EP1567179A1 - Solution d'irrigation et methode d'inhibition de l'adhesion de cellules tumorales, de douleurs et d'inflammations - Google Patents

Solution d'irrigation et methode d'inhibition de l'adhesion de cellules tumorales, de douleurs et d'inflammations

Info

Publication number
EP1567179A1
EP1567179A1 EP02793939A EP02793939A EP1567179A1 EP 1567179 A1 EP1567179 A1 EP 1567179A1 EP 02793939 A EP02793939 A EP 02793939A EP 02793939 A EP02793939 A EP 02793939A EP 1567179 A1 EP1567179 A1 EP 1567179A1
Authority
EP
European Patent Office
Prior art keywords
receptor
antagonists
agents
solution
pain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02793939A
Other languages
German (de)
English (en)
Other versions
EP1567179A4 (fr
Inventor
Gregory A. Demopulos
Pamela Pierce Palmer
Jeffrey M. Herz
Darrell Tanelian
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Omeros Corp
Original Assignee
Omeros Medical Systems Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Omeros Medical Systems Inc filed Critical Omeros Medical Systems Inc
Publication of EP1567179A1 publication Critical patent/EP1567179A1/fr
Publication of EP1567179A4 publication Critical patent/EP1567179A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/178—Lectin superfamily, e.g. selectins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • 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.
  • 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.
  • a body cavity e.g., joint, peritoneal cavity, bladder, thorax, etc.
  • surgical instruments may be placed in a body cavity, their use guided by arthroscopic visualization.
  • 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.
  • pediatric endoscopy has become routine.
  • physiologic irrigation fluid e.g., normal saline or lactated Ringer's
  • physiologic irrigation fluid e.g., normal saline or lactated Ringer's
  • U.S. Patent 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.
  • 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.
  • 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.
  • 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).
  • MMPs metalloproteinases
  • TMPs tissue inhibitors of metalloproteinases
  • 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.
  • 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.
  • 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.
  • 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.
  • cyclooxygenase inhibitors e.g., ibuprofen
  • opioids e.g., morphine, fentanyl
  • 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.
  • 5-HT applied to a human blister base has been demonstrated to cause pain that can be inhibited by 5-HT 3 receptor antagonists.
  • bradykinin receptor antagonists 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. 7(5:99-104 (1993). Peripherally applied histamine produces vasodilation, itching and pain that can be inhibited by histamine receptor antagonists.
  • 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 73:512-518 (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.
  • prostaglandms also are known to cause pain and inflammation.
  • Cyclooxygenase inhibitors e.g., ibuprofen
  • ibuprofen are commonly used in non-surgical and post-operative settings to block the production of prostaglandms, 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.
  • 5-HT, histamine, bradykinin and prostaglandms 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.
  • 5-HT and norepinephrine uptake antagonists which includes amitriptyline
  • 5-HT and norepinephrine uptake antagonists which includes amitriptyline
  • 5-HT and norepinephrine uptake antagonists which includes amitriptyline
  • amitriptyline has been used orally with moderate success for chronic pain conditions.
  • the mechanisms of chronic versus acute pain states are thought to be considerably different.
  • two studies in the acute pain setting using amitriptyline perioperatively have shown no pain-relieving effect of amitriptyline.
  • 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.
  • Hj histamine ⁇
  • 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.
  • a method for reducing or preventing tumor cell adhesion, and/or invasion and/or local metastasis 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.
  • 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.
  • 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 may provide an additive or synergistic benefit to the pharmaceutical composition.
  • This invention utilizes the approach of combining agents that act simultaneously on molecular targets associated with tumor cell adhesion, and/or tumor cell invasion, and/or metastasis, and/or inflammation, and/or pain, and/or smooth muscle spasm, and/or restenosis and delivering these agents to the operative site throughout the surgical procedure.
  • 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.
  • 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.
  • 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, selectins, protein tyrosine kinases, MMPs and MAP Kinases).
  • a particular suitable agent may interact specifically with one or more receptor subtypes (or isotypes) while exhibiting specificity for the receptor family.
  • Each suitable agent will associate with its molecular target through a specific molecular mechanism which can be characterized by a defined stoichiometry (typically 1 :1) for the ligand-receptor (or inhibitor-enzyme) complex and which can be characterized by its equilibrium binding or kinetic constant.
  • At least one second agent belongs to a class of receptor antagonists, enzyme inhibitors or cytokines that possess anti-pain, anti-inflammatory, anti-spasm, and/or anti- restenosis activity.
  • the optimal drug combination includes at least one agent drawn from a class of anti-tumor cell adhesion and/or anti-invasion agents which include receptor antagonists that act to inhibit and reduce the activity 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.
  • 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.
  • 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.
  • 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.
  • 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 - ti-inflammatory and/or anti-spasm and/or anti-restenosis agents.
  • a significant advantage of the invention is derived from the rapid onset of pharmacological action.
  • Direct, local delivery of the anti-tumor adhesion agents initiated at the outset of the surgical procedure and continued during the surgical procedure (i.e., perioperatively) has the potential to inhibit one of the earliest steps of metastasis, the initial adhesion process.
  • the immediate and constant delivery of therapeutically effective concentrations of anti-adhesion and/or invasion agents is expected to preemptively inhibit the initial attachment of tumor cells to both the extracellular matrix and other cells and subsequent invasion into these tissues. Studies have found that the highest rate of tumor implantation occurred when tumor cells were injected immediately after surgery, indicative of the critical timing for therapeutic inhibition of the adhesion process.
  • the method of delivery and pharmaceutical composition of the present invention provide distinct advantages which include: 1) a combination drug therapy directed to the multifactorial causes of tumor cell adhesion/invasion/local metastasis, inflammation, pain, smooth muscle spasm and restenosis during surgical procedures, 2) local delivery of the drug combination achieves an instantaneous therapeutic concentration of anti-tumor adhesion agents at the operative site, 3) continuous delivery of therapeutic agents in an irrigation solution provides a constant drug concentration in a therapeutically effective range at the operative site during a surgical procedure, 4) local delivery permits a reduction in total drug dose and dosing frequency compared to systemic delivery, and 5) direct, local delivery to the surgical site enables use of novel, pharmaceutically active agents, such as certain peptides and antibodies, which cannot be delivered systemically.
  • the present invention further provides a solution constituting a mixture of multiple agents in low concentrations directed at inhibiting locally the mediators of pain, inflammation, spasm and restenosis in a physiologic electrolyte carrier fluid.
  • the invention also provides a method for perioperative delivery of the irrigation solution containing these agents directly to a surgical site, where it works locally at the receptor and enzyme levels to preemptively limit pain, inflammation, spasm and restenosis at the site. Due to the local perioperative delivery method of the present invention, a desired therapeutic effect can be achieved with lower doses of agents than are necessary when employing other methods of delivery (i.e., intravenous, intramuscular, subcutaneous and oral).
  • the anti -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 j and neurokinin 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 2 isoform inhibitors and PLC ⁇ isoform inhibitors,
  • cyclooxygenase inhibitors include 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; (l l) leukotriene receptor antagonists ' including leukotriene B4 receptor subtype antagonists and leukotriene D 4 receptor subtype antagonists; (12) opioid receptor agonists, including ⁇ -opioid, ⁇ -opioid, and
  • K-opioid receptor subtype agonists (13) purinoceptor agonists and antagonists including P 2 ⁇ receptor antagonists and P 2 ⁇ 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.
  • Several preferred embodiments of the solution of the present invention also include anti-spasm agents for particular applications.
  • 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.
  • anti-spasm agents are utilized in the solution.
  • 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).
  • anti-restenosis agents include: (1) antiplatelet agents including: (a) thrombin inhibitors and receptor antagonists, (b) adenosine disphosphate (ADP) receptor antagonists (also known as purinoceptori 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) infracellular signaling inhibitors including:
  • PLC protein kinase C
  • PLC protein kinase C
  • modulators of intracellular protein tyrosine phosphatases (c) inhibitors of src homology 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.
  • 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-inflammatory agents, and for some applications anti-spasm agents and/or anti-restenosis agents, each agent included at a concentration of preferably no more than 100,000 nanomolar, and more preferably no more than 10,000 nanomolar.
  • the method of the present invention provides for the delivery of a dilute combination of at least one inhibitor of tumor cell adhesion, invasion and/or metastasis and preferably multiple receptor antagonists and agonists and enzyme inhibitors and activators directly to a wound or operative site, during therapeutic or diagnostic procedures for the inhibition of pain, inflammation, spasm and restenosis. Since the active ingredients in the solution are being locally applied directly to the operative tissues in a continuous fashion, the drugs may be used efficaciously at extremely low doses relative to those doses required for therapeutic effect when the same drugs are delivered orally, intramuscularly, subcutaneously or intravenously.
  • 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.
  • continuous encompasses uninterrupted application, repeated application at frequent intervals (e.g., repeated intravascular boluses at frequent intervals mtraprocedurally), and applications which are uninterrupted except for brief cessations such as to permit the introduction of other drugs or agents or procedural equipment, such that a substantially constant predetermined concentration is maintained locally at the wound or operative site.
  • the advantages of 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.
  • agents in the following classes are peptidic: bradykinin receptor antagonists; tachykinin receptor antagonists; opioid receptor agonists; CGRP receptor antagonists; and interleukin receptor antagonists.
  • Local, continuous delivery to the wound or operative site minimizes drug degradation or metabolism while also providing for the continuous replacement of that portion of the agent that may be degraded, to ensure that a local therapeutic concentration, sufficient to maintain receptor occupancy, is maintained throughout the duration of the operative procedure.
  • Local administration of the solution perioperatively throughout a surgical procedure in accordance with the present invention produces a preemptive analgesic, anti-inflammatory, anti-spasmodic or anti-restenotic effect.
  • the term "perioperative” encompasses application mtraprocedurally, pre- and mtraprocedurally, intra- and postprocedurally, and pre-, intra- and postprocedurally.
  • the solutions of the present invention are most preferably applied pre-, intra- and postoperatively.
  • 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.
  • 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, iriflammation, 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.
  • 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.
  • wound unless otherwise specified, is intended to include surgical wounds, operative/interventional sites, traumatic wounds and burns.
  • 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.
  • analgesic i.e., opiate
  • FIGURE 1 provides a schematic overview of a generic vascular cell showing molecular targets and flow of signaling information leading to contraction, secretion and/or proliferation.
  • the integration of extrinsic signals through receptors, ion channels and other membrane proteins 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.
  • FIGURE 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.
  • GPCR G-protein coupled receptor
  • RTK receptor tyrosine kinase
  • GPCR protein kinase C
  • PYK2 a newly discovered protein tyrosine kinase
  • PTK-X an undefined protein tyrosine kinase
  • LGR designates ligand-gated receptor
  • MAPK mitogen-activated protein kinase
  • FIGURE 3 provides a diagram of the G-Protein Coupled Receptor (GPCR) pathway. Specific molecular sites of action for some drags in a preferred arthroscopic solution of the present invention are identified.
  • GPCR G-Protein Coupled Receptor
  • FIGURE 4 provides a diagram of the G-Protein Coupled Receptor (GPCR) pathway including the signaling proteins responsible for "crosstalk" with the Growth Factor Receptor signaling pathway. Specific molecular sites of action for some drugs in a preferred cardiovascular and general vascular solution of the present invention are identified. (See also FIGURE 5.)
  • GPCR G-Protein Coupled Receptor
  • FIGURE 5 provides a diagram of the Growth Factor Receptor signaling pathway including the signaling proteins responsible for "crosstalk" with the G-Protein Coupled Receptor signaling pathway. Specific molecular sites of action for some drugs in a preferred cardiovascular and general vascular solution of the present invention are identified. (See also FIGURE 4.)
  • FIGURE 6 provides a diagram of the G-Protein Coupled Receptor pathway including the signaling proteins responsible for "crosstalk" with the Growth Factor Receptor signaling pathway. Specific molecular sites of action for some drugs in a preferred urologic solution are identified.
  • FIGURE 7 provides a diagram of the G-Protein Coupled Receptor pathway. Specific molecular sites of action for some drugs in a preferred general surgical wound solution of the present invention are identified.
  • FIGURE 8 provides a diagram of the mechanism of action of nitric oxide (NO) donor drugs and NO causing relaxation of vascular smooth muscle.
  • NO nitric oxide
  • 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.
  • GC soluble guanylate cyclase
  • PKG cGMP-dependent protein kinase
  • NO also hyperpolarizes the cell by opening potassium channels which in turn cause closure of voltage-sensitive calcium channels.
  • FIGURES 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.
  • FIGURES 11 and 12 provide charts of plasma extravasation versus dosage of amitriptyline, used in the solutions of the present invention, delivered intravenously and intra-articularly, respectively, to knee joints in which extravasation has been induced by introduction of 5-hydroxytryptamine in the animal study described in EXAMPLE VIII herein.
  • 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.
  • 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.
  • MMPs Matrix metalloproteinases
  • ECM extracellular matrix
  • This activity serves as a key control factor in the adherence, invasive capacity, metastasis, growth, and angiogenesis of tumors.
  • cloning and sequencing have identified 17 members of this family. These members are divided into subgroups of collagenases, gelatinases, stromelysins and stromelysin-like MMPs, membrane-type-MMPs, and novel MMPs.
  • the MMPs share common structural and functional characteristics. All families to date have at least three domains: a prodomain which contains a conserved cysteine residue and which is lost on enzyme activation; a catalytic domain of 106 to 119 residues which contains conserved structural metal-binding sites; and a highly conserved zinc- binding active site domain of 52 to 58 amino acids.
  • MMP-8 and MMP-9 that are stored in secretory granules of neutrophils and eosinophils
  • MMP-7 that is stored in secretory epithelial cells.
  • MMPs are 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.
  • cytokines IL-1, tumor necrosis factor, etc.
  • 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.
  • TIMPs matrix metalloproteinases
  • TACE TNF-alpha-converting enzyme
  • TIMP-2 is expressed constitutively and TIMP-4 expression is restricted.
  • TIMP-3 expression is up-regulated by mitogens, phorbol esters and TGF-beta.
  • the MMPs have been shown to facilitate tumor cell invasion and metastasis by at least three mechanisms. In a physiological sense, first, the MMPs can modulate tumor cell adhesion. This process is necessary for cells to attach and move through the ECM. Cell transfection studies altering the ratio of MMP-2 to TIMP-2 have demonstrated variation in the adhesive phenotype of tumor cells. Next, the proteinase action of MMPs can degrade ECM macromolecules such as collagens, laminins, and proteoglycans allowing tumor cell invasion. Finally, MMPs can act on ECM components or other proteins to activate biological functionality. For example, laminin-5 is degraded by MMP-2 to produce a soluble chemotactic fragment.
  • MMPs The classic role of MMPs to degrade ECM components, and in particular the basement membrane is considered critical for tumor cell invasion.
  • 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.
  • TIMPs 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.
  • 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.
  • MMP activity can inhibit adhesion, invasion, and metastasis of neoplastic cells and effectively suppress tumor growth and metastasis.
  • Prevention of tumor growth can be accomplished by inhibition of MMP expression or activity at multiple steps in the synthetic pathway of MMPs.
  • Inhibition of MMP transcription can be accomplished by blocking signaling pathways regulating MMP transcription directly or by inhibiting transcription factors responsible for activating transcription of MMP genes. Inhibition of signaling pathways involved in AP-1 activation, the MAPKs (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.
  • Another method for inhibiting expression of specific MMPs is to use antisense RNAs.
  • Antisense ohgonucleotides 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.
  • 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 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.
  • MMPIs 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.
  • BB-94 batimastat
  • marimastat BB-2516
  • results of pre-clinical studies show these agents to inhibit tumor invasion, metastasis, and growth.
  • 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.
  • non-peptidic inhibitors examples include CGS27023A (Novartis), AG3340 (Agouron), and the fenbufen derivative BAY12-9566 (Bayer).
  • CMTs chemically modified nonmicrobial tetracyclines
  • TIMPs 1, 2, 3, and 4 can inhibit invasion of malignant cells in vitro and in vivo.
  • 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.
  • 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.
  • MMP inhibitors suitable for use in the present invention are listed in the Table below.
  • Marimastat 0.1-10,000 100
  • Antisense RNA 0.5-5,000 5,000
  • TIMPS 1 0.1-10,000 1,000
  • CD44 is a transmembrane glycoprotein that comprises a family of surface receptors composed of numerous variant isoforms.
  • the CD44 receptor is found in a wide variety of tissues including the central nervous system, lung, epidermis, liver, colon and pancreas. This family of related cell surface adhesion molecules are multifunctional proteins expressed in both normal and malignant tissues.
  • CD44 is encoded by a single gene containing 20 exons, 10 of which (vl-vlO) 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.
  • CD44v CD44 which contain different combinations of exons (vl-vlO) inserted into the extracellular domain are restricted in their expression to proliferating epithelial cells, activated lymphocytes and malignant cells.
  • 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.
  • extracellular matrix ligands chondroitin sulphate, heparin sulphate, and fibronectin
  • 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.
  • HA hyaluronic acid
  • the CD44 molecule is an 85kD 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 85kD form has been identified in serum, plasma and synovial fluid. Alternative splicing creates a hematopoietic form of the molecule, CD44H, and additional subtypes. Alternative splicing results in changes in protein glycosylation and provides a mechanism for the regulation of binding activity of CD44. Thus, the CD44 molecule, by existing in several isoforms and due to its wide cellular distribution and functional association with other adhesion molecules, is a multifunctional proinflammatory molecule involved in immune cell activation as well as metastasis of certain tumor cell types.
  • CD44H 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 Abs 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. 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.
  • specific isoforms enhance tumor cell metastasis more efficiently than others do.
  • Metastatic potential can be conferred to non-metastasizing cell lines by fransfection with a variant of CD44 and high levels of CD44 are associated with several types of malignant tumors.
  • CD44 isoform expression 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.
  • 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.
  • 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.
  • PKC Protein kinase C 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.
  • 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.
  • 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.
  • 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.
  • Direct receptor antagonists include monoclonal or polyclonal antibodies made against variants of CD44, or fragments thereof, which can be used as inhibitors of HA binding to CD44 and can be employed as therapeutic agents.
  • Anti-CD44 monoclonal antibodies that can be employed include mAbs that recognize epitopes present on all isoforms of CD44 or suitable mAbs that recognize only post-translationally modified forms of CD44. Isoform (variant)-specific anti-CD44 mAbs have been described previously (F12, A1G3, A3D8) in U.S. Patent No. 5,863,540 and the mAbs IM7 and BU52.
  • Other monoclonal antibodies suitable for the present invention include anti-CD44 mAbs directed against unique CD44 isoforms that are produced as a result of alternative splicing with ten variable exons.
  • the present invention discloses the use of a recombinantly produced, chimeric soluble CD44 (CSCD44) protein, in which the extracellular domain, or a portion thereof, of a CD44 receptor is covalently linked to a constant domain of an IgG molecule which may be used as an anti-tumor, anti-cell adhesion and anti-metastasis agent.
  • CSCD44 chimeric soluble CD44
  • IgG molecule which may be used as an anti-tumor, anti-cell adhesion and anti-metastasis agent.
  • a chimeric polypeptide comprising the extracellular domain of the CD44 receptor extracellular polypeptide coupled to the CH2 and CH3 regions of a mouse IgGl heavy chain polypeptide could be employed.
  • a second example is a chimeric fusion construct comprised of the HA ligand binding domain of the CD44 receptor with portions of the Fc antibody (termed Fc fusion soluble receptors) that have been created for CD44 receptors.
  • the molecular form of the active soluble CD44 receptor can be either monomeric or dimeric. Methods have been established to test soluble CD44 antibody binding to the variant isoforms of CD44 expressed on cell surfaces.
  • 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.
  • hyaluronan oligomers injected at concentrations as low as 1 mg/ml inhibited melanoma growth.
  • 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.
  • 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.
  • the amounts of the peptide, protein or antibody to be administered for a particular treatment protocol may be a function of a particular CD44 domain associated with a pathologic clinical condition, and may be a function of a particular cell type mediating metastasis .
  • CD44 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.
  • 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.
  • the integrins have been implicated in cancer cell differentiation, tumor progression and metastasis due to their important role in mediating tumor cell interactions with the extracellular matrix and endothelial cells. Integrin expression and function are altered in many malignant cells, although changes in integrin expression vary between different tumor types.
  • Specific integrin receptor subtypes on tumor cells are capable of mediating attachment to many adhesive proteins that are components of the extracellular matrix, activated platelets and endothelial cells at sites of surgical trauma.
  • the integrins are a family of heterodimeric, transmembrane ⁇ receptors that are expressed on a wide variety of cells.
  • the receptor family includes at least 14 known -subunits and 8 ⁇ -subunits that associate with each other to form numerous subtype combinations that demonstrate differing ligand specificities.
  • the -subunit appears to be a critical determinant of ligand specificity and integrins containing ⁇ v demonstrate specificity for vitronectin, ⁇ 5 for fibronectin, and ⁇ 3 for coUagen laminin.
  • Vascular cell adhesion molecule- 1 (VCAM-1) is the endothelial cell ligand for two leukocyte integrins, 4 ⁇ l and ⁇ 4 ⁇ 7.
  • Mucosal addressin cell adhesion molecule 1 (MadCAM-1) is also recognized as a ligand for ⁇ 4 ⁇ 7 integrin.
  • 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 ⁇ 5 ⁇ l fibronectin- specific integrin binds to the central RGD/PHSRN site.
  • the ⁇ 4 ⁇ l integrin binds to the IIICS site.
  • Peptide and antibody inhibitors of fibronectin and integrin functions have been shown to be effective inhibitors of metastasis, and are potentially important agents for the control of tumor-cell adhesion.
  • Integrin-activated pathways mediated by intracellular kinases and the adapter proteins are particularly important for integrin receptor-mediated anchorage dependence.
  • 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 focal adhesion kinase
  • PTK protein-tyrosine kinase
  • FAK associates with several different cytoplasmic signaling proteins such as Src-family PTKs and several SH2-domain proteins (She, Grb2 and PI 3 -kinase).
  • a peptide sequence motif responsible for the cell attachment in the fibronectin ligand for the integrin receptor 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.
  • 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.
  • 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.
  • integrin receptor- ligand interactions by use of synthetic fibronectin fragments, peptide analogs and mimics containing the RGD sequence, or antibodies represents a useful approach for the development of clinically useful antagonists for subtype-specific integrin receptors.
  • a number of these peptides such as those disclosed within U.S. Patent No. 5,627,263, have been found to be more effective in inhibiting tumor cell adhesion and tumor metastasis than the original RGDS peptide, and are disclosed for use in the present invention in combination with other agents.
  • the present invention provides for the use of peptides and peptide analogs which are specific for fibronectin-binding integrins, in particular the 0. 5 ⁇ i integrin.
  • specific agents for inhibition of binding to ⁇ 2 , ⁇ 6 and ⁇ v integrin subunits as part of ⁇ i integrin heterodimers are also provided.
  • Disintegrins 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.
  • 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 ⁇ V ⁇ 3 and not to ⁇ 5 ⁇ l or other abundant integrins.
  • the related disintegrin echistatin specifically inhibited 125 I-labeled kistrin binding to ⁇ N ⁇ 3, while a structurally distinct disintegrin, decorsin had 1000-fold lower affinity.
  • An additional heterodimeric disintegrin, EC3 (Mr 14,762), isolated from Echis carinatus venom is a potent antagonist of ⁇ 4 integrins. Each subunit contains 67 residues and displays a high degree of homology to other disintegrins.
  • 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. TABLE 3 THERAPEUTIC AND PREFERRED CONCENTRATIONS OF INTEGRIN
  • 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),
  • GMP-140 P-selectin
  • L-selectin L-selectin
  • 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 a or Sle 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.
  • P-selectin also known as GMP-140 or PADGEM
  • 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.
  • 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. Recent studies evaluated the role of E-selectin-sialyl Lewis x (Sle x )/ sialyl Lewis a (Sle 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 (HUNEC).
  • HUNEC human umbilical cord endothelial cells
  • 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.
  • a monoclonal antibody directed against E-selectin Prior incubation of cells with two different antibodies directed against Sle x and antibodies directed against related Lewis epitopes, Le x and Le a , had no significant effect on adhesion.
  • Three antibodies directed against Sle a differed in their capacity to inhibit the adhesion of HT-29 and COLO 201 cells.
  • the Sle a epitope is important for adhesion of colon cancer cells mediated predominantly by E-selectin.
  • T ⁇ F-alpha tumor necrosis factor- ⁇
  • SW1990 cells 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 a and SLe x antigens, CD44H, and ⁇ l 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 a and against ⁇ l integrin.
  • 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 p l25 focal adhesion kinase (FAK) and paxillin. Treatment with a tyrosine kinase inhibitor, genistein, or with the protein kinase C inhibitor, staurosporine, resulted in decreased ppl25 FAK phosphorylation.
  • FAK focal adhesion kinase
  • 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 a (Sle a ) mAb in combination with either one or more anti-inflammatory and/or anti-pain and/or anti- spasm and/or anti-restenosis agents.
  • 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.
  • 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.
  • 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.
  • 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).
  • an agent such as an integrin receptor antagonist can exert its actions on any cells associated with the fluid spaces of the peritoneal cavity and structures comprising the cavity that are involved in normal function or are present due to a pathological condition.
  • These cells and structures include, but are not limited to: epithelial cells; mesothelial cells; inflammatory cells including lymphocytes, macrophages, mast cells, monocytes, eosinophils; and other cells including endothelial cells, smooth muscle cells, and fibroblasts; and all combinations of the above.
  • the present invention also provides for formulations of the active therapeutic 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.
  • 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.
  • 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.
  • 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.
  • polycations e.g., protamine, spermidine and polylysine
  • 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).
  • a homogeneous e.g., a single encapsulated microsphere
  • individual delivery vehicles e.g., a group of microspheres encapsulating one or more agents.
  • 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.
  • hydrophilic polymers e.g., polycations (e.g., protamine, spermidine, polylysine and chitosan), peptidic or synthetic ligands and antibodies capable of targeting materials to specific cell types, gels, slow release matrices, soluble and insoluble particles, as well as formulation elements not listed.
  • the present invention provides for the local delivery of a combination of tumor cell anti-adhesion 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.
  • systemic e.g., oral, intramuscular, intravenous, subcutaneous
  • systemic 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.
  • systemic methods subject the drug to second-pass metabolism and rapid degradation, thereby limiting the duration of the effective therapeutic concentration.
  • tumor cell anti-adhesion, anti-invasion and anti-metastasis 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.
  • 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.
  • the carrier is an aqueous solution which may include physiologic electrolytes, such as normal saline or lactated Ringer's solution.
  • the anti-inflammation/anti-pain agents are selected from the group consisting of:
  • serotonin receptor antagonists (I) serotonin receptor antagonists; (2) serotonin receptor agonists; (3) histamine receptor antagonists; (4) bradykinin receptor antagonists; (5) kallikrein inhibitors; (6) tachykinin receptor antagonists, including neurokinini and neurokinin 2 receptor subtype antagonists;
  • CGRP calcitonin gene-related peptide
  • interleukin receptor antagonists interleukin receptor antagonists
  • inhibitors of enzymes active in the synthetic pathway for arachidonic acid metabolites including (a) phospholipase inhibitors, including PLA isoform inhibitors and PLC ⁇ isoform inhibitors (b) cyclooxygenase inliibitors, and (c) lipooxygenase inhibitors; (10) prostanoid receptor antagonists including eicosanoid EP-1 and EP-4 receptor subtype antagonists and thromboxane receptor subtype antagonists;
  • leukotriene receptor antagonists including leukotriene B receptor subtype antagonists and leukotriene D 4 receptor subtype antagonists
  • opioid receptor agonists including ⁇ -opioid, ⁇ -opioid, and ⁇ -opioid receptor subtype agonists
  • purinoceptor agonists and antagonists including P ⁇ receptor antagonists and P ⁇ receptor agonists
  • ATP adenosine triphosphate
  • Suitable anti-inflammatory/anti-pain agents which also act as anti-spasm agents include serotonin receptor antagonists, tachykinin receptor antagonists, ATP-sensitive potassium channel openers and calcium channel antagonists.
  • Other agents which may be utilized in the solution specifically for their anti-spasm properties including endothelin receptor antagonists, calcium channel antagonists and the nitric oxide donors (enzyme activators).
  • Specific preferred embodiments of the solution of the present invention for use in cardiovascular and general vascular procedures include anti-restenosis agents, which most preferably are used in combination with anti-spasm agents.
  • Suitable anti-restenosis agents include: (1) antiplatelet agents including: (a) thrombin inhibitors and receptor antagonists, (b) adenosine disphosphate (ADP) receptor antagonists (also known as purinocepto ⁇ 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 - ti-pain/-mti-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 2 (SH2) domains, and (d) calcium channel antagonists.
  • 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.
  • concentration of each agent is determined in part based on its dissociation constant, K ⁇ .
  • 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 ⁇ nanomolar, except for cyclooxygenase inhibitors, which may be required at larger concentrations depending on the particular inhibitor selected.
  • each agent is included at a concentration of 1.0 to 1,000 times K ⁇ nanomolar and most preferably at approximately 100 times K ⁇ j nanomolar. These concentrations are adjusted as needed to account for dilution in the absence of metabolic transformation at the local delivery site.
  • the exact agents selected for use in the solution, and the concentration of the agents varies in accordance with the particular application, as described below.
  • a solution in accordance with the present invention can include just a single or multiple 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.
  • 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
  • anti-restenosis agents from the enumerated classes
  • the surgical solutions constitute a novel therapeutic approach by combining multiple pharmacologic agents acting at distinct receptor and enzyme molecular targets.
  • 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 neuiOtransmitters and hormones.
  • 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.
  • 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.
  • the surgical solutions are composed of drugs that target common molecular mechanisms operating in different cellular physiologic processes involved in the development of pain, inflammation, vasospasm, smooth muscle spasm and restenosis (see FIGURE 1). In this way, the cascading of additional receptors and enzymes in the nociceptive, inflammatory, spasmodic and restenotic pathways is minimized by the surgical solutions.
  • the surgical solutions inhibit the cascade effect both "upstream” and “downstream”.
  • upstream inhibition is the cyclooxygenase antagonists in the setting of pain and inflammation.
  • the cyclooxygenase enzymes (COX j and COX 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.
  • 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.
  • aprotinin effectively inhibits the synthesis of bradykinins, thereby providing an effective "upstream” inhibition of these inflammatory mediators.
  • the surgical solutions also make use of "downstream" inhibitors to control the pathophysiologic pathways.
  • 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
  • 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 FIGURES 2 and 4).
  • 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.
  • Serotonin (5-HT) is thought to produce pain by stimulating serotonin 2 (5-HT 2 ) and/or serotonin 3 (5-HT 3 ) receptors on nociceptive neurons in the periphery.
  • 5-HT 3 receptors on peripheral nociceptors mediate the immediate pain sensation produced by 5-HT (Richardson et al., 1985).
  • 5-HT 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 77:185-189 (1990).
  • 5-HT 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 25:216-18 (1988). Therefore, activation of 5-HT 2 receptors also may play a role in peripheral pain and neurogenic inflammation.
  • 5-HT 2 and 5-HT 3 receptor antagonists are both suitably used, either individually or together, in the solution of the present invention, as shall be described subsequently.
  • Amitriptyline (ElavilTM) is a suitable 5-HT2 receptor antagonist for use in the present invention. Amitriptyline has been used clinically for numerous years as an anti-depressant, and is found to have beneficial effects in certain chronic pain patients. Metoclopramide (ReglanTM) is used clinically as an anti-emetic drag, but displays moderate affinity for the 5-HT 3 receptor and can inhibit the actions of
  • 5-HT at this receptor possibly inhibiting the pain due to 5-HT release from platelets.
  • it also is suitable for use in the present invention.
  • 5-HT 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 (Jul. 1988).
  • Other suitable 5-HT 3 receptor antagonists include cisapride and ondansetron.
  • the cardiovascular and general vascular solution also may contain a serotoniniB (also known as serotonin ⁇ D ⁇ ) antagonist because serotonin has been shown to produce significant vascular spasm via activation of the serotonin jg receptors in humans.
  • serotoniniB also known as serotonin ⁇ D ⁇
  • Suitable serotonin ⁇ B receptor antagonists include yohimbine, N-[-methoxy-3-(4-methyl-l-piperanzinyl)phenyl]-2'-methyl-4'-(5-methyl- 1, 2, 4-oxadiazol-3-yl)[l, l-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.
  • Serotonkb 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 ⁇ 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
  • Serotonini B Human lDg Antagonists: yohimbine 0.1 - 1,000 50 - 500
  • 5-HT i A, 5-HT iB and 5-HTID receptors are known to inhibit adenylate cyclase activity.
  • serotoninjA serotoninig and serotoninirj receptor agonists in the solution should inhibit neurons mediating pain and inflammation.
  • serotonini £ and serotonin ⁇ receptor agonists because these receptors also inhibit adenylate cyclase.
  • Buspirone is a suitable 1A receptor agonist for use in the present invention.
  • Sumatriptan is a suitable 1A, IB, ID and IF receptor agonist.
  • a suitable IB and ID receptor agonist is dihydroergotamine.
  • a suitable IE receptor agonist is ergonovine. Therapeutic and preferred concentrations for these receptor agonists are provided in Table 6.
  • Serotonin ⁇ Agonists buspirone 1 - 1,000 10 - 200 sumatriptan 1 - 1,000 10 - 200
  • Serotonini B Agonists dihydroergotamine 0.1 - 1,000 10 - 100 sumatriptan 1 - 1,000 10 - 200
  • Serotonin ⁇ dihydroergotamine 0.1 - 1,000 10 - 100 sumatriptan 1 - 1,000 10 - 200
  • Serotonin ⁇ Agonists ergonovine 10 - 2,000 100 - 1,000
  • Serotoninip Agonists sumatriptan 1 - 1,000 10 - 200
  • Histamine receptors generally are divided into histamine j (Hi) and histamine 2 (H 2 ) subtypes.
  • the classic inflammatory response to the peripheral administration of histamine is mediated via the Hj receptor. Douglas, 1985. Therefore, the solution of the present invention preferably includes a histamine H ⁇ receptor antagonist.
  • Promethazine PhenerganTM
  • 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 Hi receptors, thus, the effects are believed to occur by different mechanisms.
  • the histamine receptor antagonist concentration in the solution is sufficient to inhibit Hi receptors involved in nociceptor activation, but not to achieve a "local anesthetic" effect, thereby eliminating the concern regarding systemic side effects.
  • Histamine receptors also are known to mediate vasomotor tone in the coronary arteries. In vitro studies in the human heart have demonstrated that the histamine receptor subtype mediates contraction of coronary smooth muscle. Ginsburg, R., et al., "Histamine Provocation of Clinical Coronary Artery Spasm: Implications Concerning Pathogenesis of Variant Angina Pectoris," American Heart J. 702:819-822 (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.
  • histamine receptor antagonists may be included in the cardiovascular irrigation solution.
  • Other suitable Hi receptor antagonists include terfenadine, diphenhydramine, amitriptyline, mepyramine and tripolidine. Because amitriptyline is also effective as a serotonin 2 receptor antagonist, it has a dual function as used in the present invention. Suitable therapeutic and preferred concentrations for each of these H receptor antagonists are set forth in Table 7.
  • Histamine i 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
  • Bradykinin receptors generally are divided into bradykinin i (Bi) and bradykini ⁇
  • B 2 bradykinin subtypes.
  • 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 2 receptors block bradykinin-induced acute pain and inflammation. Dray et al., 1993. Bi 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 Bi and B 2 receptor antagonists. For example, arthroscopy is performed for both acute and chronic conditions, and thus an irrigation solution for arthroscopy could include both Bi and B 2 receptor antagonists. Suitable bradykinin receptor antagonists for use in the present invention include the following bradykinini receptor antagonists: the [des-Arg 10 ] derivative of
  • Suitable bradykinin 2 receptor antagonists include: [D-Phe 7 ]-BK; D-Arg-(Hyp -Thi 5 > 8 -D-Phe 7 )-BK ("NPC 349"); D-Arg-(Hyp 3 --D-Phe ?
  • HOE 140 1 - 1,000 50 - 500
  • 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.
  • Tachykinins 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. 22:1-24 (1991). Due to the above combination of physiological actions mediated by activation of TK receptors, targeting of TK receptors is a reasonable approach for the promotion of analgesia and the treatment of neurogenic inflammation.
  • Substance P activates the neurokinin receptor subtype referred to as NKi.
  • Substance P is an undecapeptide that is present in sensory nerve terminals.
  • Substance P is known to have multiple actions which produce inflammation and pain in the periphery after C-fiber activation, including vasodilation, plasma extravasation and degranulation of mast cells.
  • a suitable Substance P antagonist is ([D-Pro 9 [spiro- gamma-lactam]Leu 10 ,Trp 11 ]physalaemin-(l-l l)) ("GR 82334").
  • Other suitable antagonists for use in the present invention which act on the NKi receptor are: 1-imino-
  • 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 . Edmonds-Alt, S., et al., "A Potent and Selective Non-Peptide Antagonist of the Neurokinin A (NK 2 ) Receptor," Life Sci. 50.PL1O1 (1992). In the urinary tract, TKs are powerful spasmogens acting through only the NK receptor in the human bladder, as well as the human urethra and ureter. Maggi, C.A., Gen. Pharmacol. 22:1-24 (1991).
  • NK 2 antagonists include: ((S)-N-methyl-N-[4-(4- acetylamino-4-phenylpiperidino)-2- (3,4-dichlorophenyl)butyl]benzamide ("( ⁇ )-SR
  • Calcitonin gene-related peptide 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.
  • CGRP Calcitonin gene-related peptide
  • An example of a suitable CGRP receptor antagonist is ⁇ -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.
  • CGRP Receptor Antagonist ⁇ -CGRP-(8-37) 1-1,000 10-500 H. INTERLEUKIN RECEPTOR ANTAGONIST
  • Interleukins are a family of peptides, classified as cytokines, produced by leukocytes and other cells in response to inflammatory mediators. Interleukins (IL) may be potent hyperalgesic agents peripherally. Ferriera, S.H., et al., "Interleukin- l ⁇ as a Potent Hyperalgesic Agent Antagonized by a Tripeptide Analogue," Nature 334:698 (1988).
  • An example of a suitable IL-l ⁇ receptor antagonist is Lys-D-Pro-Thr, which is a truncated version of IL-l ⁇ . This tripeptide inhibits the activation of IL-l ⁇ receptors. Suitable concentrations for this agent are provided in Table 13.
  • PHOSPHOLIPASE INHIBITORS The production of arachidonic acid by phospholipase A (PLA 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.
  • PLA 2 isoform inhibits the release of arachidonic acid from cell membranes, and therefore inhibits the production of prostaglandms and leukotrienes resulting in decreased inflammation and pain.
  • Glaser, K.B. "Regulation of Phospholipase A2 Enzymes: Selective Inhibitors and Their Pharmacological Potential,"
  • PLA 2 isoform inhibitor manoalide. Suitable concentrations for this agent are included in Table 14. Inhibition of the phospholipase C ⁇ (PLC ⁇ ) isoform also will result in decreased production of prostanoids and leukotrienes, and, therefore, will result in decreased pain and inflammation.
  • PLC y isoform inhibitor is l-[6-((17 ⁇ -3-methoxyestra- 1 ,3,5(10)-trien-l 7-yl)amino)hexyl]-lH-pyrrole-2,5-dione.
  • PLA 2 Isoform Inhibitor manoalide 100-100,000 500-10,000
  • Nonsteroidal anti-inflammatory drags are widely used as anti-inflammatory, anti-pyretic, anti-thrombotic and analgesic agents.
  • Lewis, R.A. "Prostaglandms 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).
  • 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 prostaglandms which contribute to pain and inflammation.
  • NSAIDs currently on the market 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 prostaglandms 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. 75:405-406 (1994).
  • ketorolac 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.
  • ketorolac or any other cyclooxygenase inhibitor(s), in the solution in substantially lower dosages than currently used perioperatively may allow the use of this drag 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.
  • 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.
  • Cyclooxygenase Inliibitors ketorolac 100 - 10,000 500 - 5,000 indomethacin 1,000 - 500,000 10,000 - 200,000
  • lipooxygenase inhibits the production of leukotrienes, such as leukotriene B4, which is l ⁇ iown to be an important mediator of inflammation and pain.
  • leukotriene B4 is l ⁇ iown to be an important mediator of inflammation and pain.
  • Lewis, R.A. "Prostaglandms 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)-l,4-benzoquinone ("AA 861”), suitable concentrations for which are listed in Table 16.
  • Lipooxygenase Inhibitor AA 861 100-10,000 500-5,000
  • prostanoids produced as metabolites of arachidonic acid mediate their inflammatory effects through activation of prostanoid receptors.
  • 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 2 receptor antagonist is 8-chlorodibenz[b,f][l,4]oxazepine-10(l lH)-carboxylic acid, 2-acetylhydrazide ("SC 19220").
  • a suitable thromboxane receptor subtype antagonist is [15-[l , 2 ⁇ (5Z), 3 ⁇ , 4 ⁇ ]-7-[3-[2-(phenylamino)-carbonyl] hydrazino] methyl]-7- oxobicyclo-[2,2,l]-hept-2-yl]-5-heptanoic acid ("SQ 29548"). Suitable concentrations for these agents are set forth in Table 17.
  • Eicosanoid EP-1 Antagonist SC 19220 100-10,000 500-5,000 K.
  • the leukotrienes 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 4 receptor is found in certain immune cells including eosinophils and neutrophils.
  • LTB 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 4 receptor involves G-protein-mediated stimulation of phosphotidylinositol (PI) metabolism and elevation of intracellular calcium (see FIGURE 2).
  • An example of a suitable leukotriene B 4 receptor antagonist is SC (+)-(S)-7-(3-(2- (cyclopropyimethyl)-3 -methoxy-4-[(methylamino)-carbonyl]phenoxy(propoxy)-3,4- dihydro-8-propyl-2H-l-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.
  • leukotriene B 4 receptor antagonists include [3-[-2(7-chloro-2- quinolinyl)ethenyl]phenyl] [[3 -(dimethylamino-3 -oxopropyl)thio] methyljthiopropanoic acid ("MK 0571”) and the drags LY 66,071 and ICI 20,3219. MK 0571 also acts as a LTD4 receptor subtype antagonist.
  • Opioid receptors include the ⁇ -, ⁇ - and ⁇ -opioid receptor subtypes.
  • the ⁇ -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).
  • ⁇ - and ⁇ -receptors are located on sympathetic efferent terminals and inhibit the release of prostaglandms, thereby inhibiting pain and inflammation.
  • the opioid receptor subtypes are members of the G-protein coupled receptor superfamily. Therefore, all opioid receptor agonists interact and initiate signaling through their cognate G-protein coupled receptor (see FIGURES 3 and 7).
  • Suitable ⁇ -opioid receptor agonists are fentanyl and Try-D-Ala-Gly-[ ⁇ - MePhe]-NH(CH 2 )-OH (“DAMGO”).
  • An example of a suitable ⁇ -opioid receptor agonist is [D-Pen 2 ,D-Pen 5 ]enkephalin (“DPDPE”).
  • An example of a suitable ⁇ -opioid receptor agonist is (trans)-3,4-dichloro-N-methyl-N-[2-(l-pyrrolidinyl)cyclohexyl]-benzene acetamide (“U50,488”). Suitable concentrations for each of these agents are set forth in Table 19.
  • Extracellular ATP acts as a signaling molecule through interactions with P 2 purinoceptors.
  • P 2x purinoceptors which are ligand-gated ion channels possessing intrinsic ion channels permeable to Na + , K + , and Ca 2+ .
  • P x 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 2 ⁇ receptors leading to depolarization of nociceptive nerve-fiber terminals.
  • the P2X 3 receptor has a highly restricted distribution (Chen, C.C, et al., Nature 377:428-431
  • Suitable antagonists of P 2 ⁇ /ATP purinoceptors for use in the present invention include, by way of example, suramin and pyridoxylphosphate-6-azophenyl-2,4-disulfonic acid ("PPADS"). Suitable concentrations for these agents are provided in Table 20.
  • Agonists of the P 2 ⁇ receptor are known to effect smooth muscle relaxation through elevation of inositol triphosphate (IP 3 ) levels with a subsequent increase in intracellular calcium.
  • IP 3 inositol triphosphate
  • Purinoceptor Antagonists suramin 100-100,000 10,000-100,000
  • ATP-sensitive potassium channels have been discovered in numerous tissues, including vascular and non- vascular smooth muscle and brain, and binding studies using radiolabeled ligands have confirmed their existence. Opening of these channels causes potassium (K + ) efflux and hyperpolarizes the cell membrane (see FIGURE 2). This hyperpolarization induces a reduction in intracellular free calcium through inhibition of voltage-dependent calcium (Ca 2+ ) channels and receptor operated Ca 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 + potassium
  • Ca 2+ voltage-dependent calcium
  • K + channel openers have been characterized as having potent antihypertensive activity in vivo and vasorelaxant activity in vitro (see FIGURE 4). K + channel openers (KCOs) also have been shown to prevent stimulus coupled secretion and are considered to act on prejunctional neuronal receptors and thus will inhibit effects due to nerve stimulation and release of inflammatory mediators. Quast, U., et al., "Cellular
  • ATP-sensitive potassium channels are expected in achieving vasorelaxation or smooth muscle relaxation.
  • KCOs ATP-sensitive potassium channels
  • 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 ⁇ receptor subsequently triggers activation of voltage-dependent channels and the entry of extracellular calcium which is required for contraction.
  • Antagonists of the ET ⁇ receptor will specifically block this receptor mediated effect but not block increases in calcium triggered by activation of other G-protein coupled receptors on the muscle cell.
  • Potassium-channel opener drags such as pinacidil, will open these channels causing K + 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 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 2+ release from intracellular sources through inhibition of inositol triphosphate (IP 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.
  • Suitable ATP-sensitive K + channel openers for the practice of the present invention include: (-)pinacidil; cromakalim; nicorandil; minoxidil; N-cyano-N'-[l,l- dimethyl-[2,2,3,3- ⁇ ]propyl]-N"-(3-pyridinyl)guanidine (“P 1075”); and N-cyano-N'-(2- nitroxyethyl)-3-pyridinecarboximidamide monbmethansulphonate ("KRN 2391"). Concentrations for these agents are set forth in Table 21.
  • ATP-Sensitive K + Channel Opener cromakalim 10-10,000 100-10,000 nicorandil 10-10,000 100-10,000 minoxidil 10-10,000 100-10,000
  • anti-pain/anti-inflammatory agents that also serve as anti-spasm agents include: serotonin receptor antagonists, particularly, serotonin 2 antagonists; tachykinin receptor antagonists and ATP-sensitive potassium channel openers.
  • 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.
  • NOS NO synthase
  • 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.
  • various agonists such as acetylcholine
  • other endothelium dependent vasodilators are key molecular targets controlling vascular tone (see FIGURE 8).
  • FIGURE 8 Muramatsu, K., et al., Coron. Artery Dis. 5:815-820 (1994).
  • Suitable nitric oxide donors for the practice of the present invention include nitroglycerin, sodium nitroprusside, the drag 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. TABLE 22
  • 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 A and ET ⁇ receptors.
  • the heart and vascular smooth muscle contain predominantly ET ⁇ receptors and this subtype is responsible for contraction in these tissues.
  • ET A receptors have often been found to mediate contractile responses in isolated smooth muscle preparations.
  • Antagonists of ET A receptors have been found to be potent antagonists of human coronary artery contractions.
  • antagonists to the ET ⁇ 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. 14:5A-60 (1993).
  • Suitable endothelin receptor antagonists include: cyclo(D-Asp-Pro-D-Val-Leu-D- Trp) ("BQ 123"); (N,N-hexamethylene)-carbamoyl-Leu-D-Trp-(CHO)-D-Trp-OH (“BQ 610"); (R)2-([R-2-[(s)-2-([l-hexahydro-lH-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-(d
  • D. CA 2+ CHANNEL ANTAGONISTS Calcium channel antagonists are a distinct group of drags 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 (NKi and NK 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, prostaglandms, 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).
  • 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.
  • 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 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 drags 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.
  • nisoldipine used alone and in combination with nitroglycerin
  • IMA internal mammary artery
  • Calcium channel antagonists also exhibit synergistic effect with endothelin receptor subtype A (ET ⁇ ) antagonists.
  • ET ⁇ endothelin receptor subtype A
  • Yanagisawa and coworkers observed that dihydropyridine antagonists blocked effects of ET-1, an endogenous agonist at the ET 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 ⁇ 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 ⁇ antagonist when combined in a surgical solution. Calcium channel antagonists and ATP-sensitive potassium channel openers likewise exhibit synergistic action.
  • K ATP ATP-sensitive
  • K AT p 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 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.
  • T-type calcium channels (2) restrains agonist induced (at receptor operated channels) Ca + release from intracellular sources through inhibition of inositol triphosphate (IP 3 ) formation, and (3) lowers the efficiency of calcium as an activator of contractile proteins.
  • IP 3 inositol triphosphate
  • NKi neurokinin]
  • NK 2 neurokinin 2 receptor
  • BK 2 bradykinin 2
  • calcium channel antagonists interfere with a common mechanism involving elevation of intracellular calcium, part of which enters through L-type channels.
  • 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.
  • ANTI-RESTENOSIS AGENTS Solutions of the present invention utilized for cardiovascular and general vascular procedures may optionally also include an anti-restenosis agent, particularly for angioplasty, rotational atherectomy and other interventional vascular uses.
  • an anti-restenosis agent particularly for angioplasty, rotational atherectomy and other interventional vascular uses.
  • the following drags 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.
  • 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.
  • anti-platelet agents can be antagonists drawn from any of the above agonist-receptor targets.
  • THROMBIN INHIBITORS AND RECEPTOR ANTAGONISTS Thrombin plays a central role in vascular lesion formation and is considered the principal mediator of thrombogenesis.
  • 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 hiradin and its synthetic peptide analogs, as well as thrombin receptor antagonist peptides (Harker, et al., Am. J. Cardiol. 75:12B (1995)).
  • Thrombin is also a potent growth factor which initiates smooth muscle cell proliferation at sites of vascular injury.
  • 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.
  • PDGF platelet-derived growth factor
  • 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 hiradin (Ciba-Geigy). Hirulog (Biogen) is a dodecapeptide modeled after hiradin, and binds to the active site of thrombin via a Phe-Pro-Arg linker molecule.
  • active anti-thrombin agents being tested which are theorized to be suitable for the present invention are argatroban (Texas Biotechnology) and efegatran (Lilly).
  • Thrombin Inhibitors and Receptor Antagonists hiradin 0.00003-3/0.0003-0.3 0.03 hirulog 0.2-20,000/2-2,000 200
  • ADP RECEPTOR ANTAGONISTS PURINOCEPTOR ANTAGONISTS Ticlopidine, an analog of ADP, inhibits both thromboxane and ADP-induced platelet aggregation. It is likely that ticlopidine blocks interaction of ADP with its receptor, thereby inhibiting signal transduction by this G-protein coupled receptor on the 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.
  • THROMBOXANE INHIBITORS AND RECEPTOR ANTAGONISTS Agents currently utilized for conventional methods of treatment of thrombosis rely upon aspirin, heparin and plasminogen activators. Aspirin irreversibly acetylates cycloxygenase and inhibits the synthesis of thromboxane A2 and prostacyclin. While data support a benefit of aspirin for PTCA, the underlying efficacy of aspirin is considered as only partial or modest. This is likely due to platelet activation through thromboxane A2 independent pathways that are not blocked by aspirin induced acetylation of cyclooxygenase. Platelet aggregation and thrombosis may occur despite aspirin treatment. Aspirin in combination with dipyridamole has also been shown to reduce the incidence of acute complication during PTCA but not the incidence of restenosis.
  • 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.
  • 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 Corporation that inhibits the activity of P-selectin.
  • the product, CY 1748, is in preclinical development, with a potential indication being restenosis.
  • the platelet glycoprotein Ilb/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.
  • 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.
  • the platelet glycoprotein Ilb/IIIa receptor blocker, integrelin is a cyclic heptapeptide that is highly specific for this molecular target. In contrast to the antibody, it has a short biologic half-life (about 10 minutes).
  • the safety and efficacy of integrelin was first evaluated in the Phase II Impact trial. Either 4 or 12 hour intravenous infusions of 1.0 ⁇ g/kg/min of integrelin were utilized (Topol, E., Am. J. Cardiol, 27B-33B (1995)). It was provided in combination with other agents (heparin, aspirin) and was shown to exhibit potent anti-platelet aggregation properties (>80%).
  • 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- ⁇ /RANTES receptors. Drags 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.
  • 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-1 AP). 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.
  • IL-1 antagonist protein IL-1 antagonist protein
  • PKC PROTEIN KINASE C
  • PKC Protein kinase C
  • G-protein coupled receptors e.g., serotonin, endothelin, etc.
  • 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.
  • 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 prohferative 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 prohferative 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 5Q of
  • G-6203 (also known as Go 6976) is a new, potent PKC inhibitor with high selectivity for certain PKC isotypes with IC 50 values in the 2-10 ⁇ M range.
  • Concentrations of these and another drug, GF 109203X, also known as Go 6850 or bisindoylmaleimide I (available from Warner-Lambert), that are believed to be suitable for use in the present invention are set forth below. TABLE 27
  • Protein Kinase C Inhibitors calphostin C 0.5-50,000/100-5,000 500
  • G-6203 (Go 6976) 0.1-10,000/1-1,000 100
  • RTK tyrosine-kinase
  • 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 50 S in vitro in the 0.5-1.0 ⁇ M range), since they have little effect on other protein kinases and other signal transduction systems.
  • IC 50 S in vitro in the 0.5-1.0 ⁇ M range IC 50 S in vitro in the 0.5-1.0 ⁇ M range
  • suitable concentrations for these agents as used in the present invention are set forth below.
  • 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.
  • Protein Kinase Inhibitors lavendustin A 10-100,000/100-10,000 10,000 tyrphostin 10-100,000/100-20,000 10,000
  • PHOSPHATASES Non-transmembrane protein tyrosine phosphatases (PTPases) containing src-homology 2 SH2 domains are known and nomenclature refers to them as SH-PTPl and SH-PTP2.
  • SH-PTPl is also known as PTP1C, HCP or SHP.
  • SH-PTP2 is also known as PTPID or PTP2C.
  • SH-PTPl is expressed at high levels in hematopoietic cells of all lineages and all stages of differentiation, and the SH-PTPl 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., J Immunol. (1994)) and PTPase activity modulates agonist induced activity by reversing the effects of tyrosine kinases activated in the initial phases of cell stimulation.
  • Agents that could stimulate PTPase activity could have potential therapeutic applications as anti-inflammatory mediators.
  • 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 drag targets.
  • PTPase blocks insulin stimulated phosphorylation of tyrosyl residues on endogenous proteins.
  • 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.
  • receptor-linked PTPases also function as extracellular ligands, similar to those of cell adhesion molecules.
  • 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.
  • RTK receptor tyrosine kinase
  • Phosphotyrosine-containing sites on autophosphorylated RTKs serve as binding sites for SH2-proteins and thereby mediate the activation of biochemical signaling pathways (see FIGURE 2) (Carpenter, G., FASEB J. 5:3283-3289 (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 FIGURE 5).
  • 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.
  • cytosolic proteins 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)).
  • 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 c " src ), abl, lck, fyn, fgr and others), phospholipase C ⁇ (PLC ⁇ ), phosphatidylinositol 3-kinase (PI-3 -kinase), p21-ras GTPase activating protein (GAP) and SH2 containing protein tyrosine phosphatases (SH- PTPases) (Songyang, et al, Cell 72:161-118 (1993)).
  • src pp60 c " src ), abl, lck, fyn, fgr and others
  • PLC ⁇ phospholipase C ⁇
  • PI-3 -kinase phosphatidylinosi
  • inhibitors which block specific SH2 protein binding are desirable as agents for a variety of potential therapeutic applications.
  • T-cell activation via the antigen specific T-cell receptor (TCR) initiates a signal transduction cascade leading to lymphokine secretion and T-cell proliferation.
  • TCR antigen specific T-cell receptor
  • One of the earliest biochemical responses following TCR activation is an increase in tyrosine kinase activity.
  • 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.
  • 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.
  • inhibitors of specific SH2 domains have the potential to block many neutrophil functions and serve as anti-inflammatory mediators.
  • CALCIUM CHANNEL ANTAGONISTS Calcium channel antagonists, previously described with relation to spasm inhibitory function, also can be used as anti-restenotic agents in the cardiovascular and general vascular solutions of the present invention. Activation of growth factor receptors, such as PDGF, is known to result in an increase in intracellular calcium (see FIGURE 2). Studies at the cellular level have shown that actions of calcium channel antagonists are effective at inhibiting mitogenesis of vascular smooth muscle cells.
  • VASCULAR SOLUTIONS Given the complexity of the disease process associated with restenosis after PTCA or other cardiovascular or general vascular therapeutic procedure and the multiplicity of molecular targets involved, blockade or inhibition of a single molecular target is unlikely to provide adequate efficacy in preventing vasospasm and restenosis (see FIGURE 2). Indeed, a number of animal studies targeting different individual molecular receptors and or enzymes have not proven effective in animal models or have not yielded efficacy for both pathologies in clinical trials to date. (Freed, M., et al, "An Intensive Poly-pharmaceutical Approach to the Prevention of Restenosis: the Mevacor, Ace Inhibitor, Colchicine (BIG-MAC) Pilot Trial," J Am.
  • GPCRs G-protein coupled 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 2+ which triggers contraction in smooth muscle cells (see FIGURE 2).
  • Each GPCR transmembrane receptor activates a specific class of trimeric G proteins, including G q , Gj or many others.
  • G ⁇ and/or G ⁇ ⁇ subunits activate phospholipase CR, resulting in activation of protein kinase C (PKC) and an increase in the levels of cytoplasmic calcium by release of calcium from intracellular stores.
  • PKC protein kinase C
  • 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 ⁇ , 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 FIGURE 2).
  • Calcium channel antagonists 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 She activation. Conversely, the PKC inhibitor acts on part of the pathway leading to contraction.
  • the 5-HT 2 receptor family contains three members designated 5-HT 2A , 5-HT 2 B . and 5-HT 2 c . 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 A receptor activation requires extracellular calcium and is at least partially blocked by nicardipine.
  • 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.
  • thrombin mediates its action via the thrombin receptor, another member of the
  • GPCR superfamily Binding to the receptor stimulates platelet aggregation, smooth muscle cell contraction and mitogenesis. Signal transduction occurs through multiple pathways: activation of phospholipase (PLC) through G proteins and activation of tyrosine kinases. The activation of tyrosine kinase activity is also essential for mitogenesis of the vascular smooth muscle cells.
  • PLC phospholipase
  • tyrosine kinase activity is also essential for mitogenesis of the vascular smooth muscle cells.
  • inhibition with a specific tyrosine kinase inhibitor was effective in blocking thrombin-induced mitosis, although there were no effects on the PLC pathway as monitored by measurement of intracellular calcium (Weiss and Nucitelli, J Biol. Chem. 2(57:5608-5613
  • Nonsteroidal anti-inflammatory drags are widely used as anti- inflammatory, anti-pyretic, anti-thrombotic and analgesic agents (Lewis, R.A., "Prostaglandms 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.
  • COX-1 isozymes are also known as Prostaglandin H Synthase (PGHS)-l and PGHS-2, respectively.
  • PGHS Prostaglandin H Synthase
  • Both enzymes catalyze the conversion of arachidonic acid to unstable intermediates, PGG and PGH 2 , which are intermediates in the biosynthesis of prostaglandms 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.
  • the COX-2 isozyme is induced in settings of inflammation by cytokines and inflammatory mediators, such as IL-1, TNF- ⁇ and endotoxin, and its expression is upregulated at sites of inflammation.
  • cytokines and inflammatory mediators such as IL-1, TNF- ⁇ and endotoxin
  • 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).
  • nonselective NSAID drugs e.g., clotting time, bleeding, renal and gastrointestinal
  • 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 IC50 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).
  • NSAIDs 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.
  • 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.
  • PGE2 the endogenous prostanoid ligands
  • 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.
  • 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.
  • selective COX-2 inhibitors such as celecoxib
  • these compounds are expected to exhibit anti-inflammatory action when applied intraoperatively in an irrigation solution directly to a tissue or a joint.
  • it is expected to be an effective drug delivered by an irrigation solution during an arthroscopic, urologic, cardiovascular or general surgical procedure (periprocedurally).
  • a selective COX-2 inhibitor may replace ketorolac, a relatively non-selective cyclooxygenase inhibitor, in Examples IN, V & VI.
  • the selective COX-2 inhibitor may be delivered alone, or in combination with other small molecule drags, 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.
  • the selective COX-2 inhibitor can exert its actions on any cells associated with the fluid spaces of the joint and structures comprising the j oint, and which are involved in the normal function of the j oint 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.
  • 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.
  • the selective COX-2 inhibitor may be delivered in a formulation useful for introduction and administration of the drag 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.
  • formulation molecules include, but are not limited to, lipids capable of forming liposomes or other ordered lipid structures, cationic lipids, hydrophilic polymers, polycations (e.g., protamine, spermidine, polylysine), peptide or synthetic ligands and antibodies capable of targeting materials to specific cell types, gels, slow release matrices, soluble and insoluble particles, as well as formulation elements not listed.
  • the present invention describes the local delivery of a selective COX-2 inhibitor drag using an irrigation solution containing the drug which is present at low concentration and which enables the drag to be delivered directly to the affected tissue or joint.
  • the drug-containing irrigation solution is employed perioperatively during a surgical procedure.
  • systemic administration e.g., intramuscular, intravenous, subcutaneous, oral
  • COX-2 drags and higher total dose
  • 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).
  • 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 drag 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.
  • Compounds suitable for the invention are listed in the Table below.
  • 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.
  • the term "perioperative” encompasses application intraprocedurally, pre- and intraprocedurally, intra- and postprocedurally, and pre-, intra- and postprocedurally.
  • the solution is applied preprocedurally and/or postprocedurally as well as intraprocedurally.
  • irrigation 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/anti-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.
  • 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.
  • the term “continuously” is intended to also include situations in which there is repeated and frequent irrigation of wounds at a frequency sufficient to maintain a predetermined therapeutic local concentration of the applied agents, and applications in which there may be intermittent cessation of irrigation fluid flow necessitated by operating technique.
  • the concentrations listed for each of the agents within the solutions of the present invention are the concentrations of the agents delivered locally, in the absence of metabolic transformation, to the operative site in order to achieve a predetermined level of effect at the operative site. It is understood that the drug concentrations in a given solution may need to be adjusted to account for local dilution upon delivery. For example, in the cardiovascular application, if one assumes an average human coronary artery blood flow rate of 80 cc per minute and an average delivery rate for the solution of 5 cc per minute via a local delivery catheter (i.e., a blood flow-to-solution delivery ratio of 16 to 1), one would require that the drag concentrations within the solution be increased 16-fold over the desired in vivo drag concentrations. Solution concentrations are not adjusted to account for metabolic transformations or dilution by total body distribution because these circumstances are avoided by local delivery, as opposed to oral, intravenous, subcutaneous or intramuscular application.
  • Arthroscopic techniques for which the present solution may be employed include, by way of non-limiting example, partial meniscectomies and ligament reconstructions in the knee, shoulder acromioplasties, rotator cuff debridements, elbow synovectomies, and wrist and ankle arthroscopies.
  • the irrigation solution is continuously supplied intraoperatively to the joint at a flow rate sufficient to distend the joint capsule, to remove operative debris, and to enable unobstructed infra-articular visualization.
  • Example I A suitable irrigation solution for control of pain and edema during such arthroscopic techniques is provided in Example I herein below.
  • the solution include a combination, and preferably all, or any of the following: a serotonin 2 receptor antagonist, a serotonin receptor antagonist, a histaminei receptor antagonist, a serotonin receptor agonist acting on the 1A, IB, ID, IF and/or IE receptors, a bradykinini receptor antagonist, a bradykinin 2 receptor antagonist, and a cyclooxygenase inhibitor.
  • This solution utilizes extremely low doses of these pain and inflammation inhibitors, due to the local application of the agents directly to the operative site during the procedure.
  • amitriptyline a suitable serotonin 2 and histaminei "dual" receptor antagonist
  • irrigation fluid to provide the desired effective local tissue concentrations that would inhibit 5-HT and Hi 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.
  • nisoldipine a suitable calcium channel antagonist
  • irrigation fluid 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.
  • 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 drag administration since drugs given systemically are subject to first- and second-pass metabolism.
  • Example XII a 5-HT2 antagonist
  • Example XII compared the therapeutic dosing of amitriptyline delivered locally (i.e., intra-articularly) at the knee and intravenously.
  • the difference in plasma drag levels between the two routes of administration is much greater than the difference in total amitriptyline dosing levels.
  • Practice of the present invention should be distinguished from conventional intra- articular injections of opiates and/or local anesthetics at the completion of arthroscopic or "open" joint (e.g., knee, shoulder, etc.) procedures.
  • the solution of the present invention is used for continuous infusion throughout the surgical procedure to provide preemptive inhibition of pain and inflammation.
  • 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.
  • 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 2 receptor antagonist (Saxena, P. R., et al.,
  • the cardiovascular and general vascular solution also preferably will contain a serotonini ⁇ (also known as serotonin ⁇ D ⁇ ) antagonist because serotonin has been shown to produce significant vascular spasm via activation of the serotoniniB receptors in humans.
  • serotonini ⁇ also known as serotonin ⁇ D ⁇
  • Kaumann, A.J., et al. "Variable Participation of 5-HTl-Like Receptors and 5-HT2 Receptors in Serotonin-Induced Contraction of Human Isolated Coronary Arteries," Circulation 90:1141-53 (1994).
  • the cardiovascular and general vascular solution of the present invention also may suitably include one or more of the anti-restenosis agents disclosed herein that reduce the incidence and severity of post-procedural restenosis resulting from, for example, angioplasty or rotational atherectomy.
  • the solution of the present invention also has utility for reducing pain and inflammation associated with urologic procedures, such as trans-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.
  • 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. 140:535-539 (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 histaminei receptor antagonist to inhibit histamine- induced pain and inflammation; a 5-HT 3 receptor antagonist to block activation of these receptors on peripheral C-fiber nociceptive neurons; a bradykinini antagonist; a bradykinin antagonist; and a cyclooxygenase inhibitor to decrease pain/inflammation produced by prostaglandins at the tissue injury sites.
  • an anti-spasm agent is also included to prevent spasm in the urethral canal and bladder wall.
  • Some of the solutions of the present invention may suitably also include a gelling agent to produce a dilute gel. This gellable solution may be applied, for example, within the urinary tract or an arterial vessel to deliver a continuous, dilute local predetermined concentration of agents.
  • the solution of the present invention may also be employed perioperatively for the inhibition of pain and inflammation in surgical wounds, as well as to reduce pain and inflammation associated with burns.
  • Burns result in the release of a significant quantity of biogenic amines, which not only produce pain and inflammation, but also result in profound plasma extravasation (fluid loss), often a life-threatening component of severe 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 23:861- 871 (1983).
  • 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.
  • composition is suitable for use in anatomic joint irrigation during arthroscopic procedures.
  • Each drag 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.
  • physiologic electrolytes such as normal saline or lactated Ringer's solution
  • Preferred serotonin 2 antagonist amitriptyline 0.1-1,000 50-500 100 serotonin 3 antagonist metoclopramide 10-10,000 200-2,000 1,000 histaminei antagonist amitriptyline 0.1-1,000 50-500 200 serotonin 1A; 1B> 1D> IF sumatriptan 1-1,000 10-200 50 agonist bradykinini antagonist [des-Arg 10 ] 1-1,000 50-500 200 derivative of
  • HOE 140 bradykinin 2 antagonist HOE 140 1-1,000 50-500 200 B.
  • IRRIGATION SOLUTION FOR CARDIOVASCULAR AND GENERAL VASCULAR THERAPEUTIC AND DIAGNOSTIC PROCEDURES The following drugs and concentration ranges in solution in a physiologic carrier fluid are suitable for use in irrigating operative sites during cardiovascular and general vascular procedures.
  • Class of Agent Drag (Nanomolar): Most Therapeutic Preferred Preferred serotonin 2 antagonist trazodone 0.1-2,000 50-500 200 serotonin antagonist metoclopramide 10-10,000 200-2,000 1,000 serotoniniB antagonist yohimbine 0.1-1,000 50-500 200 bradykinini antagonist [des-Arg 10 ] 1-1,000 50-500 200 derivative of
  • HOE 140 cyclooxygenase inhibitor ketorolac 100-10,000 500-5,000 3,000
  • Therapeutic Preferred Preferred histaminei antagonist terfenadine 0.1-1,000 50-500 200 serotonin antagonist metoclopramide 10-10,000 200-2,000 1,000 bradykinini antagonist [des-Arg 10 ] 1-1,000 50-500 200 derivative of
  • HOE 140 bradykinin 2 antagonist HOE 140 1-1,000 50-500 200 cyclooxygenase inhibitor 100-10,000 500-5,000 3,000 D.
  • EXAMPLE IV
  • 1F agonist cyclooxygenase ketorolac 100 - 10,000 500 - 5,000 3,000 inhibitor neurokinin ! antagonist GR 82334 1 - 1,000 10 - 500 200 neurokinin 2 antagonist ( ⁇ ) SR 48968 1 - 1,000 10 - 500 200 purine 2 ⁇ antagonist PPADS 100 - 100,000 10,000- 50,000 100,000
  • ALTERNATE IRRIGATION SOLUTION FOR CARDIOVASCULAR AND GENERAL VASCULAR THERAPEUTIC AND DIAGNOSTIC PROCEDURES The following drugs and concentration ranges in solution in a physiologic canier fluid are prefened for use in inigating 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.
  • PROCEDURES The following drags and concentration ranges in solution in a physiologic carrier fluid are prefened for use in inigating operative sites during urologic procedures. The solution is believed to have even higher efficacy than the solution set forth in prior Example III. Concentration
  • Histaminei terfenadine 0.1 - 1,000 50 - 500 200 antagonist cyclooxygenase ketorolac 100 - 10,000 500 - 5,000 3,000 inhibitor neurokinin 2 SR 48968 1 - 1,000 10 - 500 200 antagonist purine 2x antagonist PPADS 100 - 100,000 10,000-100, 50,000 000
  • Preferred Preferred thrombin hhiirruulloogg 0 0..22--2200,,000000 2 2--22,,000000 200 inhibitor glycoprotein integrelin 0.1-10,000 x Kd 1-1000 x Kd 100 x Kd
  • 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
  • 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:
  • NO donor FK 409 (NOR-3) 1-1,000 10-500 250 NO donor FR 144420 (NOR-4) 10-10,000 100-5,000 1,000 I.
  • amitriptyline is replaced by mepyramine as the Hi antagonist; 2) the kallikrein inhibitor, aprotinin, is deleted;
  • HOE 140 a bradykinin 2 antagonist
  • SIN-1 is replaced as the NO donor by a combination of two agents: a) FK 409 (NOR-3); and b) FR 144420 (NOR-4); 2) the kallikrein inhibitor, aprotinin, is deleted;
  • HOE 140 a bradykinin antagonist
  • the test solution included: (a) the serotonin antagonist metoclopramide at a concentration of 16.0 ⁇ M; (b) the serotonin 2 antagonist trazodone at a concentration of 1.6 ⁇ M; and (c) the histamine antagonist promethazine at concentrations of 1.0 ⁇ M, all in normal saline.
  • Drag concentrations within the test solution were 16-fold greater than the drag 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.
  • the iliac arteries of 3-4 kg male New Zealand white rabbits fed a regular rabbit chow were studied.
  • the animals were sedated using intravenous xylazine (5 mg/kg) and ketamine (35 mg/kg) dosed to effect and a cutdown was performed in the ventral midline of the neck to isolate a carotid artery.
  • the artery was ligated distally, an arteriotomy performed and a 5 French sheath was introduced into the descending aorta.
  • Baseline blood pressure and heart rate were recorded and then an angiogram of the distal aorta and bilateral iliac arteries was recorded on 35 mm cine film (frame rate 15 per second) using hand injection of iopamidol 76% (Squibb Diagnostics, Princeton, NJ) into the descending aorta.
  • a calibration object was placed in the radiographic field of view to allow for conection for magnification when diameter measurements were made.
  • a 2.5 French infusion catheter (Advanced Cardiovascular Systems, Santa Clara, CA) was placed through the carotid sheath and positioned 1-2 cm above the aortic bifurcation.
  • Infusion of the test solution ⁇ either saline alone or saline containing the histamine/serotonin receptor antagonists - was started at a rate of 5 cc per minute and continued for 15 minutes.
  • a second angiogram was performed using the previously described technique then a 2.5 mm balloon angioplasty catheter (the Lightning, Cordis Corp., Miami, FL) was rapidly advanced under fluoroscopic guidance into the left and then the right iliac arteries.
  • 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.
  • the infusion was stopped (a total of 75 cc of solution had been infused) and the infusion catheter was removed.
  • 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.
  • ANGIOGRAPHIC ANALYSIS The angiograms were recorded on 35 mm cine film at a frame rate of 15 per second. For analysis, the angiograms were projected from a Vanguard projector at a distance of 5.5 feet. Iliac artery diameters at prespecified locations relative to the balloon angioplasty site were recorded based on hand held caliper measurement after correction for magnification by measurement of the calibration object.
  • % Vasoconstriction ⁇ (Baseline area - Later time point area)/Baseline area ⁇ xlOO. 4. STATISTICAL METHODS All values are expressed as mean ⁇ 1 standard enor of the mean. The time course of vasomotor response in control arteries was assessed using one way analysis of variance with conection for repeated measures. Post hoc comparison of data between specific time points was performed using the Scheffe test.
  • control and histamine/serotonin receptor antagonist treated arteries were compared at those time points where significant vasoconstriction occuned 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, OK). 5.
  • 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 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).
  • 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
  • the histamine/serotonin receptor blockade solution was infused into 16 arteries (8 animals); angiographic data was available at all time points in 12 arteries.
  • 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
  • FIGURE 10A shows the effects of the histamine/serotonin infusion on proximal segment vasoconstriction relative to the vasoconstriction present in the control arteries.
  • amitriptyline The following study was undertaken in order to compare two routes of administration of the 5-HT 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 drag dose of amitriptyline delivered via each route was determined.
  • Rats were housed under controlled lighting conditions (lights on 6 A.M. to 6 P.M.), with food and water available ad libitum.
  • 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 ⁇ l/min
  • a 25-gauge needle was also inserted into the joint space and perfusate fluid was extracted at 250 ⁇ l/min, controlled by a Sage Instruments Syringe pump (Model 351).
  • the rats were randomly assigned to three groups: 1) those receiving only intra- articular (IA) 5-HT (1 ⁇ M), 2) those receiving amitriptyline intravenously (IV) (doses ranging from 0.01 to 1.0 mg/kg) followed by IA 5-HT (1 mM), and 3) those receiving amitriptyline intra-articularly (I A) (concentrations ranging from 1 to 100 ⁇ M) followed by IA 5-HT (1 ⁇ M) plus IA amitriptyline.
  • IA intra- articular
  • IV amitriptyline intravenously
  • I A concentrations ranging from 1 to 100 ⁇ M
  • 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 (Can and Wilhelm, 1964). The IV amitriptyline group was administered the drag during the tail vein injection of the Evans Blue dye. The knee joints were then perfused for 15 min with saline (baseline), followed by 25 min perfusion with 5-HT (1 ⁇ M). Perfusate samples were collected every 5 min for a total of 25 min. Samples were then analyzed using spectrophotometry.
  • 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.
  • A. 5-HT-INDUCED PLASMA EXTRAVASATION Baseline plasma extravasation was measured in all knee joints tested (total n 22). Baseline plasma extravasation levels were low, averaging 0.022 ⁇ 0.003 absorbance units at 620 nm (average ⁇ standard enor of the mean). This baseline extravasation level is shown in FIGURES 11 and 12 as a dashed line.
  • 5-HT (1 ⁇ M) perfused into the rat knee joint produces a time-dependent increase in plasma extravasation above baseline levels.
  • maximum levels of plasma extravasation were achieved by 15 min and continued until the perfusion was terminated at 25 min (data not shown). Therefore, 5-HT-induced plasma extravasation levels reported are the average of the 15, 20 and 25 min time points during each experiment.
  • 5-HT-induced plasma extravasation averaged 0.192 ⁇ 0.011, approximately an 8-fold stimulation above baseline. This data is graphed in FIGURES 11 and 12, conesponding to the "0" dose of IV amitriptyline and the "0" concentration of IA amitriptyline, respectively.
  • 5-HT-INDUCED PLASMA EXTRAVASATION Amitriptyline administered via tail vein injection produced a dose-dependent decrease in 5-HT-induced plasma extravasation as shown in FIGURE 11.
  • the IC50 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.
  • 5-HT-induced plasma extravasation in the presence of 3 ⁇ M IA amitriptyline was not significantly different from that produced by 5-HT alone, however, 30 ⁇ M amitriptyline co-perfused with 5-HT produced a greater than 50% inhibition, while 100 ⁇ M amitriptyline produced complete inhibition of 5-HT-induced plasma extravasation.
  • the IC5 0 for IA amitriptyline inhibition of 5-HT- induced plasma extravasation is approximately 20 ⁇ M.
  • 5-HT (1 ⁇ M) perfused intra- articularly in the rat knee joint produces a stimulation of plasma extravasation that is approximately 8-fold above baseline levels and that either intravenous or intra-articular administration of the 5-HT 2 receptor antagonist, amitriptyline, can inhibit 5-HT-induced plasma extravasation.
  • the total dosage of administered amitriptyline differs dramatically between the two methods of drag delivery.
  • the IC5 0 for IV amitriptyline inhibition of 5-HT-induced plasma extravasation is 0.025 mg/kg, or 7.5 x 10 "3 mg in a 300 g adult rat.
  • the IC 50 for IA amitriptyline inhibition of 5-HT-induced plasma extravasation is approximately 20 ⁇ M. Since 1 ml of this solution was delivered every five minutes for a total of 35 min during the experiment, the total dosage perfused into the knee was 7 ml, for a total dosage of 4.4 x 10 "5 mg perfused into the knee. This IA amitriptyline dose is approximately 200-fold less than the IV amitriptyline dose. Furthermore, it is likely that only a small fraction of the IA perfused drug is systemically absorbed, resulting in an even greater difference in the total delivered dose of drug.
  • 5-HT may play an important role in surgical pain and inflammation
  • 5-HT antagonists such as amitriptyline may be beneficial if used during the peri-operative period.
  • a recent study attempted to determine the effects of oral amitriptyline on post-operative orthopedic pain (Kenick 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 postoperative patients. Whether this was due to the overall unpleasantness produced by oral amitriptyline is not known.
  • an intra-articular route of administration allows an extremely low concentration of drag to be delivered locally to the site of inflammation, possibly resulting in maximal benefit with minimal side-effects.
  • Example V Example V above, with the following exceptions. Nitroprusside replaced SIN-1 as the nitric oxide donor and nicardipine replaced nisoldipine as the Ca 2+ channel antagonist.
  • the concentration of nitroprusside was selected based on its previously-defined pharmacological activity (EC 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.
  • An angiogram of the distal aorta and bilateral iliac arteries was recorded on 35 mm cine film (frame rate 15 per second) using hand injection of iopamidol 76% (Squibb Diagnostics, Princeton, NJ) into the descending aorta.
  • a calibration object was placed in the radiographic field of view to allow for conection 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.
  • a second angiogram was performed using the previously described technique.
  • a 1.25 mm or a 1.50 mm rotational atherectomy bun (Heart Technology/Boston Scientific Inc.) was advanced to the iliac arteries. The rotational.
  • the 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.
  • the rotational atherectomy bun was advanced from the distal aorta to the mid portion of the iliac artery between the first and second deep femoral branches.
  • the rotational atherectomy burr was rapidly removed and another angiogram was recorded on cine film at a mean of 8 minutes after the infusion was begun.
  • the infusion was continued until the 20 minute time point, and another angiogram (the fourth) was performed. Then the infusion was stopped. A total of about 95 cc of the control or test solution had been infused. At the 30 minute time point (15 minutes after the infusion was stopped), a final angiogram was recorded as before. Blood pressure and heart rate were recorded at the 15 and 30 minute time points immediately before the angiograms. After the final angiogram, the animal was euthanized with an overdose of the anesthetic agents administered intravenously.
  • ANGIOGRAPHIC ANALYSIS The angiograms were recorded on 35 mm cine film at a frame rate of 15 per second. Angiograms were reviewed in random order without knowledge of treatment assignment. For analysis, the angiograms were projected from a 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.
  • 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 occuned 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).
  • 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 ⁇ xlOO.
  • RA rotational atherectomy l
  • Proximal iliac artery measurement site proximal to the first deep femoral branch 2
  • Distal iliac artery measurement site near or distal to the second deep femoral branch
  • 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.
  • 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.
  • 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
  • test solution treated animals sustained substantial hypotension and significant tachycardia during the solution infusion.
  • test solution treated animals showed some partial, but not complete, return of blood pressure towards baseline.
  • 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.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cell Biology (AREA)
  • Zoology (AREA)
  • Oncology (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Molecular Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

L'invention concerne une méthode et une solution permettant d'inhiber de manière périopératoire une adhésion de cellules tumorales et une palette de douleurs et de processus inflammatoires au niveau de blessures liées à des interventions chirurgicales générales, notamment des interventions orales/dentaires. La solution comprend, de préférence, au moins un agent anti-adhésion de cellules tumorales et plusieurs agents d'inhibition de douleurs et d'inflammations dans une concentration diluée dans un véhicule physiologique, tel qu'une solution saline ou de Ringer lactée. La solution est appliquée par irrigation continue d'une blessure pendant une intervention chirurgicale aux fins d'inhibition préventive de douleurs et de manière à éviter simultanément des effets secondaires non souhaités associés à l'application orale, intramusculaire, sous-cutanée ou intraveineuse de doses importantes des agents. Une solution préférée permettant d'inhiber l'adhésion de cellules tumorales, des douleurs et des inflammations comprend au moins un agent anti-adhésion de cellules tumorales, un antagoniste de la sérotonine2, un antagoniste de la sérotonine3, un antagoniste de l'histamine, un agoniste de la sérotonine, un inhibiteur de la cyclo-oxygénase, un antagoniste de la neurokininel, un antagoniste de la neurokinine2, un antagoniste du purinocepteur, un ouvreur des canaux potassiques sensibles à ATP, un antagoniste du canal calcique, un antagoniste de la bradykininel, un antagoniste de la bradykinine2 et un agoniste mu -opioïde.
EP02793939A 2002-11-12 2002-11-12 Solution d'irrigation et methode d'inhibition de l'adhesion de cellules tumorales, de douleurs et d'inflammations Withdrawn EP1567179A4 (fr)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2002/036675 WO2004043479A1 (fr) 2002-11-12 2002-11-12 Solution d'irrigation et methode d'inhibition de l'adhesion de cellules tumorales, de douleurs et d'inflammations

Publications (2)

Publication Number Publication Date
EP1567179A1 true EP1567179A1 (fr) 2005-08-31
EP1567179A4 EP1567179A4 (fr) 2007-06-27

Family

ID=32311656

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02793939A Withdrawn EP1567179A4 (fr) 2002-11-12 2002-11-12 Solution d'irrigation et methode d'inhibition de l'adhesion de cellules tumorales, de douleurs et d'inflammations

Country Status (6)

Country Link
EP (1) EP1567179A4 (fr)
JP (1) JP2006508961A (fr)
CN (1) CN1694717A (fr)
AU (1) AU2002359404B2 (fr)
CA (1) CA2502363A1 (fr)
WO (1) WO2004043479A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI821204B (zh) * 2017-10-20 2023-11-11 日商大塚製藥工場股份有限公司 沾黏防止材
WO2020051543A1 (fr) * 2018-09-07 2020-03-12 Heron Therapeutics, Inc. Traitement post-chirurgical de la douleur
CN113384702A (zh) * 2021-05-20 2021-09-14 温州医科大学附属第一医院 H1组胺受体拮抗剂在制备治疗神经胶质瘤的药物中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996006622A1 (fr) * 1991-07-03 1996-03-07 Hyal Pharmaceutical Corporation Acide hyaluronique et derives destines a la modulation de l'activite cellulaire

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL110597A (en) * 1994-08-09 2002-11-10 Micro Tag Temed Ltd Method of marking, verifying and / or identifying an object and an instrument for performing the method
US6492332B1 (en) * 1995-12-12 2002-12-10 Omeros Corporation Irrigation solution and methods for inhibition of tumor cell adhesion, pain and inflammation
JPH10510540A (ja) * 1994-12-12 1998-10-13 オメロス メディカル システムズ，インコーポレーテッド 灌注用溶液並びに疼痛、炎症及びけいれんの抑制法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996006622A1 (fr) * 1991-07-03 1996-03-07 Hyal Pharmaceutical Corporation Acide hyaluronique et derives destines a la modulation de l'activite cellulaire

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
L REMY: "Données récentes sur les metalloproteinases, acteurs incontournables de la progression tumorale" PATHOLOGIE BIOLOGIE, vol. 45, no. 9, November 1997 (1997-11), pages 759-765, XP008079088 *
See also references of WO2004043479A1 *

Also Published As

Publication number Publication date
EP1567179A4 (fr) 2007-06-27
JP2006508961A (ja) 2006-03-16
AU2002359404B2 (en) 2008-06-05
WO2004043479A1 (fr) 2004-05-27
AU2002359404A1 (en) 2004-06-03
CN1694717A (zh) 2005-11-09
CA2502363A1 (fr) 2004-05-27

Similar Documents

Publication Publication Date Title
US6492332B1 (en) Irrigation solution and methods for inhibition of tumor cell adhesion, pain and inflammation
US6254585B1 (en) Surgical irrigation solution and method for inhibition of pain and inflammation
US5858017A (en) Urologic irrigation solution and method for inhibition of pain, inflammation and spasm
US20100029712A1 (en) Irrigation solution and method for inhibition of pain, inflammation, spasm and restenosis
CA2240256C (fr) Solution d'irrigation vasculaire et methode d'inhibition de la douleur, de l'inflammation, du spasme et de la restenose
US20060205658A1 (en) Irrigation solution and method for inhibition of pain and inflammation
WO2000023072A1 (fr) Solution d'irrigation contenant des inhibiteurs de mapk et son utilisation pour traiter la douleur et l'inflammation
EP1227807B1 (fr) Solution d'irrigation et procede d'inhibition de la douleur et de l'inflammation
WO2000023062A2 (fr) Solution et methode d'irrigation destinees a l'inhibition d'une douleur et d'une inflammation
EP1206275B1 (fr) Solution d'irrigation et methode pour l'inhibition de la douleur et de l'inflammation
AU2002359404B2 (en) Irrigation solution and method for inhibition of tumor cell adhesion, pain and inflammation
WO2000023061A2 (fr) Solution et methode d'irrigation destinees a l'inhibition d'une douleur et d'une inflammation
US20040214854A1 (en) Cardiovascular compositions and methods using combinations of anti-platelet agents
EP1563838A2 (fr) Solution d'irrigation et méthodes pour inhiber la douleur et l'inflammation
AU752132B2 (en) Irrigation solution and method for inhibition of pain, inflammation and spasm

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20050610

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LI LU MC NL PT SE SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

A4 Supplementary search report drawn up and despatched

Effective date: 20070530

17Q First examination report despatched

Effective date: 20071019

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20100601