1B, 1D, 1F and/or 1E receptors,
a bradykinin.sub.1 receptor antagonist, a bradykinin.sub.2 receptor
antagonist, and a cyclooxygenase inhibitor.
[0247] 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 serotonin.sub.2 and
histamine.sub.1 "dual" receptor antagonist) are needed per liter of
irrigation fluid to provide the desired effective local tissue
concentrations that would inhibit 5-HT.sub.2 and H.sub.1 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.
[0248] 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.
[0249] For example, using a rat model of arthroscopy, the inventors
examined the ability of amitriptyline, a 5-HT.sub.2 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-articularly) at the
knee and intravenously. The results demonstrated that
intra-articular administration of amitriptyline required total
dosing levels approximately 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-articularly
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.
[0250] 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.
[0251] 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 and inflammation inhibitors as used
in the irrigation solution at the operative site, as an alternative
or supplement to opiates.
[0252] 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-HT.sub.2 receptor antagonist (Saxena, P. R., et al.,
Cardiovascular Effects of Serotonin Inhibitory Agonists and
Antagonists, J Cardiovasc Pharmacol 15 (Suppl. 7), pp. S17-S34
(1990); Douglas, 1985); a 5-HT.sub.3 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
bradykinin.sub.1 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 serotonin.sub.1B (also known as
serotonin.sub.1D.beta.) antagonist because serotonin has been shown
to produce significant vascular spasm via activation of the
serotonin.sub.1B 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, Circulation 90, pp. 1141-53 (1994). This excitatory
action of serotonin.sub.1B receptors in vessel walls, resulting in
vasoconstriction, is in contrast to the previously-discussed
inhibitory action of serotonin.sub.1B 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.
[0253] The solution of the present invention also has utility for
reducing pain and inflammation associated with urologic procedures,
such as trans-urethral 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 Bradykinin, Proc Soc Exp Biol Med 140, pp. 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.sub.1 receptor antagonist to inhibit histamine-induced
pain and inflammation; a 5-HT.sub.3 receptor antagonist to block
activation of these receptors on peripheral C-fiber nociceptive
neurons; a bradykinin.sub.1 antagonist; a bradykinin.sub.2
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 urethral canal and bladder wall.
[0254] 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.
[0255] 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 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 Trauma 23, pp. 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
[0256] 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.
[0257] A. Example I
Irrigation Solution for Arthroscopy
[0258] 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.
30 Concentration (Nanomolar): Class of Agent Drug Therapeutic
Preferred Most Preferred serotonin.sub.2 antagonist amitriptyline
0.1-1,000 50-500 100 serotonin.sub.3 antagonist metoclopramide
10-10,000 200-2,000 1,000 histamine.sub.1 antagonist amitriptyline
0.1-1,000 50-500 200 serotonin.sub.1A,1B,1D,1F sumatriptan 1-1,000
10-200 50 agonist bradykinin.sub.1 antagonist [des-Arg.sup.10]
1-1,000 50-500 200 derivative of HOE 140 bradykinin.sub.2
antagonist HOE 140 1-1,000 50-500 200
B. Example II
Irrigation Solution for Cardiovascular and General Vascular
Therapeutic and Diagnostic Procedures
[0259] 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.
31 Concentration (Nanomolar): Class of Agent Drug Therapeutic
Preferred Most Preferred serotonin.sub.2 antagonist trazodone
0.1-2,000 50-500 200 serotonin.sub.3 antagonist metoclopramide
10-10,000 200-2,000 1,000 serotonin.sub.1B antagonist yohimbine
0.1-1,000 50-500 200 bradykinin.sub.1 antagonist [des-Arg.sup.10]
1-1,000 50-500 200 derivative of HOE 140 cyclooxygenase inhibitor
ketorolac 100-10,000 500-5,000 3,000
C. Example III
Irrigation Solution for Urologic Procedures
[0260] The following drugs and concentration ranges in solution in
a physiologic carrier fluid are suitable for use in irrigating
operative sites during urologic procedures.
32 Concentration (Nanomolar): Class of Agent Drug Therapeutic
Preferred Most Preferred histamine.sub.1 antagonist terfenadine
0.1-1,000 50-500 200 serotonin.sub.3 antagonist metoclopramide
10-10,000 200-2,000 1,000 bradykinin.sub.1 antagonist
[des-Arg.sup.10] 1-1,000 50-500 200 derivative of HOE 140
bradykinin.sub.2 antagonist HOE 140 1-1,000 50-500 200
cyclooxygenase inhibitor 100-10,000 500-5,000 3,000
D. Example IV
Irrigation Solution for Arthroscopy, Burns, General Surgical Wounds
and Oral/Dental Applications
[0261] 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.
33 Concentration (Nanomolar): Most Class of Agent Drug Therapeutic
Preferred Preferred serotonin.sub.2 antagonist amitriptyline
0.1-1,000 50-500 200 serotonin.sub.3 antagonist metoclopramide
10-10,000 200-2,000 1,000 histamine.sub.1 amitriptyline 0.1-1,000
50-500 200 antagonist serotonin .sub.1A,1B,1D, sumatriptan 1-1,000
10-200 100 .sub.1F agonist cyclooxygenase ketorolac 100-10,000
500-5,000 3,000 inhibitor neurokinin.sub.1 GR 82334 1-1,000 10-500
200 antagonist neurokinin.sub.2 (.+-.) SR 48968 1-1,000 10-500 200
antagonist purine.sub.2X antagonist PPADS 100-100,000
10,000-100,000 50,000 ATP-sensitive K.sup.+ (-) pinacidil 1-10,000
100-1,000 500 channel agonist Ca.sup.2+ channel nifedipine 1-10,000
100-5,000 1,000 antagonist kallikrein inhibitor aprotinin 0.1-1,000
50-500 200
E. Example V
Alternate Irrigation Solution for Cardiovascular and General
Vascular Therapeutic and Diagnostic Procedures
[0262] 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.
34 Concentration (Nanomolar): Most Class of Agent Drug Therapeutic
Preferred Preferred serotonin.sub.2 trazodone 0.1-2,000 50-500 200
antagonist cyclooxygenase ketorolac 100-10,000 500-5,000 3,000
inhibitor endothelin BQ 123 0.01-1,000 10-1,000 500 antagonist
ATP-sensitive K.sup.+ (-) pinacidil 1-10,000 100-1,000 500 channel
agonist Ca.sup.2+ channel nisoldipine 1-10,000 100-1,000 500
antagonist nitric oxide SIN-1 10-10,000 100-1,000 500 donor
F. Example VI
Alternate Irrigation Solution for Urologic Procedures
[0263] 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.
35 Concentration (Nanomolar): Class of Agent Drug Therapeutic
Preferred Most Preferred Serotonin.sub.2 LY 53857 0.1-500 1-100 50
antagonist Histamine.sub.1 terfenadine 0.1-1,000 50-500 200
antagonist cyclooxygenase ketorolac 100-10,000 500-5,000 3,000
inhibitor neurokinin.sub.2 SR 48968 1-1,000 10-500 200 antagonist
purine.sub.2X antagonist PPADS 100-100,000 10,000-100,000 50,000
ATP-sensitive K.sup.+ (-) pinacidil 1-10,000 100-1,000 500 channel
agonist Ca.sup.2+ channel nifedipine 1-10,000 100-5,000 1,000
antagonist kallikrein inhibitor aprotinin 0.1-1,000 50-500 200
nitric oxide donor SIN-1 10-10,000 100-1,000 500
G. Example VII
Cardiovascular and General Vascular Anti-Restenosis Irrigation
Solution
[0264] 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.
36 Concentration (Nanomolar): Most Class of Agent Drug Therapeutic
Preferred Preferred thrombin inhibitor hirulog 0.2-20,000 2-2,000
200 glycoprotein IIb/IIIa integrelin 0.1-10,000 .times. Kd 1-1000
.times. Kd 100 .times. Kd receptor blocker PKC inhibitor GF
109203X* 0.1-10,000 1-1,000 200 protein tyrosine tyrphostin
10-100,000 100-20,000 10,000 kinase inhibitor AG1296 *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
[0265] 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:
37 Concentration Class of (Nanomolar): Most Agent Drug Therapeutic
Preferred Preferred NO donor FK 409 (NOR-3) 1-1,000 10-500 250 NO
donor FR 144420 10-10,000 100-5,000 1,000 (NOR-4)
I. Example IX
Alternate Irrigation Solution for Arthroscopy, General Surgical
Wounds, Burns and Oral/Dental Applications
[0266] 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:
38 Concentration Class of (Nanomolar): Most Agent Drug Therapeutic
Preferred Preferred H.sub.1 antagonist mepyramine 0.1-1,000 5-200
100 bradykinin.sub.1 [leu.sup.9][des-Arg.sup.10] 0.1-500 10-200 100
antagonist kalliden bradykinin.sub.2 HOE 140 1-1,000 50-500 200
antagonist .mu.-opioid fentanyl 0.1-500 10-200 100 agonist
[0267] 1) amitriptyline is replaced by mepyramine as the H.sub.1
antagonist;
[0268] 2) the kallikrein inhibitor, aprotinin, is deleted;
[0269] 3) a bradykinin.sub.1 antagonist, [leu.sup.9]
[des-Arg.sup.10] kalliden, is added;
[0270] 4) a bradykinin.sub.2 antagonist, HOE 140, is added; and
[0271] 5) a .mu.-opioid agonist, fentanyl, is added.
J. Example X
Alternate Irrigation Solution for Urologic Procedures
[0272] 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:
[0273] 1) SIN-1 is replaced as the NO donor by a combination of two
agents:
[0274] a) FK 409 (NOR-3); and
[0275] b) FR 144420 (NOR-4);
[0276] 2) the kallikrein inhibitor, aprotinin, is deleted;
[0277] 3) a bradykinin.sub.1 antagonist, [leu.sup.9]
[des-Arg.sup.10] kalliden, is added; and
[0278] 4) a bradykinin.sub.2 antagonist, HOE 150, is added.
39 Concentration Class of (Nanomolar): Most Agent Drug Therapeutic
Preferred Preferred NO donor FK 409 (NOR-3) 1-1,000 10-500 250 NO
donor FR 144420 10-10,000 100-5,000 1,000 (NOR-4) bradykinin.sub.1
[leu.sup.9][des-Arg.sup.10] 0.1-500 10-200 100 antagonist kalliden
bradykinin.sub.2 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 Histamine/Serotonin Receptor
Blockade on the Response
[0279] 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
[0280] 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). 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 serotonin.sub.3
antagonist metoclopramide at a concentration of 16.0 .mu.M; (b) the
serotonin.sub.2 antagonist trazodone at a concentration of 1.6
.mu.M; and (c) the histamine antagonist promethazine at
concentrations of 1.0 .mu.M, 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
[0281] 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 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, N.J.) 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, Calif.) 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 Corp., Miami, Fla.) 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
[0282] 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 Oust 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.
[0283] The diameter measurements were then converted to area
measurements by the formula:
Area=(Pi)(Diameter.sup.2)/4.
[0284] For calculation of vasoconstriction, baseline values were
used to represent the maximum area of the artery and percent
vasoconstriction was calculated as: % Vasoconstriction={(Baseline
area-Later time point area)/Baseline area}.times.100.
4. Statistical Methods
[0285] 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, Okla.).
5. Results
[0286] 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 1.82.+-.0.05 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 slight increase in lumen
diameter from baseline to the 30 minute time point (1.82.+-.0.05 mm
to 1.94.+-.0.07 mm, p=NS by post hoc testing).
40TABLE 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 Proximal.sup.1 2.18 .+-. 0.7 2.03 .+-. 0.7 1.81 .+-. 0.08*
2.00 .+-. .08 2.23 .+-. .08 Balloon.sup.2 1.82 .+-. .05 1.77 .+-.
.03 1.79 .+-. .05 1.70 .+-. .04 1.94 .+-. .07 Distal.sup.3 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. .sup.1p = 0.0002 (ANOVA within group
comparison), .sup.2p = 0.03 (ANOVA within group comparison),
.sup.3p < 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.
[0287] 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 FIG. 9; the changes in the amount of
vasoconstriction over time are 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 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.
[0288] 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.
[0289] The histamine/serotonin receptor blockade solution was
infused into 16 arteries (8 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 (-14.+-.5 mm Hg, p=0.04) and a lower heart
rate (-26.+-.10, p=0.05). Within the control animals, there was no
change in heart rate or systolic blood pressure over the duration
of the experiment.
41TABLE 31 Systolic Blood Pressure and Heart Rate Measurements in
Control and Histamine/serotonin Treated Animals Baseline 5 Minute
15 Minute 30 Minute Group (N) (N) (N) (N) Systolic Blood Pressure
83 .+-. 4 (8) 84 .+-. 4 (8) 82 .+-. 6 (8) 80 .+-. 4 (8) Control
Histamine/Serotonin 93 .+-. 5 (6) 87 .+-. 9 (4) 82 .+-. 9 (6) 80
.+-. 8 (6)* Heart Rate 221 .+-. 18 (5) 234 .+-. 18 (4) 217 .+-. 23
(5) 227 .+-. 22 (5) Control Histamine/Serotonin 232 .+-. 8 (5) 232
.+-. 8 (5) 209 .+-. 14 (5) 206 .+-. 12 (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.
[0290] The proximal and distal segments of histamine/serotonin
treated arteries were compared to control arteries using the
percent vasoconstriction measurement. FIG. 10A 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 FIG. 10B. 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). Lack of
statistical significance may be attributed to smaller than expected
vasoconstriction values in the control vessels.
L. Example XII
Amitriptyline Inhibition of 5-Hydroxytryptamine-Induced Knee Joint
Plasma Extravasation--Comparison of Intra-Articular Versus
Intravenous Routes of Administration
[0291] The following study was undertaken in order to compare two
routes of administration of the 5-HT.sub.2 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
[0292] 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,
Calif.) 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
[0293] 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 .mu.l/min) was controlled by a Sage Instruments Syringe
pump (Model 341B, Orion Research Inc., Boston, Mass.). A 25-gauge
needle was also inserted into the joint space and perfusate fluid
was extracted at 250 .mu.l/min, controlled by a Sage Instruments
Syringe pump (Model 351).
[0294] The rats were randomly assigned to three groups: 1) those
receiving only intra-articular (IA) 5-HT (1 .mu.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 .mu.M) followed by IA 5-HT (1 .mu.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 5 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 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
.mu.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
intra-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.
[0295] 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
[0296] 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 FIGS. 11 and 12 as a dashed line.
[0297] 5-HT (1 .mu.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 intra-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 FIGS. 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
[0298] Amitriptyline administered via tail vein injection produced
a dose-dependent decrease in 5-HT-induced plasma extravasation as
shown in FIG. 11. The IC.sub.50 for 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
[0299] 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 FIG. 12. 5-HT-induced plasma
extravasation in the presence of 3 .mu.M IA amitriptyline was not
significantly different from that produced by 5-HT alone, however,
30 .mu.M amitriptyline co-perfused with 5-HT produced a greater
than 50% inhibition, while 100 .mu.M amitriptyline produced
complete inhibition of 5-HT-induced plasma extravasation. The
IC.sub.50 for IA amitriptyline inhibition of 5-HT-induced plasma
extravasation is approximately 20 .mu.M.
[0300] The major finding of the present study is that 5-HT (1
.mu.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-HT.sub.2 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 IC.sub.50 for IV amitriptyline inhibition of
5-HT-induced plasma extravasation is 0.025 mg/kg, or
7.5.times.10.sup.-3 mg in a 300 g adult rat. The IC.sub.50 for IA
amitriptyline inhibition of 5-HT-induced plasma extravasation is
approximately 20 .mu.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 knee was 7 ml, for a total dosage of
4.4.times.10.sup.-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.
[0301] 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 perioperative
period. A recent study attempted to determine the effects of oral
amitriptyline on post-operative orthopedic pain (Kerrick et al.,
1993). An oral dose as 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 in Rabbit Arteries
1. Solution Tested
[0302] 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 Ca.sup.2+ channel
antagonist.
[0303] The concentration of nitroprusside was selected based on its
previously-defined pharmacological activity (EC.sub.50). 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
[0304] 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 kg 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
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 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 iopamidol 76% (Squibb Diagnostics,
Princeton, N.J.) into the descending aorta.
[0305] 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.
[0306] 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. Angiographic Analysis
[0307] 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 Vanguard 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. 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, R.I.). 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 15 minutes after the
infusion was stopped (30 minutes after the rotational atherectomy
was begun). The calibration object was measured in each
angiogram.
[0308] The diameter measurements were then converted to area
measurements by the formula:
Area=(Pi)(Diameter.sup.2)/4.
[0309] For calculation of vasoconstriction, baseline values were
used to represent the maximum area of the artery and percent
vasoconstriction was calculated as:
% Vasoconstriction={(Baseline area-Later time point area)/Baseline
area}.DELTA.100.
4. Statistical Methods
[0310] 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, Okla.).
5. Results
[0311] 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.
[0312] 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.
42TABLE 32 Iliac Artery Lumen Diameters at Specified Time Points in
Saline Treated Arteries 5 Minutes into Immediate 15 Minute 30
Minutes Baseline Infusion Post RA after RA after RA Segment N = 8 N
= 8 N = 8 N = 8 N = 8 Proximal.sup.1 2.40 .+-. .18 2.32 .+-. .14
2.32 .+-. 0.13 2.38 .+-. .13 2.34 .+-. .07* Mid.sup.2 2.01 .+-. .08
1.84 .+-. .09 1.57 .+-. .15 1.24 .+-. .13 1.87 .+-. .06**
Distal.sup.3 2.01 .+-. .10 1.86 .+-. .08 1.79 .+-. .08 1.81 .+-.
.09 1.96 .+-. .06*** RA = rotational atherectomy .sup.1Proximal
iliac artery measurement site, proximal to the first deep femoral
branch .sup.2Mid iliac artery at the site of maximal vasospasm
.sup.3Distal 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.
[0313] 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.
43TABLE 33 Iliac Artery Lumen Diameters at Specified Time Points in
Test Solution Treated Arteries 5 Minutes Base- into Immediate 15
Minute 30 Minutes line Infusion Post RA after RA after RA Segment N
= 13 N = 10 N = 13 N = 13 N = 11 Proximal.sup.1 2.28 .+-. .06 2.07
.+-. .07 2.22 .+-. .05 2.42 .+-. .06 2.39 .+-. .08 Mid.sup.2 1.97
.+-. .06 1.79 .+-. .06 1.74 .+-. .04 1.95 .+-. .07 1.93 .+-. .08
Distal.sup.3 2.00 .+-. .06 1.92 .+-. .04 1.90 .+-. .04 2.00 .+-.
.06 2.01 .+-. .07 RA = rotational atherectomy .sup.1Proximal iliac
artery measurement site, proximal to the first deep femoral branch
.sup.2Mid iliac artery at the site of maximal vasospasm
.sup.3Distal iliac artery measurement site, near or distal to the
second deep femoral branch 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.
[0314] 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.
[0315] In the proximal segment (FIG. 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 postrotational
atherectomy time point. Test solution did not alter the results of
rotational atherectomy compared to saline treatment in this
segment. In the mid-vessel (FIG. 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 (FIG. 15), there was little
vasoconstriction in the saline treated arteries and test solution
did not significantly alter the response to rotational
atherectomy.
44TABLE 34 Amount of Vasoconstriction (Negative Values) or
Vasodilatation (Positive Values) at Specified Time Points in Saline
Treated Arteries 5 Minutes Immediate 15 Minute 30 Minutes into
Infusion Post RA after RA after RA Segment N = 8 N = 8 N = 8 N = 8
Proximal.sup.1 -3% .+-. .8% -1% .+-. 10% .sup. 3% .+-. 8% 3% .+-.
13% Mid.sup.2 -14% .+-. 7% -35% .+-. 10% -58% .+-. 7% -11% .+-. .9%
Distal.sup.3 -9% .+-. .10% -14% .+-. .14% -14% .+-. 10% 2% .+-.
.12% .sup.1Proximal iliac artery measurement site, proximal to the
first deep femoral branch .sup.2Mid iliac artery at the site of
maximal vasospasm .sup.3Distal iliac artery measurement site, near
or distal to the second deep femoral branch
[0316]
45TABLE 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 Proximal.sup.1 -17% .+-. .5% -4% .+-. 3% 14% .+-. 6% 7%
.+-. 9% Mid.sup.2 -14% .+-. 5% -20% .+-. 5% 0.3% .+-. 7% -5% .+-.
.5% Distal.sup.3 -8% .+-. .4% -9% .+-. .4% 1% .+-. 4% 3% .+-. .6%
.sup.1Proximal iliac artery measurement site, proximal to the first
deep femoral branch .sup.2Mid iliac artery at the site of maximal
vasospasm .sup.3Distal iliac artery measurement site, near or
distal to the second deep femoral branch
[0317] 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 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.
46TABLE 36 Blood Pressure and Heart Rates During the Protocol
Baseline 5 Minute 15 Minute 30 Minute Group (N) (N) (N) (N)
Systolic Blood Pressure Saline 83 .+-. 9 (4) 93 .+-. 6 (3) .sup. 92
.+-. 11(4).sup. 83 .+-. 10 (4)* Test 92 .+-. 5 (7) 35 .+-. 5 (7) 35
.+-. 5 (7) 46 .+-. 5 (7)** Solution Heart Rate Saline 202 .+-. 16
(3) 204 .+-. 3 (3) 198 .+-. 22 (3) 193 .+-. 29 (3)* Test .sup. 187
.+-. 111(7).sup. 246 .+-. 11 (7) 240 .+-. 5 (7) 247 .+-. 16 (7)**
Solution *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
[0318] 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.
[0319] 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.
[0320] 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.
[0321] 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-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.
* * * * *