JP2006508961A - Irrigation solutions and methods for inhibiting tumor cell adhesion, pain, and inflammation - Google Patents

Abstract

Methods and solutions for perioperatively inhibiting tumor cell adhesion and various pain and inflammatory processes in wounds resulting from common surgical procedures including oral / dental procedures. The solutions of the present invention preferably comprise at least one tumor cell adhesion inhibitor and a number of pain and inflammation inhibitors in rare concentrations in a physiological carrier such as physiological saline or lactated Ringer's solution. The solution of the present invention is used for surgical treatment to prophylactically inhibit pain and while avoiding undesirable side effects associated with oral, intramuscular, subcutaneous or intravenous application of larger doses of drugs. Applied by continuous irrigation of the wound during the period. One preferred solution for inhibiting tumor cell adhesion, pain and inflammation comprises at least one tumor cell adhesion inhibitor, as well as a serotonin ₂ antagonist, a serotonin 3 antagonist, and the like.

Description

  The present invention uses perioperative local delivery of a combination of therapeutic agents to simultaneously treat pain and / or inflammation and / or smooth muscle spasm and / or restenosis during surgical procedures. However, it relates to a method of inhibiting tumor cell adhesion and / or invasion and / or local tumor cell metastasis.

  Endoscopy is performed in a body cavity (eg, joint, peritoneal cavity, bladder, thoracic cavity, etc.) through a small entrance incision in the skin and body wall covering the remote light source and video monitor. Is a surgical procedure inserted into the Through a similar entrance incision, various surgical devices can be placed in the body cavity and their use can be guided by arthroscopic visualization. As the skills of endoscopists have improved, a growing number of surgical procedures, once performed by “open” surgical techniques, can now be achieved endoscopically. Such techniques include, for example, appendectomy, cholecystectomy and cardiac surgery. As a result of the expansion of surgical indications and the development of small diameter endoscopes, endoscopy in pediatrics has become routine.

  Throughout each endoscopic procedure, physiological irrigation fluid (eg, normal saline or lactated Ringer's) is continuously flushed from the joint, thereby inflating the body cavity, removing the surgical piece, and thus , Has resulted in a clearer visualization. US Pat. No. 4,504,493 (Marshall) discloses an equimolar aqueous solution of glycerol for a non-conductive, optically clear irrigation solution for arthroscopy.

  Irrigation is also used in other procedures such as cardiovascular and general vascular diagnostic and therapeutic procedures, urological procedures, and the treatment of burns and any surgical wounds. In each case, physiological fluid is used to irrigate the wound or body cavity or passage. Conventional physiological irrigation solutions do not inhibit tumor cell adhesion and / or invasion and / or local tumor cell metastasis, and provide analgesic, anti-inflammatory, anticonvulsant and anti-restenosis effects. Not.

  Various cell adhesion molecules are important in cell-cell interactions and in interactions between cells and components of the extracellular matrix. While such adhesion molecules are essential for a variety of biological processes and regulate intracellular signaling events, these molecules are highly metastatic to tumor metastases that require tumor cell adhesion and / or invasion. This is particularly important in the early stages. Specific cell adhesion molecules and proteinases are associated with the attachment and transplantation of free tumor cells present at the time of metastasis in a wide variety of human malignancies including breast cancer, prostate cancer, liver cancer, ovarian cancer and bladder cancer Yes. Such diverse adhesion molecules, which mediate adhesion interactions, are composed of cell surface receptor-ligand pairs. Cell surface receptors constitute a group of transmembrane proteins that can be classified as members of an associated biological family or superfamily based on homologous structures and functional characteristics they have together. Cancer cell adhesion at secondary sites is known to be regulated by several families of adhesion proteins including CD44 proteoglycans, integrins and selectins. The main function of these receptors is to mediate cell binding to the specific structural proteins that make up the extracellular matrix (ECM) and to recognize membrane-bound ligands that mediate cell-cell adhesion. . Cell adhesion and invasion can also be facilitated by proteinases such as metalloproteinases (MMPs) and their natural inhibitors (tissue inhibitors of metalloproteinases (TIMP)).

  Surgical trauma has been found to increase the frequency of tumor implantation at the site of surgical injury and wound healing. One result of surgical trauma to malignant tissue is the spread of locally free tumor cells at the surgical site. For example, pancreatic cancer patients often exhibit peritoneal dispersal and liver metastases after undergoing surgery. Various adhesion molecules and receptors that mediate free floating tumor cell attachment and transplantation at surgical and wound healing sites are often present in many other normal cell types. Surgical trauma also stimulates and enhances the production and release of proteinases, inflammatory cells, and extracellular matrix components from tumor cells. Therefore, pharmaceutical methods aimed at interfering with the function of cell adhesion molecules to reduce tumor cell adhesion and inhibit proteinase production and activation are at risk for local tumor metastasis Delivering a therapeutic agent only to the tissue. The process of free tumor cells adhering during surgery and invading the extracellular matrix or other cells is a dynamic process with specific interactions that are temporally related to surgical trauma, so The best time for is during surgery. Drugs that prevent the attachment of tumor cells to extracellular matrix proteins and / or inhibit the invasion of tumor cells during surgical procedures by discovering the role of numerous adhesion proteins, adhesion receptors and proteinases in the spread of cancer metastasis Development of the composition is possible.

  Relieving pain and distress in patients after surgery is an area of particular focus in clinical medicine, especially as an increasing number of outpatient surgeries are performed each year. The most widely used drugs, namely cyclooxygenase inhibitors (eg ibuprofen) and opioids (eg morphine, fentanyl) have significant side effects including gastrointestinal hypersensitivity / bleeding and respiratory depression. The high incidence of opioid-related nausea and vomiting is particularly problematic in the post-operative period. Therapeutic agents aimed at treating post-operative pain while avoiding harmful side effects because molecular targets for these agents are widely distributed throughout the body and mediate a variety of physiological effects. Not easily developed. Despite pain and inflammation and the clinical need to inhibit vasospasm, smooth muscle spasms and restenosis, pain and inflammation with minimal adverse systemic side effects No method has been developed to deliver effective dosages of inhibitors of convulsions and restenosis. As an example, conventional methods of administering opiates at therapeutic doses (ie, intravenous, oral, subcutaneous or intramuscular) have severe respiratory depression, mood changes, consciousness opacities, marked nausea and Often accompanied by significant adverse side effects including vomiting.

  Previous studies have shown that endogenous factors that cause pain and inflammation such as serotonin (5-hydroxytryptamine, which may be referred to herein as “5-HT”), bradykinin and histamine, etc. The ability is revealed. Sicuteri, F.M. Et al., “Serotonin-Bradykinin Potentiation in the Pain Receptors in Man”, Life Sci. 4: 309-316 (1965); Rosenthal, S .; R. “Histamine as the Chemical Mediator for Cutaneous Pain”, J. et al. Invest. Dermat. 69: 98-105 (1977); Richardson, B .; P. Et al., “Identification of Serotonin M-Receptor Subtypes and the Speci fi c Blockade by a New Class of Drugs”, Nature 316: 126-131 (1985); T.A. Et al., “The Effect of Kinin Agons and Antagonists”, Naunyn-Schmiedeb Arch. Pharmacol. 36: 652-57 (1987); Lang, E .; Et al., “Chemo-Sensitivity of Fine Afferents from Rat Skin In Vitro”, J. et al. Neurophysiol. 63: 887-901 (1990).

For example, 5-HT applied to the base of human blisters (exposed skin) has been shown to cause pain that can be inhibited by 5-HT ₃ receptor antagonists. Richardson et al. (1985). Similarly, bradykinin applied to the periphery causes pain that can be blocked by bradykinin receptor antagonists. Sicuteri et al., 1965; Whallley et al., 1987; Et al., “Bradykinin and Inflammatory Pain”, Trends Neurosci. 16: 99-104 (1993). Histamine applied to the periphery produces vasodilation, pruritus and pain that can be inhibited by histamine receptor antagonists. Rosenthal, 1977; Douglas, W .; W. "Histamine and 5-Hydroxytryptamine (Serotonin) and ther Antagonists" [Goodman, L .; S. Other, The Pharmaceutical Basis of Therapeutics, MacMillan Publishing Company, New York, pages 605-638 (1985)]; M.M. Et al., “Analysis Effects of Antihistamics”, Life Sci. 36: 403-416 (1985). The combination of these three agonists applied together (5-HT, bradykinin and histamine) has been shown to exhibit a synergistic action that causes pain, thereby resulting in a long-lasting intense pain signal. Yes. Sicuteri et al., 1965; Richardson et al., 1985; Kessler, W .; “Excitation of Cutaneous Affective Nerve Endings In Vitro by a Combination of Infrastructure Mediators and Conditioning Effect of Substance P”, Exp. Brain Res. 91: 467-476 (1992).

  In the body, 5-HT is present in platelets and central nerve neurons, histamine is found in mast cells, and bradykinin is produced from larger precursor molecules during tissue trauma, pH changes and temperature changes. 5-HT is released in large quantities from platelets at the site of tissue injury and can result in plasma levels that are 20 times greater than resting levels (Ashton, JH et al., “Serotonin as a Mediator of Cyclic Flow Variations in Stained Canon Arteries ", Circulation 73: 572-578 (1986)), so endogenous 5-HT may play a role in causing post-surgical pain, hyperalgesia and inflammation. Indeed, activated platelets have been shown to excite peripheral nociceptors in vitro. Ringkamp, M.M. Et al., “Activated Human Platelets in Plasma Excite Nociceptors in Rat Skin, In Vitro”, Neurosci. Lett. 170: 103-106 (1994). Similarly, histamine and bradykinin are also released into tissues during trauma. Kimura, E .; Others, “Changes in Bradykinin Level in Coronary Sinus Blood After the Experimental Occurrence of a Coronary Art”, Am Heart J. et al. 85: 635-647 (1973); Douglas, 1985; Dray et al. (1993).

  Prostaglandins are also known to cause pain and inflammation. Cyclooxygenase inhibitors (eg, ibuprofen) are commonly used in non-surgical and post-surgical situations to block the production of prostaglandins and thereby reduce prostaglandin-mediated pain and inflammation. Yes. Flower, R.A. J. et al. Et al., “Analgesic-Antipyretics and Anti-Inflammatory Agents; Drugs Implemented in the Treatment of Gout” [Goodman, L. et al. S. Other, The Pharmaceutical Basis of Therapeutics, MacMillan Publishing Company, New York, pages 674-715 (1985)]. Cyclooxygenase inhibitors are associated with several deleterious systemic side effects when applied conventionally. For example, indomethacin or ketorolac is widely recognized for adverse gastrointestinal and renal side effects.

  As discussed, 5-HT, histamine, bradykinin and prostaglandins cause pain and inflammation. Various receptors through which these factors mediate their effects on peripheral tissues have been known and / or discussed over the last 20 years. Most studies are done in rats or other animal models. However, there are differences in pharmacology and receptor sequences between human and animal species. No studies have been done to concludely reveal the importance of 5-HT, bradykinin or histamine in causing post-surgical pain in humans.

  Moreover, antagonists of these mediators are not currently used to treat post-surgical pain. A group of drugs called 5-HT and norepinephrine uptake antagonists, which include amitriptyline, have been used orally and have achieved moderate success for chronic pain conditions. However, the mechanism of the acute and chronic pain state appears to be quite different. In fact, two studies in the acute pain situation using amitriptyline in the perioperative period do not show any pain-reducing effects of amitriptyline. Levine, J .; D. Et al., “Desiplane Enhanced Operative Analgesia”, Pain 27: 45-49 (1986); Kerrick, J. et al. M.M. Et al., “Low-Dose Amitropyline as an Adjunct to Opioids for Postoperative Organic Pain: a Placebo-Controlled Trial Period”, Pain 52: 325-30 (1930). In both studies, the drug was administered orally. In the second study, it was observed that orally administered amitriptyline actually reduced the overall feeling that it was a satisfactory condition in patients after surgery. This may be due to the drug's affinity for multiple amine receptors in the brain.

  Amitriptyline is a potent 5-HT receptor antagonist in addition to blocking 5-HT and norepinephrine uptake. Thus, the lack of efficacy in reducing post-surgical pain in the previous study appears to contradict the proposed role for endogenous 5-HT in acute pain. There are many reasons for the lack of acute pain relief found with amitriptyline in these two studies. (1) In the first study (Levine et al., 1986), amitriptyline was used preoperatively for one week until the night before surgery, whereas in the second study (Kerrick et al., 1993), amitriptyline was used postoperatively. It was only used for. Thus, amitriptyline was not present in the surgical site tissue during the acute tissue injury phase, i.e. during the period when 5-HT is claimed to be released. (2) It is known that a large amount of amitriptyline is metabolized by the liver. In the case of oral administration, the concentration of amitriptyline in the surgical site tissue is not considered high enough for a long enough period in the second study to inhibit the activity of 5-HT released after surgery. (3) Blocking only one factor (5-HT) as there are numerous inflammatory mediators and research has shown synergy between inflammatory mediators Does not sufficiently inhibit the inflammatory response to tissue injury.

A study to reveal the ability of very high concentrations (1% to 3% solution, ie 10 mg / ml to 30 mg / ml) of histamine ₁ (H ₁ ) receptor antagonists to act as local anesthetics for surgical procedures There are few. This anesthetic effect is not thought to be mediated through the H ₁ receptor, but rather due to non-specific interactions with neuronal membrane sodium channels (which is similar to the action of lidocaine). Conceivable. Given the side effects (eg, sedation) associated with such high “anesthetic” concentrations of histamine receptor antagonists, local administration of histamine receptor antagonists is not currently used in perioperative situations.

(Summary of the Invention)
In one aspect, the present invention relates to preventing and treating tumor cell adhesion and / or invasion and / or local metastasis in a patient undergoing surgical treatment. The present invention inhibits tumor cell attachment and / or invasion and / or local metastasis during surgical procedures and at the same time pain and / or inflammation associated with surgery and / or smooth muscle spasm and / or relapse. Methods and pharmaceutical compositions based on drug combinations that inhibit stenosis are described. According to one aspect of the present invention there is provided a method for reducing or preventing tumor cell adhesion and / or invasion and / or local metastasis, at least one of which is a tumor adhesion inhibitor or an invasion agent. Or a composition comprising a combination of two or more metabolically active agents that are local metastasis inhibitors in a pharmaceutically effective carrier for delivery in an irrigation solution is administered directly to the surgical site Including that. Metabolically active agents include, but are not limited to, all that act directly or indirectly to modulate or alter the biological or biochemical or biophysical state of a cell Compounds are included. For example, plasma membrane potential, ligand binding to the receptor, agents that alter cellular receptor enzyme activity or expression levels, enzyme inhibitors or activators, protein-protein interactions or RNA-protein interactions or DNA-protein interactions Inhibitors of action are included. For example, functional receptor antagonists include monoclonal antibodies that competitively inhibit receptor binding sites, soluble receptors that reduce the concentration of free ligands that can be utilized to activate the receptor, inhibitors of receptor signaling pathways, cells Activators and inhibitors of internal or extracellular enzymes, and agents that modulate the binding of transcription factors to DNA can be included.

  Specifically, the present invention provides a combination of agents delivered locally and perioperatively to achieve tumor cell adhesion and / or invasion and / or local metastasis to achieve maximum therapeutic benefit. Pharmacological methods of use and treatment are provided. The use of a combination of drugs allows only one to block the multifactorial process of tumor cell adhesion and invasion regulated by numerous pro-inflammatory cytokines and receptors implicated in the inflammatory response resulting from surgical procedures The limitations of existing therapeutic approaches that rely on the use of drugs are overcome. Pro-inflammatory cytokines have been reported to stimulate the expression of adhesion receptors and metalloproteinases present in both tumor cells and normal cells, thereby increasing their adhesion and invasion properties. For example, the combination of an anti-adhesive agent and an anti-inflammatory agent can provide additional or synergistic benefits to the pharmaceutical composition.

  In the present invention, 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 A method is used that combines drugs that act simultaneously on and deliver these drugs to the surgical site throughout the surgical procedure. Such combinations of agents can achieve prophylactic inhibition of tumor cell adhesion and / or invasion throughout the surgical procedure, while also pain and / or inflammation, and / or Smooth muscle spasm and / or restenosis can be prophylactically inhibited. Inhibition of only one adhesion receptor-ligand interaction by systemic delivery can cause adverse reactions. This is because these molecules also function to deliver signals to normal cells. For example, CD44 receptor binding plays a physiological role in the immune response of normal T cells and other hematopoietic cells, and thus inhibition of CD44 binding can cause undesirable effects on the immune system.

  Specifically, the present invention provides a pharmaceutical composition of metabolically active agents based on a combination of at least two agents that act simultaneously on different molecular targets. At least one agent directly reduces or directly prevents tumor cell adhesion to either the extracellular matrix or other cells (both normal and tumor) or prevent invasion Agent or local metastasis inhibitor. Suitable agents for inclusion in a pharmaceutical composition are only one family (or class) receptor or only one enzyme family (eg CD44, integrins, selectins, protein tyrosine kinases, MMPs and MAP kinases) Is further characterized by a requirement for pharmacological selectivity and specificity that limits the interaction of the drug to Certain suitable agents are capable of specifically interacting with one or more receptor subtypes (or isoforms) and at the same time exhibit specificity for that receptor family. Each suitable agent can be characterized by the stoichiometry (typically 1: 1) defined for the ligand-receptor (or inhibitor-enzyme) complex and its equilibrium binding constant or equilibrium It associates with its molecular target by a specific molecular mechanism that can be characterized by kinetic constants.

  The at least one second agent belongs to the class of receptor antagonists, enzyme inhibitors or cytokines having anti-pain activity, anti-inflammatory activity, anti-convulsant activity and / or anti-restenosis activity. Optimal drug combinations include tumor cell adhesion inhibitors and / or receptor antagonists that act to inhibit or reduce the activity or expression of cell adhesion receptor molecule targets (eg, integrin receptor antagonists, CD44 receptor antagonists and selectin receptor antagonists) and / or Or at least one agent selected from the class of invasion inhibitors, or a proteinase inhibitor.

  Also, other suitable agents included in optimal drug combinations that are functional tumor cell adhesion inhibitors act to inhibit cellular signaling pathways activated by cell adhesion receptors. Molecular targets that make up these pathways include membrane-bound enzymes and intracellular localized enzymes that transduce and integrate signals generated by activated cell surface adhesion receptors. By way of example, these agents include inhibitors of enzymes belonging to the following families: protein tyrosine kinase, protein kinase C and mitogen activated protein kinase (MAPK). Such enzyme inhibitors can be characterized by a defined stoichiometry (typically 1: 1) of the enzyme-inhibitor complex and can be characterized by their equilibrium binding constants. A mechanism may interact with specific member (s) of the kinase family.

  Despite numerous experimental studies, effective treatments have not been previously developed to prevent tumor cell adhesion and / or invasion during surgical procedures. It is an object of the present invention to provide a method for prophylactically treating tumor cell adhesion and / or invasion and / or local metastasis during surgical procedures with irrigation fluid for continued delivery of therapeutic agents. Is the purpose. Existing delivery methods provide a means to initiate the delivery of a pharmaceutical agent directly to the surgical site and to maintain drug levels throughout the surgical procedure at therapeutically effective concentrations or higher Not. Large volumes of irrigation fluid are used during certain surgical procedures (eg, laparoscopic surgery), so that the irrigation fluid contains the drug to prevent drug dilution and maintain a therapeutic level A method for providing is provided. Various problems associated with systemic delivery of therapeutic peptides, proteins and small molecules as potential tumor cell adhesion / invasion and metastasis inhibitors, including toxicity, pharmacological stability and poor pharmacokinetic profile Can be reduced or avoided by local delivery of these therapeutic agents in the irrigation fluid directly to the surgical site.

  Numerous drug combinations can be delivered locally and perioperatively by irrigation (ie, intraoperative delivery alone or in conjunction with preoperative and / or postoperative delivery). The present invention also provides tumor adhesion inhibitors and / or invasion inhibitors and / or local metastasis inhibitors, one or more analgesics and / or anti-inflammatory and / or anticonvulsants and / or restenosis prevention. Delivery in combination with an agent is provided.

  A significant advantage of the present invention is obtained from the rapid onset of pharmacological action. Direct and local delivery of the tumor adhesion inhibitor starting from the beginning of the surgical procedure and continuing during the surgical procedure (ie during the perioperative period) is a very early stage of metastasis. One has the potential to inhibit the initial adhesion process. Immediate and consistent delivery of therapeutically effective concentrations of anti-adhesive and / or anti-intrusion agents is the first attachment of tumor cells to both the extracellular matrix and other cells, and subsequent to these tissues It is expected to prevent invasion prophylactically. Studies have found that the maximum rate of tumor transplantation occurred when tumor cells were injected immediately after surgery. This indicates that immediately after surgery is a critical time to therapeutically inhibit the adhesion process.

  The delivery methods and pharmaceutical compositions of the present invention provide a variety of different advantages. Such benefits include: 1) Mixed drug therapy directed at multifactorial causes of tumor cell adhesion / invasion / local metastasis, inflammation, pain, smooth muscle spasm and restenosis during surgical procedures, 2) The local delivery of the drug combination achieves an instantaneous therapeutic concentration of the tumor adhesion inhibitor at the surgical site, and 3) the continuous delivery of the therapeutic agent in the irrigation solution allows a therapeutically effective range at the surgical site. A constant drug concentration is provided during the surgical procedure, 4) local delivery allows for a reduction in overall drug dose and dosing frequency when compared to systemic delivery, and 5) to the surgical site Direct and local delivery includes allowing the use of new pharmaceutically active agents (eg, certain peptides and antibodies) that cannot be delivered systemically.

The present invention further provides a solution comprising a mixture of multiple drugs at low concentrations directed to locally inhibit pain, inflammation, convulsions and restenosis mediators in a physiological electrolyte carrier fluid. provide. The present invention also provides these agents directly against surgical sites where the irrigation solution functions locally at the receptor and enzyme levels to prophylactically limit pain, inflammation, convulsions and restenosis at the site. A method is provided for delivering a containing irrigation solution in the perioperative period. Due to the local perioperative delivery method of the present invention, the desired therapeutic effect is greater than that of the drug required when using other delivery methods (ie, intravenous, intramuscular, subcutaneous and oral). It can be achieved at low doses. Anti-pain / anti-inflammatory agents in solution include drugs selected from the following classes of receptor antagonists and receptor agonists and enzyme activators and enzyme inhibitors, each class differing in pain and inflammation inhibition: Acting through a molecular mechanism of action): (1) serotonin receptor antagonist; (2) serotonin receptor agonist; (3) histamine receptor antagonist; (4) bradykinin receptor antagonist; (5) kallikrein inhibitor; (6) tachykinin receptor antagonists This includes the neurokinin ₁ receptor subtype antagonists 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 (PLA ₂ isoform inhibitors and PLC containing _γ isoform inhibitors), (b) include cyclooxygenase inhibitors, and (c) lipoxygenase inhibitors; (10) prostanoid receptor antagonists, including, eicosanoid EP-1 receptor subtype antagonists and eicosanoid EP-4 Receptor subtype antagonists as well as thromboxane receptor subtype antagonists); (11) leukotriene receptor antagonists Agonist, This includes the leukotriene B ₄ receptor subtype antagonists and leukotriene D ₄ receptor subtype antagonists; (12) opioid receptor agonists, including, .mu.-opioid receptor subtype agonists, .delta.-opioid receptor subtype Agonists and kappa-opioid receptor subtype agonists; (13) purine receptor agonists and purine receptor antagonists, including P _2X receptor antagonists and P _2Y receptor agonists; and (14) adenosine triphosphate ( ATP) sensitive potassium channel opener. Each of the above drugs functions either as an anti-inflammatory agent and / or as an antinociceptive agent (ie, a pain-preventing or analgesic agent). The selection of drugs from these compound classes is tailored for specific applications.

  In some preferred embodiments of the solutions of the present invention, anticonvulsants are also included for specific applications. For example, an anticonvulsant can be included alone in a solution used for vascular treatment to limit vasospasm, or in combination with an anti-pain agent / anti-inflammatory agent, Can be included for urological procedures to limit convulsions in the urinary tract and bladder wall. For such applications, anticonvulsants are utilized in solution. For example, it can include an anti-pain / anti-inflammatory agent that also acts as an anticonvulsant. Suitable anti-pain / anti-inflammatory agents that also act as anticonvulsants include serotonin receptor antagonists, tachykinin receptor antagonists and ATP sensitive potassium channel openers. Other agents that can be utilized in solution, particularly for their anticonvulsant properties, include calcium channel antagonists, endothelin receptor antagonists and nitric oxide donors (enzyme activators).

Certain preferred embodiments of the solutions of the invention used in cardiovascular and general vascular procedures include anti-restenosis agents most preferably used in combination with anticonvulsants. Suitable anti-restenosis agents include: (1) antiplatelet agents, which include (a) thrombin inhibitors and thrombin receptor antagonists, (b) adenosine diphosphate (ADP) receptor antagonists (which are also , Known as receptor antagonists of purine receptor ₁ ), (c) thromboxane inhibitors and thromboxane receptor antagonists, and (d) platelet membrane glycoprotein receptor antagonists; (2) inhibitors of cell adhesion molecules, which (A) selectin inhibitors and (b) integrin inhibitors; (3) anti-chemotactic agents; (4) interleukin receptor antagonists (which also serve as pain / anti-inflammatory agents); And ( ) Intracellular signaling inhibitors, in this, (a) protein kinase C (PKC) inhibitors and protein tyrosine kinase inhibitors, (b) modulators of intracellular protein tyrosine phosphatases, (c) src homology ₂ (SH2) domain And (d) a calcium channel antagonist. Such agents are useful in preventing restenosis of arteries treated by angioplasty, rotational atherectomy, or other therapeutic or diagnostic procedures of cardiovascular or general blood vessels.

  The present invention also provides a method for producing a pharmaceutical formulated as a dilute irrigation solution for use in continually irrigating a surgical site or wound during a surgical procedure. In this method, at least one inhibitor of tumor cell adhesion, invasion and / or metastasis, preferably an anti-pain agent / anti-inflammatory agent, and for some applications an anti-convulsant and / or Involving the dissolution of an anti-restenosis agent in a physiological electrolyte carrier fluid, wherein each agent is preferably included at a concentration of at most 100,000 nanomolar, more preferably at most 10, 000 nanomolar concentration.

  In the method of the present invention, a diluted combination of at least one inhibitor of tumor cell adhesion, invasion and / or metastasis, and preferably a number of receptor antagonists and receptor agonists and enzyme inhibitors and enzyme activators, In order to inhibit inflammation, convulsions and restenosis, it is provided to deliver directly to the wound or surgical site during therapeutic or diagnostic treatment. Since the active ingredient in the solution is topically applied directly to the surgical tissue in a continuous manner, the drug has a therapeutic effect when the same drug is delivered orally, intramuscularly, subcutaneously or intravenously It can be used effectively at very low doses compared to such doses required for. As used herein, the term “topical” includes application of a drug at or around a wound site or other surgical site, and does not include oral, subcutaneous, intravenous, and intramuscular administration. . As used herein, the term “continuous” refers to uninterrupted application, repeated application at frequent intervals (eg, intravascular bolus repeated during treatment at frequent intervals), and substantially constant. Includes applications that are uninterrupted except for short-term outages, such as allowing the introduction of other drugs or agents or treatment devices so that a predetermined concentration is maintained locally at the wound or surgical site .

  The advantages of low dose application of the drug consist of three points. Most importantly, there are no systemic side effects that often limit the usefulness of these drugs. Also, the drugs selected for a particular application in the solutions of the present invention are very specific with respect to the mediators on which they function. This specificity is maintained by the low dosage utilized. Finally, the cost of these active agents per surgical procedure is low.

  The advantages of local administration of the drug by luminal irrigation or other fluid application are as follows: (1) Due to local administration, known concentrations at the target site, despite variations between patients in metabolism, blood flow, etc. (2) because of the direct delivery mode, therapeutic concentrations are obtained instantaneously, thus providing improved dosage control; and (3) for wound or surgical sites Direct topical administration of the active agent is also an extracellular process that occurs when the agent is administered orally, intravenously, subcutaneously or intramuscularly (eg, first pass metabolism and two passes). Substantially reduces the degradation of the drug by metabolism). This is especially true for such active agents that are rapidly metabolized peptides. Thus, topical administration allows the use of compounds or drugs that cannot otherwise be used therapeutically. For example, some drugs in the following classes are peptidic: bradykinin receptor antagonists; tachykinin receptor antagonists; opioid receptor agonists; CGRP receptor antagonists; and interleukin receptor antagonists. Local and continued delivery to the wound or surgical site minimizes drug degradation or metabolism, while also providing continuous replacement of such portions of the drug that can be degraded, locally High therapeutic concentrations (which are sufficient to maintain receptor occupancy) are reliably maintained throughout the duration of the surgical procedure.

  According to the present invention, topical administration of the solution in the perioperative period throughout the surgical procedure provides a prophylactic analgesic, anti-inflammatory, anticonvulsant or anti-restenosis effect. As used herein, the term “perioperative” encompasses application during, before and during treatment, before and after treatment, and before, during and after treatment. To do. In order to maximize the prophylactic anti-inflammatory effect, analgesic effect (for a specific application), anticonvulsant effect (for a specific application) and anti-restenosis effect (for a specific application), the solution of the present invention Is most preferably applied before, during and after surgery. Prior to the onset of significant surgical trauma, the solution agents of the present invention can be selected for specific routes by locally occupying the target receptor or by locally inactivating or activating the targeted enzyme. To prevent and prevent targeted pathological processes. If the inflammatory mediators and processes are prophylactically inhibited according to the present invention before they can cause tissue damage, the benefits are more substantial than if they are obtained after damage is initiated.

  Inhibiting two or more mediators of tumor cell adhesion, invasion and / or metastasis, inflammation or convulsions or restenosis by applying the multidrug solution of the present invention, cell adhesion, invasion and / or metastasis, It is useful to dramatically reduce the degree of inflammation, pain and convulsions and should theoretically reduce restenosis. The irrigation solution of the present invention comprises a combination of drugs, each solution acting on a number of receptors or enzymes. Thus, drug agents are simultaneously effective against a combination of various pathological processes including tumor cell adhesion, invasion and / or metastasis, pain and inflammation, vasospasm, smooth muscle spasms and restenosis. The actions of these agents are considered to be synergistic in that many receptor antagonists and inhibitory agonists of the present invention provide increased potency that is not proportional to the potency of the individual drugs. Some synergistic actions of the agents of the present invention are discussed below as examples in the detailed description of such agents.

  In addition to arthroscopy, the solutions of the present invention may also be applied to any human body cavity or passage, surgical wound, traumatic wound (eg, burn), or any surgery / intervention where irrigation can be performed. Can be applied topically in the procedure. These procedures include urological procedures, cardiovascular and general vascular diagnostic and therapeutic procedures, endoscopic procedures, and oral, dental and periodontal procedures, It is not limited to these. The term “wound” as used herein below is intended to encompass surgical wounds, surgical / intervention sites, traumatic wounds and burns unless otherwise indicated.

  When used in the perioperative period, the solution of the present invention results in a clinically significant reduction in pain and inflammation at the surgical site compared to currently used irrigation solutions, thereby reducing the patient's post-surgery It should reduce the demand for analgesics (ie, opiates) and, if appropriate, allow for faster mobilization of the surgical site. No additional effort on the department of surgeons and operating room personnel is required to use the solution of the present invention compared to conventional irrigation solutions.

(Detailed description of preferred embodiments)
In accordance with the present invention, various methods and solutions are provided to reduce or prevent tumor cell adhesion and / or invasion and / or local metastasis, which are two or more metabolically active agents. A composition comprising a combination of (at least one of which is a tumor adhesion inhibitor or an invasion inhibitor or a local metastasis inhibitor) in a pharmaceutically effective carrier for delivery in an irrigation solution to the surgical site Including direct administration.

I. Inhibitors of cell adhesion and cell entry molecules Metalloproteinase antagonists and tissue inhibitors of metalloproteinases (TIMP)
Matrix metalloproteinases (MMPs) are a family of zinc-dependent neutral endopeptidases whose enzyme activity is capable of controlled degradation of the extracellular matrix (ECM). This activity is used as an important regulator in tumor adhesion, invasion ability, metastasis, growth and angiogenesis. In humans, 17 members of this family have been identified by cloning and sequencing. These members are divided into collagenase, gelatinase, stromelysin and stromelysin-like MMPs, membrane-type MMPs, and novel MMP subgroups.

  These MMPs have both common structural and functional characteristics. All families to date have at least three domains: a conserved cysteine residue and a prodomain that is lost upon enzyme activation; a conserved structural metal binding site A catalytic domain of 106 to 119 residues; and a highly conserved zinc-binding active site domain of 52 to 58 amino acids.

  Cells generally do not constitutively express MMPs, but rather their expression is responsive to exogenous signals, cytokines or growth factors, and altered cell-matrix and cell-cell interactions. To be guided quickly. Exceptions to this rule are MMP-8 and MMP-9 stored in secretory granules of neutrophils and eosinophils, and MMP-7 stored in secretory epithelial cells.

  MMP expression is primarily regulated at the transcriptional level, and its proteolytic activity is regulated by zymogen activation and inhibition of activity. Extracellular signals such as cytokines (IL-1, tumor necrosis factor, etc.) activate the AP-1 transcription factor complex composed of members of Jun protein and Fos protein, in which case Jun protein and Fos The protein binds to the cis element of AP-1 and activates the transcription of the corresponding MMP gene. Most MMPs are secreted as latent precursors (zymogens) that are proteolytically activated in the extracellular space. However, as an exception, MMP-11 and MT1-MMP are activated before their secretion by furin-like proteases bound to the Golgi.

  The activity of MMPs in the extracellular space is controlled by matrix proteinase tissue inhibitors (TIMPs), a family of natural inhibitors. To date, four members of this family have been identified (TIMP-1, 2, 3, 4) and are well characterized. These TIMPs have similar inhibitory activity against most of the MMPs, but differ with respect to their interaction with pro-MMP, solubility, regulation of expression, and tissue-specific expression. TIMP-3, unlike other TIMPs, inhibits the activity of TNF-α converting enzyme (TACE), suggesting a role in regulating inflammation. TGF-β, IL-1, IL-6, TNF-α, EGF and glucocorticoids regulate TIMP-1 at the transcriptional level. TIMP-2 is constitutively expressed and TIMP-4 expression is limited. TIMP-3 expression is upregulated by mitogenic factors, phorbol esters and TGF-β.

  MMP has been shown to promote tumor cell invasion and metastasis by at least three mechanisms. In a physiological sense, initially, MMPs can regulate tumor cell adhesion. This process is necessary for the cells to attach and pass through the ECM. Cell transfection studies with varying ratios of MMP-2 to TIMP-2 reveal changes in tumor cell adhesion phenotypes. The proteinase action of MMP can then degrade ECM macromolecules such as collagens, laminins and proteoglycans, thereby allowing tumor cells to enter. Finally, MMPs can act on ECM components or other proteins to activate biological functionality. For example, laminin-5 is degraded by MMP-2 resulting in a soluble chemotactic fragment.

  The classical role of MMPs to degrade ECM components and in particular the basement membrane is considered very important for tumor cell invasion. Experimentally, the need for protease action in tumor cell invasion has been demonstrated using a chemoinvasion assay. Abundant expression of different MMPs occurs in invasive primary tumors or metastases thereof. MMP-1 expression levels correlate with poor prognosis in colorectal and esophageal cancer, and MMP-2 and MMP-3 expression is 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 reduced tumor development in the intestine, and MMP-2 deficient mice show reduced angiogenesis and tumor progression. Inflammatory cell infiltration is also a hallmark of many malignant tumors, and inflammatory cells may be a source of MMPs that contribute to tumor cell invasion. These inflammatory cells also produce cytokines, which can enhance the expression of MMPs by tumor cells and stromal cells, resulting in further tumor cell invasion.

  TIMPs also play a role in tumor cell invasion. The ability of TIMP-1, 2, 3 and 4 to inhibit tumor growth has been shown by overexpressing them in various human and rodent models. In melanoma cells, overexpression of TIMP-2 inhibits the growth of tumors transplanted into the skin of SCID mice. This growth inhibition does not appear to depend on angiogenesis but appears to depend on the collagen matrix. Currently accepted ideas for the role of TIMPs in cancer currently focus on inhibiting tumor invasion, metastasis, and regulation of ECM homeostasis.

  Based on in vitro, in vivo, in situ and clinical studies, inhibition of MMP activity can inhibit neoplastic cell adhesion, invasion and metastasis, and effectively suppress tumor growth and metastasis be able to. Prevention of tumor growth can be achieved by inhibiting MMP expression or activity at multiple stages in the MMP synthesis pathway.

  Inhibition of MMP transcription is such signaling by directly blocking signaling pathways that regulate MMP transcription or by inhibiting transcription factors involved in activating MMP gene transcription. It can be achieved by blocking the path. Inhibition of signaling involved in AP-1 activation, ie inhibition of MAPK (ERK1, ERK2, SAPK / JNK or p38), can significantly inhibit MMP expression and malignant cell invasion in vitro. Expression of a dominant negative transcription factor, which requires an effective method of gene therapy in vivo, can be used to inhibit MMP production. For example, the expression of dominant negative c-Jun has been shown to inhibit the induction of MMP-1, 2, 3, 10 and 14 as well as the in vitro entry of keratinocytes. Retinoids as inhibitors of AP-1 activity can also be used to prevent expression of the MMP gene in selected human carcinomas.

  Another method for inhibiting the expression of specific MMPs is to use antisense RNA. Antisense oligonucleotides to MMP-7 have been shown to inhibit colon carcinoma cell invasion in vitro and in vivo. An antisense-ribozyme expression construct of MMP-9 has been shown to inhibit MMP-9 expression and rat bone cancer cell invasion.

  The most extensively studied area of inhibiting tumor cell invasion by inhibiting MMPs has so far been the development of compounds to block MMP activity. Synthesized MMP inhibitors (MMPI), such as hydroxymart inhibitors, are small peptide analogs of fiber collagen that specifically interact with zinc at the MMP catalytic site and inhibit MMP activity. . Currently, some of these drugs are in clinical trials to treat invasive tumors. The first class to be developed was broad spectrum MMPI that nonspecifically inhibits many MMPs. Examples include batimastat (BB-94), which has low oral bioavailability and requires topical administration, and marimastat (BB-2516), which can be administered orally. The results of preclinical studies indicate that these drugs inhibit tumor invasion, metastasis and growth. Next, the development of selective drugs to target specific MMPs was undertaken. Because peptide-based MMP inhibitors are expensive to manufacture and generally have poor bioavailability, non-peptide inhibitors are developed by rational design based on X-ray crystallographic information of the MMP active site Has been. Examples of non-peptide inhibitors are CGS27023A (Novartis), AG3340 (Agouron), and the fenbufen derivative BAY12-9565 (Bayer). In addition, tetracycline compounds having no antibacterial action have been developed to block host-derived MMPs. This class of chemically modified non-antibacterial tetracycline compounds (CMT) has been shown to block the expression of MMP-2, 3 and 8 by a number of mechanisms and also induces apoptosis of malignant cells It is also shown. Most recently, bisphosphonate compounds have been identified as MMP inhibitors and aggrecanase-1 inhibitors, and SE206, BB-16 and XS309 have been shown to have good potency. Finally, the ability of TIMP to potently and specifically inhibit MMP activity is of increasing interest in its use. TIMP-1, 2, 3 and 4 can inhibit invasion of malignant cells in vitro and in vivo.

  In one aspect, the present invention provides one or more MMP inhibitors that are effective in preventing or inhibiting tumor cell adhesion, invasion and metastasis, at least one additional pain and / or anti-inflammatory agent. And / or in combination with an anticonvulsant and / or an anti-restenosis agent for direct administration to the surgical site by an irrigation solution. Various inhibitors and antagonists can act at the level of transcription, translation, secretion, activation or degradation of MMPs.

  One of ordinary skill in the art will be able to determine, in part, the amount of inhibitor administered for any particular treatment protocol, and the amount of these will be easily determined by the choice of MMP inhibitor as a therapeutic agent for the patient. Understand that can be determined.

  Suitable MMP inhibitors for use in the present invention are shown in the table below.

B. CD44 receptor antagonists and inhibitors of CD44 signaling CD44 is a transmembrane glycoprotein that constitutes a family of surface receptors composed of numerous variant isoforms. CD44 receptors are found in a wide variety of tissues including the central nervous system, lungs, epithelium, liver, colon and pancreas. The related cell surface adhesion molecules of this family are multifunctional proteins that are expressed in both normal and malignant tissues. CD44 is a variant exon encoded by a single gene containing 20 exons, 10 of which (v1-v10) are inserted by alternative splicing. The standard isoform of CD44 (CD44s) is ubiquitously expressed and does not contain the sequence encoded by these variant exons. Numerous variant isoforms of CD44 (CD44v) containing various combinations of exons (v1 to v10) inserted into the extracellular domain, their expression was activated in proliferating epithelial cells Limited to lymphocytes and malignant cells.

  Recent studies have revealed that CD44 protein is the primary receptor for hyaluronic acid in humans. CD44 is also called the hyaluronic acid receptor because CD44 is the major transmembrane receptor for glycosaminoglycans in this extracellular matrix. CD44 can also bind with a lower affinity to several other extracellular matrix ligands (chondroitin sulfate, heparin sulfate and fibronectin).

  CD44 receptor-ligand interactions are required for many normal cellular processes including cell adhesion (aggregation and migration), hyaluronic acid degradation, lymphocyte activation, lymphocyte recruitment to inflammatory sites, and cytokine release It is. Further specialized functions include assembly of the pericellular matrix during cartilage formation and wound healing. Interaction of the CD44 receptor with hyaluronic acid (HA) delivers important signals to normal and transformed CD44-bearing cells. In common with many other cell surface receptors, in cells expressing CD44, the expression of this receptor is regulated in response to ligand binding.

  The CD44 molecule is an 85 kD glycosylated molecule with N-terminal sequence homology to cartilage binding protein. CD44 isoforms with various sizes have been described for many cell types. Various changes in the size of the CD44 isoform occur due to glycosylation differences and the addition of chondroitin sulfate molecules to CD44. An 85 kD form has been identified in serum, plasma and synovial fluid. Alternative splicing has resulted in a hematopoietic form of this molecule (CD44H) and additional subtypes. Alternative splicing results in altered protein glycosylation and provides a mechanism for modulating the binding activity of CD44. Thus, the CD44 molecule, due to its presence in several isoforms and because of its broad cellular distribution and functional relevance to other adhesion molecules, activates immune cells as well as certain tumor cell types. It is a multifunctional pro-inflammatory molecule involved in the metastasis.

  The predominant isoform found in human leukocytes is CD44H. Both hyaluronic acid binding and CD44 mAb binding to monocytes increase IL-1 release. In T cells, the binding of CD44 hyaluronic acid and CD44 mAb has essentially different effects: CD44 mAb enhances T cell trigger function, whereas hyaluronic acid suppresses T cell trigger function. Finally, CD44 mAb and polyclonal anti-CD44 serum have been shown to inhibit lymphocyte binding to endothelial venules at sites of inflammation such as synovium.

  The physiological function of CD44 indicates that CD44 plays a role in tumor metastasis expansion. Various properties of CD44, such as hyaluronic acid-mediated adhesion, cell motility, and adhesion to lymphoid tissue are some of several properties required by invasive and metastatic tumor cells. . In vivo experiments show that tumor cells transfected to overexpress specific CD44 isoforms exhibit increased metastatic potential. Among many CD44 isoforms, certain isoforms enhance tumor cell metastasis more efficiently than other isoforms. The ability to metastasize can be conferred on a non-metastatic cell line by transfection with a variant of CD44. High levels of CD44 are associated with several types of malignancy.

  Numerous studies have reported that human tumors exhibit specific changes in the expression of CD44 isoforms, and that the degree of clinical disease in a particular tumor correlates with the expression pattern of CD44 isoforms. Further shown. As an example, it was found that expression of a splice variant of CD44 was required for the adenocarcinoma cells to metastasize and metastasis was prevented by a monoclonal antibody (mAb) against this particular CD44 variant. A wide variety of epithelial and mesenchymal malignancies also express high levels of CD44H and various variant isoforms of CD44. Specifically, the report reports that CD44 variant expression is a common feature of epithelial ovarian cancer.

  Many studies have examined the CD44 distribution pattern in tumors. It has been found that the presence of CD44 standard and CD44-9v isoforms on the surface of gastric cancer cells is significantly associated with greater tumor-induced mortality and shorter survival for patients. Intestinal type gastric carcinoma expresses abundant CD44-6v isoforms that provide the ability to metastasize to these tumor cells. There is convincing evidence that integrin receptors, and various receptors of the CD44 isoform, change after malignant transformation of the colonic mucosa to adenomas and invasive carcinomas and correlate with their metastatic potential. Expression of the CD44-6v isoform is associated with an adverse prognosis of colorectal cancer due to tumor metastasis and adhesion development. These data suggest that CD44 plays an important role in tumor metastasis and that various molecules that inhibit the interaction of the CD44 receptor with its natural ligand can be used for therapy. .

  At the cellular level, recent studies have characterized the cytoplasmic signaling pathway for CD44 in activated endothelial cells involving tyrosine kinases. The HA binding activity of CD44 is linked to cell activation in some cell types. Addition of hyaluronan degradation products (3-10 disaccharides) to cells induces rapid tyrosine phosphorylation of many sustained proteins. Increased phosphorylation of the CD44 receptor was also observed. Pretreatment of cells with either an anti-CD44 receptor antibody or a non-selective inhibitor of tyrosine kinase activity inhibited both induced protein tyrosine phosphorylation and proliferative responses. Protein kinase C (PKC) activity increased 2- to 3-fold in the membranes of treated cells. Furthermore, PKC activation was shown to initiate a cytoplasmic kinase cascade with Raf-1 kinase, MAP kinase (MEK-1), and a kinase regulated by extracellular signals (ERK-1). ERK is a bispecific kinase that controls not only tumor cell growth, but also the expression of proteins related to tumorigenesis and locomotor activity.

  In summary, phosphorylation of the CD44 receptor results in increased tyrosine phosphorylation, which results in activation of the cytoplasmic cascade, activation of early response genes, and cell proliferation. Thus, an alternative indirect method for inhibiting CD44 activation and signaling is to use inhibitors of cell signaling pathways activated by CD44, rather than utilizing direct CD44 receptor antagonists. It is thought that. Tyrosine kinase inhibitors, PKC inhibitors (eg, calphostin) or ERK inhibitors can be used as agents that block signaling proteins mediated by CD44 and provide novel targets for therapeutic agents.

  One very important event in tumor growth and metastasis is the interaction between tumor cells and host tissue stroma mediated by various combinations of various adhesion receptors in different tumor cell types. Several lines of evidence indicate that the interaction between CD44 receptor expressed in tumor cells and tissue stromal HA can enhance the growth and invasiveness of certain tumors. Transformation of colonic mucosa to carcinoma is associated with overexpression of several CD44 alternative splice variants. The importance of CD44-hyaluronic acid interaction in tumor development is shown to be that of CD44-hyaluronic acid by soluble recombinant CD44, which was shown to inhibit tumor formation by lymphoma and melanoma cells transfected with CD44. It is revealed by breaking the interaction. Mutant non-hyaluronic acid-binding CD44 whose differential inhibitory effect of soluble wild-type and mutant CD44-Ig fusion proteins on melanoma growth in vivo had no effect on subcutaneous melanoma growth in mice -Revealed by topical administration of -Ig fusion protein, whereas infusion of wild-type CD44-Ig prevented tumor development.

  The present invention prevents or inhibits tumor cell adhesion and metastasis, while being effective in inhibiting one or more of the other undesirable processes associated with surgical procedures. It is provided that the receptor antagonist is administered in combination with at least one additional pain-preventing agent and / or anti-inflammatory and / or anti-convulsant and / or anti-restenosis agent. Various inhibitors and antagonists can act directly by physical association or binding to cell surface receptors to prevent ligand-activating binding, or inhibit different molecules associated with CD44 signaling Can act as an indirect inhibitor.

  Direct receptor antagonists include monoclonal or polyclonal antibodies raised against variants of CD44 or fragments thereof, which can be used as inhibitors of HA binding to CD44 and therapeutic agents Can be used as Anti-CD44 monoclonal antibodies that can be used 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 previously described in US Pat. No. 5,863,540 (F12, A1G3, A3D8) and IM7 and BU52 mAbs have been described. Other monoclonal antibodies suitable for the present invention include anti-CD44 mAbs directed against the unique CD44 isoform produced as a result of alternative splicing with 10 variable exons.

  The present invention is recombinantly produced wherein the extracellular domain of CD44 receptor or a part thereof is covalently linked to the constant domain of an IgG molecule, which can be used as an anti-tumor agent, cell adhesion inhibitor and metastasis inhibitor. Disclosed is the use of a chimeric soluble CD44 (CSCD44) protein. Specifically, and as a first example, a chimeric polypeptide (recombinant chimera) comprising the extracellular domain of a CD44 receptor extracellular polypeptide coupled to the CH2 and CH3 regions of a mouse IgG1 heavy chain polypeptide. Body) can be used. The second example is a chimeric fusion construct composed of the HA ligand binding domain of the CD44 receptor along with a portion of the Fc antibody generated for the CD44 receptor, which is termed the soluble receptor of the Fc fusion. The molecular form of the active soluble CD44 receptor can be either monomeric or dimeric. Various methods have been established for testing the binding of soluble CD44 antibodies to CD44 variant isoforms expressed on the cell surface.

  Another method for inhibiting CD44-hyaluronan receptor-ligand binding is to use a defined size of hyaluronan oligosaccharides (hyaluronan oligomers) to inhibit tumor formation by saturating the CD44 receptor with excess ligand That is. One study found that hyaluronan oligomers injected at concentrations as low as 1 mg / ml inhibited melanoma growth. Thus, local delivery of such oligomers can inhibit the interaction of CD44 with its natural substrate and thus can provide a suitable method for suppressing local tumor development.

  One skilled in the art will determine, in part, the amount of agent administered for any particular treatment protocol by selection of a specific anti-CD44 monoclonal antibody or CD44 peptide sequence as a therapeutic agent for the patient. It is understood that these amounts can easily be determined. One skilled in the art will recognize that the amount of peptide, protein, or antibody administered for a particular treatment protocol can be a function of a particular CD44 domain associated with a pathological clinical condition, and that it mediates metastasis. Understand that it can be a function of the cell type.

  Suitable CD44 receptor antagonists for use as anti-adhesive and / or invasion and / or metastasis inhibitors in the surgical application of the present invention, pain inhibitors and / or anti-inflammatory and / or anti-convulsants and / or Alternatively, CD44 receptor antagonists delivered in combination with an anti-restenosis agent are shown in the table below.

C. Integrin receptor antagonists and inhibitors of integrin signaling Integrins are a superfamily of cell surface receptors involved in cell-cell adhesion and cell-matrix adhesion. These receptors play fundamental roles in the regulation of numerous cell functions such as cell differentiation and migration, maintenance of tissue structure, clot formation and withdrawal, and programmed cell death. Integrins are also implicated in cancer cell differentiation, tumor progression and metastasis because of their important role in mediating tumor cell interactions with extracellular matrix and endothelial cells. Integrin expression and function is altered in many malignant cells, but changes in integrin expression differ between different cell types. Specific integrin receptor subtypes in tumor cells can mediate attachment at the site of surgical trauma to many adhesion proteins, activated platelets and endothelial cells that are components of the extracellular matrix.

  Integrins are a family of heterodimeric transmembrane αβ receptors that are expressed in a wide variety of cells. This receptor family includes at least 14 known α subunits and 8 β subunits, which bind to each other and numerous subtypes that reveal different ligand specificities. Form a combination. The α subunit appears to be a very important determinant of ligand specificity, integrin containing αv reveals specificity for vitronectin, integrin containing α5 reveals specificity for fibronectin, and α3 The contained integrin reveals its specificity for collagen / laminin. Numerous integrin heterodimeric subunit combinations have been found in various tumor cells, which promote their binding to fibronectin, laminin, vitronectin, fibrinogen, collagen and thrombospondin. Vascular cell adhesion molecule-1 (VCAM-1) is an endothelial cell ligand for two leukocyte integrins (α4β1 and α4β7). Mucosal addressin cell adhesion molecule 1 (MadCAM-1) is also recognized as a ligand for α4β7 integrin.

  The interaction between fibronectin and several subtypes of integrin receptors that bind to it plays a particularly important role in some stages of tumor development and influences the process of malignant metastasis. Fibronectin is an extracellular glycoprotein consisting of repeating units of amino acids. A single gene encodes fibronectin, and alternative splicing allows the formation of multiple isoforms. Specific protein domains allow molecules to interact with various cells through both integrin and non-integrin receptors. Fibronectin has at least two independent cell adhesion regions with different receptor specificities. The cell adhesion domain in the central part of fibronectin is composed of at least two minimal amino acid sequences (Arg-Gly-Asp (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) sequences). The fibronectin specific integrin of α5β1 binds to the central RGD / PHSRN site. α4β1 integrin binds to the IIICS site. Peptide inhibitors and antibody inhibitors of fibronectin and integrin function have been shown to be effective inhibitors of metastasis and are potentially important agents for inhibiting tumor cell adhesion.

  The pathway by integrin activation mediated by intracellular kinases and adapter proteins is particularly important for anchor dependence mediated by integrin receptors. Recently, signal transduction studies in a variety of cell types have shown that integrin receptor binding to extracellular matrix proteins rapidly generates intracellular signals via increased tyrosine phosphorylation. The role of focal adhesion kinase (FAK) protein tyrosine kinase (PTK) in linking to intracellular signaling pathways has been elucidated. FAK associates with several different cytoplasmic signaling proteins, such as the Src family of PTKs and several SH2 domain proteins (Shc, Grb2 and PI3-kinase). This allows FAK to function within a network of integrin-stimulated signaling pathways that result in the activation of targets such as the ERK pathway and the JNK / mitogen activated protein kinase pathway. These signaling mechanisms are the rationale for the therapeutic potential of tyrosine kinase inhibitors as anti-adhesive agents based on direct inhibitors of integrin receptor-mediated signaling in the signaling cascade close to the receptor .

  A peptide sequence motif (arginine-glycine-aspartic acid) involved in cell attachment in fibronectin ligands to integrin receptors allows the integrin binding activity of adhesion proteins to be reproduced by short synthetic peptides containing RGD sequences, thus Is the basis for. Peptides containing RGD sequences have been shown to inhibit tumor cell attachment in vitro. Agents that selectively bind to only one or a few of the RGD-directed integrins are prepared by cyclizing peptides having a selected sequence adjacent to the RGD motif and by synthesizing RGD mimetics Has been. Development of a series of peptide analogs of the RGD sequence of fibronectin has been synthesized and their anti-metastatic effect in mice and their inhibitory effect on tumor cell invasion in vitro have been investigated.

  Cellular and animal studies have shown that anti-adhesive peptides and polypeptides are useful for preventing the peritoneal dispersal of certain forms of cancer. For example, studies using the peptide-based integrin antagonists tyrosylisoleucylglycylserylarginine (YIGSR), acetylarginylglycylaspartylserinamide (Ac-RGDS-NH2) and arginylglycylaspartic acid (RGD) The effect on gastric cancer cell line adhesion and invasion was examined using the peritoneal disseminated cell line OCUM-2MD3. It is reported that the expression of both α2β1 and α3β1 integrin is significantly increased in OCUM-2MD3 cells, and the ability of these cells to bind to the extracellular matrix is also significantly higher than that of control cells It was done. The adhesion polypeptides YIGSR and RGD, and the two RGD derivatives, markedly inhibited the adhesion and invasiveness of OCUM-2MD3 cells to the mid-subcutaneous ECM in a cell-specific manner. Survival of nude mice with peritoneal dispersal was longer than that of untreated mice when YIGSR or RGD sequences were given intraperitoneally. Treatments based on interfering with tumor cell attachment using integrin-binding peptides have been shown to be effective metastasis prevention in animal experiments, and the cells are fibrinogen, laminin, or When pre-exposed to peptides containing the RGDS motif, the frequency of tumor transplantation at the surgical trauma site is also reduced.

These and other studies indicate that inhibition of specific integrin receptor-ligand interactions through the use of synthesized fibronectin fragments, peptide analogs and peptidomimetics, or antibodies containing RGD sequences is subtype-specific. It is suggested that it represents a useful method for developing clinically useful antagonists for integrin receptors. Many of these peptides, such as those disclosed in US Pat. No. 5,627,263, may be more effective than the original RGDS peptide in inhibiting tumor cell adhesion and tumor metastasis. Have been found and disclosed for use in the present invention in combination with other agents. In particular, the present invention provides the use of peptides and peptide analogs that are specific for fibronectin binding integrins (especially α ₅ β ₁ integrin). Also provided are specific agents for inhibiting the binding of α ₂ , α ₆ and α _v to integrin subunits as part of the β ₁ integrin heterodimer.

A further class of inhibitors are the naturally occurring disintegrins, which include a family of small, highly homologous cysteine-rich polypeptides purified from snake venom containing RGD sequences. Disintegrins can competitively inhibit integrin-ligand interactions and have been shown to inhibit cell adhesion interactions in various systems. Specific snake venom-derived disintegrins have been shown to be over 2000 times more potent than short synthesized linear RGD-containing peptides in blocking fibrinogen-dependent platelet aggregation. This greater potency of the disintegrin molecule comes from the amino acid sequence surrounding the RGD sequence and the interchain disulfide bridge that forces the RGD sequence to the proper conformation. Specific disintegrins block the integrin cell surface receptor adhesion function, thereby inhibiting integrin-dependent cellular responses in various cell types and tissues. The disintegrin cistrin has been shown by affinity cross-coupling to bind specifically to αVβ3 but not to α5β1 or other high abundance integrins with high affinity. The related disintegrin echistatin specifically inhibited the binding of ¹²⁵ I-labeled cistrin to αVβ3, while the structurally distinct disintegrin decorcin had a 1/1000 affinity. .

Two disintegrins recently isolated from the venom of the Chinese viper (Agkistrodon usuriensis), usristatin 1 (US-1) and usristatin 2 (US-2), have been characterized as potent inhibitors of cell adhesion. Yes. The two polypeptides are composed of 71 amino acids and are characterized by sequences that show strong similarity to other disintegrins. US-1 had a typical Arg-Gly-Asp (RGD) sequence related to blocking fibrinogen binding to the receptor. In US-2, the corresponding sequence was Lys-Gly-Asp (KGD). US-2 also inhibited platelet aggregation, but the IC ₅₀ was about 10 times greater. US-1 also _is a IC 50 = 17~33nM, the adhesion of human melanoma cells to fibrinogen and fibronectin dose dependently inhibited, whereas, US-2 did not inhibit cell adhesion to fibronectin.

An additional heterodimeric disintegrin, EC3 (Mr = 14,762), was isolated from the venom of the Shark snake (Echis carinatus), which is a potent antagonist of α4 integrins. Each subunit contains 67 residues and shows a high degree of homology to other disintegrins. EC3 inhibits adhesion of cells expressing α4β1 integrin and α4β7 integrin to natural ligands such as vascular cell adhesion molecule 1 (VCAM1) and mucosal addressin cell adhesion molecule 1 (MadCAM-1) with an IC ₅₀ = 6-30 nM. Then, the adhesion of K562 cells (α5β1) to fibronectin was inhibited with IC ₅₀ = 150 nM, and the adhesion of αIIbβ3 Chinese hamster ovary cells to fibrinogen was inhibited with IC ₅₀ = 500 nM.

  Examples of suitable integrin antagonists for the present invention are shown below. As an example, for each of the indicated drugs, preferred and most preferred concentrations of irrigation solutions containing the indicated drug are shown, and such concentrations are expected to be therapeutically effective.

D. Selectin Receptor Antagonists and Inhibitors of Selectin Signaling Adherence of malignant cells to the vascular endothelium surface involves a family of adhesion receptors similar to those involved in attracting inflammatory cells to tissue sites. Selectins are involved in a cascade of sequential molecular steps following endothelial cell (EC) activation. The selectin family consists of E-selectin (ELAM-1), P-selectin (GMP-140) and L-selectin (LECAM-1). The carbohydrate determinants sialyl Lewis A (SLe ^a ) and sialyl Lewis X (SLe ^x ) are frequently expressed in human cancer cells, which act as ligands for E-selectin expressed in vascular endothelial cells. These carbohydrate determinants are involved in cancer cell adhesion to the vascular endothelium and thus contribute to cancer metastasis. Selectins are inducible endothelial-expressed adhesion molecules involved in leukocyte recruitment. The initial adhesion mediated by these molecules initiates the activation of integrin molecules by the action of several cytokines. The extent of carbohydrate ligand expression on the surface of cancer cells correlates well with the frequency of metastasis. The monoclonal antibody against Sle ^a or sle ^x, tumor cells to EC and platelets (leukemia cells, colon cancer cells, and histiocytic lymphoma cells) adhesion is prevented. In addition, selectin expression is frequently upregulated in breast cancer endothelium.

  P-selectin, also known as GMP-140 or PADGEM, is a membrane glycoprotein present in quiescent platelets and endothelial secretory granules. When these cells are activated by various mediators, P-selectin is rapidly redistributed to the plasma membrane. Selectins constitute a family of structurally and functionally related molecules. A structural motif common to each of these molecules is an N-terminal lectin-like domain followed by a series of consensus repeats associated with repeats in complement-binding proteins, a transmembrane domain, and a short cytoplasmic tail. Including. P-selectin is a receptor for neutrophils and monocytes when expressed in activated platelets and endothelium. This property facilitates rapid adhesion of leukocytes to the endothelium in the area of tissue injury and platelet-leukocyte interactions at sites of inflammation and bleeding. Studies have also found that P-selectin binds to tumor cells in various tissue sections and that P-selectin binds to the cell surface of numerous cell lines derived from carcinoma. By acting in concert with other adhesion molecules, P-selectin has been implicated in tumor metastasis and is therefore a target for drugs that block adhesion receptor function.

  Studies supporting the role for selectins in metastasis are based on the adhesion of human colon cancer cell lines to selectins using an in vitro flow model. Recombinant forms of P-selectin and P-selectin expressed in Chinese hamster ovary cells helped KM12-L4 colon cancer cell attachment and rolling. This effect was lost by pretreating KM12-L4 cells with neuraminidase. KM12-L4 cells interact with P-selectin through an adhesion pathway that is not dependent on PSGL-1. E-selectin-IgG chimeras have also been found to help sialylation component-dependent adhesion of colon cancer cells under fluid flow. Thus, various sialylated components are involved in selectin-mediated adhesion of human cancer cells to IL-1-stimulated endothelium under flow conditions. Furthermore, it has been found that the efficiency of E-selectin mediated binding of colon cancer cells to human and mouse ECs correlates with the metastatic potential of these cancer cells.

In a recent study, E-selectin-sialyl Lewis x (Sle ^x ) / sialyl Lewis a (Sle in mediating in vitro adhesion of two human colon cancer cell lines (HT-29 and COLO201) to human umbilical cord endothelial cells (HUVEC). The role of ^a ) interaction was evaluated. The colon cancer cell line had a strong expression of carbohydrate epitopes. It has been shown that the adhesion of HT-29 and COLO201 cells to IL-1 stimulated HUVEC can be inhibited by a monoclonal antibody to E-selectin. Preincubation of cells with two different antibodies to Sle ^x and antibodies to related Lewis epitopes (Le ^x and Le ^a ) did not have a major effect on adhesion. The three antibodies against Sle ^a differed in their ability to inhibit HT-29 and COLO201 cell adhesion. Thus, the Sle ^a epitope is important for colon cancer cell adhesion mediated primarily by E-selectin.

Further studies have shown that pretreatment of HUVEC with tumor necrosis factor-α (TNF-α) enhanced binding of the COLO205 carcinoma cell line. Increased adhesion was dependent on both concentration and time, and maximum tumor cell attachment was found at 4 hours. This result was consistent with increased expression of the adhesion molecule E-selectin in the endothelium. Incubation of TUV-stimulated HUVEC with BB11 (anti-E-selectin mAb) resulted in complete inhibition of tumor cell adhesion prior to the addition of COLO205. These studies confirm the utility of specific antibodies against E-selectin to inhibit tumor cell attachment to endothelial cell targets. In other studies, the anti-P- selectin monoclonal antibody or anti-L- selectin monoclonal antibody (i.e., MAbPB1.3 and MAbDREG-200) pretreated with either or sialyl Lewis ^x containing oligosaccharides ^(Sle x -OS), the cell It has been found to be effective in inhibiting adhesion.

Similar in vitro and in vivo studies have demonstrated the role of selectins in the metastatic process using SW1990 cells derived from human pancreatic malignancies. SW1990 cells also strongly express Sle ^a antigen and Sle ^x antigen, CD44H, and β1 integrins. The cells show binding activity against IL-1 activated HUVEC cells and human peritoneal mesothelial cells. Bonding resulting in implantation of cancer cells to endothelial cells was inhibited by treatment with antibodies to antibodies and β1 integrin against Sle ^a. In animal studies, respectively by the process antibodies to antibodies and β1 integrin against Sle ^a is, to inhibit the development of liver metastasis in nude mice with SW1990 cells, extended survival of nude mice.

  Evaluation of the signal transduction properties of selectins has elucidated a common mechanism shared by other cell adhesion receptors. In studies using HT-29 human colon cancer cells that specifically adhere to chimeras of E-selectin-IgG, tyrosine phosphorylation of several proteins in HT-29 cell lysates when attached to E-selectin. It has been shown that the amount of increases. This effect is specific for adhesion to E-selectin because the addition of an E-selectin blocking monoclonal antibody caused a significant decrease in tyrosine phosphorylation compared to E-selectin alone. The kinase assay showed a dose-dependent and significant reduction in c-src activity when attached to E-selectin. This correlates with phosphorylation of Tyr527 (negative regulatory tyrosine). These studies identify signaling pathways with E-selectin ligands in HT-29 cells and c-src.

  Other studies have shown that lymphocyte binding to P-selectin induces tyrosine phosphorylation of different proteins. Activation appears to occur upon first contact with the surface adhesion molecule. The effect of P-selectin was time dependent, with an initial response revealed after 10 minutes and a maximum effect revealed at 30 minutes. Identified proteins included pp125 focal adhesion kinase (FAK) and paxillin. Treatment of the tyrosine kinase inhibitor with genistein or the protein kinase C inhibitor with staurosporine resulted in decreased pp125FAK phosphorylation. These signaling mechanisms are the rationale for the therapeutic potential of tyrosine kinase inhibitors as anti-adhesive agents based on their action as direct inhibitors of selectin receptor-mediated signaling. The rapid signaling properties of the selectin receptor emphasize that the delivery of therapeutic agents at the beginning of the surgical procedure is necessary to provide a prophylactic inhibitory effect.

The present invention provides therapeutically effective amounts of anti-E-selectin specific mAb or antibody fragment and / or anti-P-selectin specific mAb or antibody fragment and / or anti-L-selectin specific mAb or antibody fragment and / or anti-sialyl Lewis ^a Administering to the patient a pharmaceutical composition comprising (Sle ^a ) mAb in combination with one or more of an anti-inflammatory agent and / or an anti-pain agent and / or an anti-convulsant and / or an anti-restenosis agent Thereby providing a method for inhibiting the spread of metastatic tumor cells.

  Studies have also found that heparin inhibited the binding of selectins to their carbohydrate ligands. Heparin (enoxaparin) inhibition of selectin interactions is expected to reduce tumor cell adhesion associated with selectins.

  Examples of suitable selectin antagonists for the present invention are shown below. For each of the indicated drugs, preferred and most preferred concentrations of irrigation solution containing the indicated drug are indicated, and such concentrations are expected to be therapeutically effective.

  Due to the molecular and cellular mechanisms of action defined for the class of drugs identified in the present invention, these drugs, when applied perioperatively in irrigation solutions, prevent tumor cell adhesion and prevent invasion. It is expected to show an effect and / or a metastasis preventing effect. These agents are expected to be effective drugs when delivered in an irrigation solution at the time of a surgical procedure with identified or potential malignancy. Compositions included in the delivery methods and irrigation include peritoneal cavity, abdominal cavity, thoracic cavity, including but not limited to surgery adapted for oncological treatment of ovarian cancer, gastric cancer, pancreatic cancer and colon cancer It is expected to be useful in procedures involving the pleural space, mediastinal space, urogenital space, epidural space, subarachnoid space and joint space. Each metabolically active tumor adhesion inhibitor can be delivered in combination with one or more other pain inhibitors and / or anti-inflammatory and / or anti-convulsants and / or anti-restenosis agents. Suitable anti-tumor agents to be included in the pharmaceutical composition are only one family (class) receptor or only one enzyme family (eg CD44, integrins, selectins, protein tyrosine kinases, MMPs and MAPs). It is further characterized by requirements for pharmacological selectivity and specificity that limit drug interactions with kinases). Certain suitable agents may specifically interact with one or more receptor subtypes (or isoforms) while exhibiting specificity for the receptor family. Each suitable agent can be characterized by the stoichiometry (typically 1: 1) defined for the ligand-receptor (or inhibitor-enzyme) complex and its equilibrium binding constant or equilibrium It associates with its molecular target through a specific mechanism that can be characterized by kinetic constants. These agents 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 non-viral) Can be.

  For example, an agent such as an integrin receptor antagonist constitutes a body cavity whose action is associated with any cell associated with the fluid space of the peritoneal cavity and normal function or exists for pathological conditions This can be exerted on the structure. These cells and structures include, but are not limited to, epithelial cells; mesothelial cells; inflammatory cells (including lymphocytes, macrophages, mast cells, monocytes, eosinophils); and endothelium Included are cells, smooth muscle cells and other cells whose fibers contain cells; and all combinations described above.

  The present invention is also useful for surgical site introduction and administration, which enhances the delivery, uptake, stability or pharmacokinetics of anti-adhesive, intrusion and / or metastasis inhibitors and other drug combinations. Formulations of active therapeutic agents that can be delivered in a formulation are provided. Such formulations can include, but are not limited to, microparticles, microspheres or nanoparticles composed of proteins, carbohydrates, synthetic organic compounds or inorganic compounds. Examples of formulated molecules include lipids that can form liposomes or other aligned lipid structures, cationic lipids, hydrophilic polymers, polycations (eg, protamine, spermidine, and polylysine), substances specific to certain cell types Peptides or synthetic ligands and antibodies that can be targeted to, gels, sustained release matrices, soluble and insoluble particles, and other formulation elements known to those skilled in the art.

  The present invention provides for multiple pharmaceutically active substances contained in a homogeneous mixture (eg, a single encapsulated microsphere) or as a separate mixture (eg, one or more individual delivery vehicles). It provides for the delivery of a combination of anti-tumor and / or invasion and / or local metastasis drugs, both present as microspheres encapsulating the drug). Examples of formulated molecules include hydrophilic polymers, polycations (eg, protamine, spermidine, polylysine and chitosan), peptides or synthetic ligands and antibodies that can target substances to specific cell types, gels, sustained release Including but not limited to matrices, soluble and insoluble particles, and formulation elements not shown.

  The present invention provides for local delivery with an irrigation solution of a combination of tumor cell adhesion inhibitor drug (s). In this case, such an irrigation solution contains a tumor cell adhesion preventing drug present at a therapeutically effective low concentration, allowing the drug to be delivered directly to the desired tissue. The drug-containing irrigation solution can be used in conjunction with surgical procedures, during surgery, or during and before surgery, or before and after surgery, or before, during and after surgery.

  Conventional methods used for drug delivery involve higher concentrations (and larger total doses) of drug administered to patients to achieve significant therapeutic concentrations in pathologically affected tissues. Necessary systemic (eg, oral, intramuscular, intravenous, subcutaneous) administration is required. With systemic administration, high concentrations also occur in tissues other than the undesired targeted tissues, and depending on the dose, adverse side effects may occur. These systemic methods expose the drug to two-pass metabolism and rapid degradation, thereby limiting the duration of effective therapeutic concentrations. A combination of tumor cell anti-adhesion, invasion and metastasis agents (which may involve one or more anti-pain and / or anti-inflammatory and / or anti-convulsants and / or anti-restenosis agents Vascular perfusion is not required to deliver the drug to the targeted site because it is administered directly to the surgical site by irrigation. This remarkable advantage allows for local delivery of various tumor cell adhesion prevention drugs, local metastasis prevention drugs, and lower therapeutically effective total doses than would otherwise be possible by other routes of administration. .

  In another aspect, the irrigation solution of the present invention comprises the physiology of at least one inhibitor of tumor cell adhesion, invasion and / or metastasis, preferably a plurality of pain / inflammatory inhibitors, anticonvulsants and anti-restenosis agents. A dilute solution in a typical carrier. The carrier is a liquid and the carrier, as used herein, is intended to include biocompatible solvents, suspensions, polymerizable and non-polymerizable gels, pastes and salves. . Preferably, the carrier is an aqueous solution that may contain a physiological electrolyte (eg, normal saline or lactated Ringer's solution).

Anti-inflammatory / pain-preventing agents are: (1) serotonin receptor antagonist; (2) serotonin receptor agonist; (3) histamine receptor antagonist; (4) bradykinin receptor antagonist; (5) kallikrein inhibitor; (6) tachykinin receptor antagonist; This includes neurokinin ₁ receptor subtype antagonists and neurokinin ₂ receptor subtype antagonists; (7) calcitonin gene related peptide (CGRP) receptor antagonists; (8) interleukin receptor antagonists; (9) arachidonic acid metabolism Inhibitors of enzymes active in the synthetic pathway of products, including (a) phospholipase inhibitors (PLA ₂ isoform inhibitors and PLC _gamma isoform inhibitors), (b) cyclooxygenase inhibitors, and (c) lipooxygenase inhibitors; (10) prostanoid receptor antagonists, including eicosanoid EP-1 receptor subtype antagonists and eicosanoid EP-4 receptors contains subtypes antagonists and thromboxane receptor subtype antagonists; (11) leukotriene receptor antagonists, This includes the leukotriene B ₄ receptor subtype antagonists and leukotriene D ₄ receptor subtype antagonists; (12) opioid receptor agonists This includes μ-opioid receptor subtype agonists, δ-opioid receptors, Puta include subtypes agonist and κ- opioid receptor subtype agonists; (13) purine receptor agonist and purine receptor antagonists, This includes the P _2X receptor antagonists and P _2Y receptor agonists; and (14) adenosine-tri Selected from the group consisting of phosphate (ATP) sensitive potassium channel openers.

  Suitable anti-inflammatory / pain-preventing agents that also act as anticonvulsants include serotonin receptor antagonists, tachykinin receptor antagonists, ATP sensitive potassium channel openers and calcium channel antagonists. Other agents that can be utilized in solution, particularly for their anticonvulsant properties, include endothelin receptor antagonists, calcium channel antagonists and nitric oxide donors (enzyme activators).

A specific preferred embodiment of the solution of the present invention used in cardiovascular and general vascular procedures comprises an anti-restenosis agent, most preferably used in combination with an anticonvulsant. The Suitable anti-restenosis agents include (1) antiplatelet agents, including (a) thrombin inhibitors and thrombin receptor antagonists, (b) adenosine diphosphate (ADP) receptor antagonists (also purine receptor ₁ (C) thromboxane inhibitors and thromboxane 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 pain / anti-inflammatory agents); and (5) ) Intracellular signal Transduction inhibitors, including, (a) protein kinase C (PKC) inhibitors and protein tyrosine phosphatases, (b) modulators of intracellular protein tyrosine kinase inhibitors, (c) src homology ₂ (SH2) domain of the inhibitor, and (D) Calcium channel antagonists are included. Such agents are useful in preventing restenosis of arteries treated by angioplasty, rotational atherectomy, or other therapeutic procedures of cardiovascular or general blood vessels.

In each of the surgical solutions of the present invention, the drug is included at a lower concentration than the concentration and dose required using conventional methods of drug administration to achieve the desired therapeutic effect, and at lower doses Delivered locally. Because systemically administered drugs are exposed to first-pass metabolism and two-pass metabolism, they have equivalent therapeutic effects and other routes of drug administration (ie, intravenous route, subcutaneous route, intramuscular route) Route or oral route). The concentration of each drug is determined in part based on its dissociation constant K _d . As used herein, the term dissociation constant refers to the equilibrium dissociation constant for its respective agonist-receptor or antagonist-receptor interaction and its respective activator-enzyme or inhibitor-enzyme interaction. It is intended to encompass both equilibrium inhibition constants for action. Each agent is preferably included at a low _Kd nanomolar concentration of 0.1 to 10,000 times, except for cyclooxygenase inhibitors which may be required at higher concentrations depending on the particular inhibitor selected. Preferably, each agent is included at a concentration of 1.0 to 1,000 times _Kd nanomolar, most preferably about 100 times a concentration of _Kd nanomolar. These concentrations are adjusted as necessary to compensate for dilution in the absence of metabolic transformation at the local delivery site. The exact drug selected and the concentration of the drug used in the solution will vary according to the particular application, as described below.

  The solution according to the invention is derived from one or more of the listed classes, one or more inhibitors of tumor cell adhesion, invasion and / or metastasis, pain / inflammatory inhibitor (s), only one or more It may only contain low concentrations of anticonvulsants, inhibitors of tumor cell adhesion, invasion and / or metastasis and anticonvulsants and pain / inflammatory inhibitors or anti-restenosis agents. However, due to the above synergistic effects of multiple drugs and the desire to extensively prevent pain and inflammation, convulsions and restenosis, it is preferable to utilize multiple drugs.

The surgical solution of the present invention constitutes a novel therapeutic method by combining multiple pharmacological agents acting on different molecular targets of receptors and enzymes. To date, various pharmacological efforts have focused on the development of highly specific drugs that are selective for individual receptor subtypes and enzyme isoforms that mediate responses to individual signaling neurotransmitters and hormones is doing. As an example, endothelin peptide is one of the most potent vasoconstrictors known. Selective antagonists specific for the endothelin (ET) receptor subtype are being sought by several pharmaceutical companies for use in the treatment of numerous disorders with elevated endothelin levels in the body. By recognizing the potential role of the receptor subtype ET _A in hypertension, these drug companies are specifically selective for ET _A receptor subtype for the anticipated treatment of vasospasm of the coronary arteries Aims to develop new antagonists. Such standard pharmacological approaches are widely accepted but not optimal. This is because many other vasoconstrictor factors (eg, serotonin, prostaglandins, eicosanoids, etc.) can be simultaneously involved in initiating and maintaining vasospasm episodes (see FIGS. 2 and 4). ) Furthermore, despite the inactivation of only one receptor subtype or enzyme, the activation of other receptor subtypes or enzymes, and the resulting signaling, can often initiate a cascade effect. This explains that it is extremely difficult to use only one receptor-specific drug to block pathophysiological processes in which multiple transmitters play a role. Therefore, specific individual receptor subtype (e.g., such as ET _A) alone may not be effective to target.

  In contrast to standard methods for pharmacological treatment, the therapeutic solutions of the surgical solutions of the present invention are based on the development of pathophysiological conditions, where combinations of drugs that act simultaneously on different molecular targets. It is based on the rationale that it is required to inhibit the full range of events that are present. In addition, instead of targeting specific receptor subtypes alone, the surgical solutions of the present invention enable different physiological cellular processes involved in the development of pain, inflammation, vasospasm, smooth muscle spasms and restenosis (see FIG. (See 1)) composed of drugs that target a common molecular mechanism that operates in In this way, further receptor and enzyme cascading in the nociceptive, inflammatory, convulsive and restenotic pathways is minimized by the surgical solution of the present invention. In these pathophysiological pathways, surgical solutions inhibit cascade effects both “upstream” and “downstream”.

An example of “upstream” inhibition is a cyclooxygenase antagonist in the context of pain and inflammation. Cyclooxygenase enzymes (COX ₁ and COX ₂ ) are responsible for the arachidonic acid to plastaglandin H, an intermediate in the biosynthesis of inflammatory and nociceptive mediators including prostaglandins, leukotrienes and thromboxanes. Catalyze conversion. Cyclooxygenase inhibitors block the formation of these inflammatory and nociceptive mediators “upstream”. In this way, it is not necessary to block the interaction of the seven described subtypes of prostanoid receptors with their natural ligands. A similar “upstream” inhibitor contained in surgical solutions is aprotinin (kallikrein inhibitor). The enzyme kallikrein is a serine protease that cleaves high molecular weight kininogens in plasma, leading to bradykinins that are important mediators of pain and inflammation. By inhibiting kallikrein, aprotinin effectively inhibits the synthesis of bradykinins, thereby providing effective “upstream” inhibition of these inflammatory mediators.

  Surgical solutions also use “downstream” inhibitors to control pathophysiological pathways. In vascular smooth muscle preparations pre-contracted with various neurotransmitters involved in coronary vasospasm (eg, serotonin, histamine, endothelin and thromboxane), ATP-sensitive potassium channel opener (KCO) It results in smooth muscle relaxation that is dependent (Quast et al., 1994; Kashiwabara et al., 1994). Thus, KCO provides vasospasm and smooth muscle spasms by providing a “downstream” anticonvulsant effect that is not related to the physiological combination of agonists that initiate convulsive events (see FIGS. 2 and 4). Provides significant advantages over the surgical solution of the present invention in the context of Similarly, NO donors and voltage-gated calcium channel antagonists can limit vasospasm and smooth muscle spasms initiated by multiple mediators known to act earlier in the convulsive pathway.

II. Anti-inflammatory / pain-preventing agents The following is a description of the preferred drugs included in the above classes of anti-inflammatory / pain-preventing agents of the present invention, as well as the preferred concentrations used in the solutions of the present invention. While not wishing to be limited by theory, there is also evidence to support the selection of various classes of drugs and to make these drugs effective.

A. Serotonin receptor antagonist Serotonin (5-HT) causes pain by stimulating serotonin ₂ (5-HT ₂ ) and / or serotonin ₃ (5-HT ₃ ) receptors in peripheral nociceptive neurons It is thought that there is. Most researchers agree that 5-HT ₃ receptors in peripheral nociceptors mediate the immediate pain sensation caused by 5-HT (Richardson et al., 1985). In addition to inhibiting 5-HT induced pain, 5-HT ₃ receptor antagonists may also inhibit neurogenic pain by inhibiting activation of nociceptors. Barnes P.M. J. et al. Et al., “Modulation of Neuroinformatics: Novel Approaches to Inframolecular Diseases”, Trends in Pharmaceutical Sciences 11: 185-189 (1990). However, a study in the ankle joint of rat, be argued that the 5-HT ₂ receptor is involved in the infringement receptor by 5-HT. Grubb, B.M. D. Et al., "A Study of 5-HT-Receptors Associated with Affected Nerves Located in Normal and Infrared Rat Angle Joints", Agents Actions 25:16 (18:18). Thus, activation of 5-HT ₂ receptors may also play a role in peripheral pain and neurogenic inflammation.

One purpose of the solution of the present invention is to prevent pain and numerous inflammatory processes. Accordingly, both 5-HT ₂ receptor and 5-HT ₃ receptor antagonists are preferably used either individually or together in the solutions of the present invention, as described below. Amitriptyline (Elavil ™) is a preferred 5-HT ₂ receptor antagonist for use in the present invention. Amitriptyline has been used clinically as an antidepressant for many years and has been found to have beneficial effects in certain types of chronic pain patients. Metoclopramide (Reglan ™) has been used clinically as an antiemetic, but exhibits moderate affinity for the 5-HT ₃ receptor and can inhibit the action of 5-HT at this receptor, Probably inhibits pain due to 5-HT release from platelets. This is therefore also suitable for use in the present invention.

Other suitable 5-HT ₂ receptor antagonists include imipramine, trazodone, desipramine and ketanserin. Ketanserin has been used clinically for its antihypertensive effect. Hedner, T .; Et al., "Effects of a New Serotonin Antagonist, Kesanserin, in Experimental and Clinical Hypertension", Am. J. et al. of Hypertension, 317s-23s (July 1988). Other suitable 5-HT ₃ receptor antagonists include cisapride and ondansetron. Cardiovascular and general vascular solutions may also contain an antagonist of serotonin _1B (also known as serotonin _1Dβ ). This is because serotonin is known to cause significant vasospasm in humans due to the activation of serotonin _1B receptor. Kaumann, A.M. J. et al. Et al., “Variable Participation of 5-HT1-Like Receptors and 5-HT2 Receptors in Serotonin-Induced Contrast of Human Isolated Coronaries 53” (Circle 114). Suitable serotonin _1B receptor antagonists include yohimbine, N-[-methoxy-3- (4-methyl-1-piperazinyl) phenyl) -2'-methyl-4 '-(5-methyl-1,2,4- Oxadiazol-3-yl) [1,1-biphenyl] -4-carboxamide ("GR127935") and methiothepine. The therapeutic and preferred concentrations for using these drugs in the solutions of the present invention are shown in Table 5.

B. Serotonin receptor agonists 5-HT _1A receptor, 5-HT _1B receptor and 5-HT _1D receptor are known to inhibit adenylate cyclase activity. Therefore, including low doses of these serotonin _1A receptor, serotonin _1B receptor and serotonin _1D receptor agonists in the solution should inhibit neurons that mediate pain and inflammation. The same effect is expected from serotonin _1E receptor and serotonin _1F receptor agonists. This is because these receptors also inhibit adenylate cyclase.

  Buspirone is a preferred 1A receptor agonist for use in the present invention. Sumatriptan is the preferred receptor agonist for 1A, 1B, 1D and 1F. A preferred receptor agonist for 1B and 1D is dihydroergotamine. A preferred 1E receptor agonist is ergonobin. The therapeutic concentrations and preferred concentrations for these receptor agonists are shown in Table 6.

C. Histamine receptor antagonists Histamine receptors are generally divided into histamine ₁ (H ₁ ) and histamine ₂ (H ₂ ) subtypes. The classical inflammatory response to peripheral administration of histamine is mediated by the H ₁ receptor (Douglas, 1985). Accordingly, the solution of the present invention preferably comprises a histamine H ₁ receptor antagonist. Promethazine (Phenergan ™) is a commonly used antiemetic that potently blocks the H ₁ receptor and is suitable for use in the present invention. Interestingly, this drug has also been shown to have a local anesthetic effect. However, the concentration required for this effect is orders of magnitude greater than that required to block the H ₁ receptor, and therefore the effect is thought to be caused by a different mechanism. The concentration of histamine receptor antagonist in solution is sufficient to inhibit the H ₁ receptor involved in the activation of nociceptors, but achieves a “local anesthetic” effect, thereby eliminating problems with systemic side effects. Not enough to remove.

Histamine receptors are also known to mediate coronary vasomotor tone. In vitro studies in the human heart have shown that the histamine ₁ receptor subtype mediates coronary smooth muscle contraction. Ginsburg, R.A. Et al., “Histamine Promotion of Clinical Coronary Arty Spasm: Implications Concerning Pathology of Varian Angina Pectoris”, American Heart J. et al. 102: 819-822 (1980). Several studies suggest that histamine-induced hypercontraction in the human coronary arterial system is most prominent in the proximal artery in the context of atherosclerosis and associated epidermal exfoliation of the arterial endothelium. Keitoku, M .; Et al., “Differential Histamine Actions in Proximal and Distal Human Coronary Arts in Vitro”, Cardiovascular Research 24: 614-622 (1990). Thus, a histamine receptor antagonist can be included in the cardiovascular irrigation solution.

Other suitable H ₁ receptor antagonists include terfenadine, diphenhydramine, amitriptyline, mepyramine and tripolyzine. Amitriptyline is also effective as a serotonin ₂ receptor antagonist and therefore has a dual function when used in the present invention. Suitable therapeutic and preferred concentrations for each of these H ₁ receptor antagonists are shown in Table 7.

D. Bradykinin receptor antagonists Bradykinin receptors are generally divided into bradykinin ₁ (B ₁ ) subtype and bradykinin ₂ (B ₂ ) subtype. The study pain and inflammation of peripheral acute caused by bradykinin are mediated by the B ₂ subtype, whereas that pain from bradykinin induced in the context of chronic inflammation is mediated by B ₁ subtype shows Has been. Perkins, M.M. N. Others, “Antinoctive Activity of the Bradykinin B1 and B2 Receptor Antagonists, des-Arg ⁹ , [Leu ⁸ ] -BK and HOE 140, in Two. Dray, A .; Et al., “Bradykinin and Inflammatory Pain”, Trends Neurosci. 16: 99-104 (1993), each of which is specifically incorporated herein by reference.

Currently, bradykinin receptor antagonists are not clinically used. These drugs are peptides (small proteins) and are therefore digested and cannot be taken orally. Antagonists to B ₂ receptors block the pain and inflammation of acute induced by bradykinin (Dray et al., 1993). B ₁ receptor antagonists inhibit pain in chronic inflammatory conditions (Perkins et al., 1993; Dray et al., 1993). Thus, depending on the application, the solution of the present invention preferably comprises either or both of a bradykinin B ₁ receptor antagonist and a bradykinin B ₂ receptor antagonist. For example, arthroscopy is performed for both acute and chronic pain symptoms, and therefore the irrigation solution for arthroscopy can contain both a B ₁ receptor antagonist and a B ₂ receptor antagonist.

Suitable bradykinin receptor antagonists for use in the present invention include the following bradykinin ₁ receptor antagonists: D-Arg- (Hyp ³ -Thi ⁵ -D-Tic ⁷ -Oic ⁸ ) -BK [des-Arg ¹⁰ ] derivatives (“[des-Arg ¹⁰ ] derivatives of HOE140”, which are available from Hoechst Pharmaceuticals); and [Leu ⁸ ] des-Arg ⁹ -BK. Suitable bradykinin ₂ receptor antagonists include [D-Phe ⁷ ] -BK; D-Arg- (Hyp ³ -Thi ^5,8- D-Phe ⁷ ) -BK (“NPC349”); D-Arg- (Hyp ^{3 -D-Phe} 7) -BK ( "NPC567"); and ^{D-Arg- (Hyp 3 -Thi 5} -D-Tic 7 -Oic 8) -BK ( "HOE140"). These compounds are described in more detail in the previously incorporated references of Perkins et al. (1993) and Dray et al. (1993). Suitable therapeutic concentrations and preferred concentrations are shown in Table 8.

E. The kallikrein inhibitor peptide bradykinin is an important mediator of pain and inflammation, as previously noted. Bradykinin is produced as a cleavage product by the action of kallikrein on high molecular weight kininogens in plasma. Thus, kallikrein inhibitors are believed to be therapeutic in inhibiting bradykinin production and the resulting pain and inflammation. A preferred kallikrein inhibitor for use in the present invention is aprotinin. Suitable concentrations for use in the solutions of the present invention are shown in Table 9 below.

F. Tachykinin receptor antagonists Tachykinins (TK) s are a family of structurally related peptides that include substance P, neurokinin A (NKA) and neurokinin B (NKB). Neurons are the main source of TKs in the periphery. An important general action of TKs is neuronal stimulation, but other actions include endothelium-dependent vasodilation, plasma protein extravasation, mast cell recruitment, and inflammatory cell degranulation And stimulation. Maggi, C.I. A. Gen. Pharmacol. 22: 1-24 (1991). Because of the above combination of physiological effects mediated by activation of the TK receptor, targeting the TK receptor is a reasonable method for promoting analgesia and treating neurogenic inflammation.

1. Neurokinin ₁ receptor subtype antagonist Substance P activates the neurokinin receptor subtype referred to as NK _1. Substance P is an undecapeptide present at the end of sensory nerves. Substance P may have a number of actions that cause inflammation and pain in the periphery after C fiber activation, including vasodilation, plasma extravasation, and mast cell degranulation. Are known. Levine, J .; D. Et al., “Peptides and the Primary Affiliate Nociceptor”, J. Am. Neurosci. 13: 2273 (1993). A preferred substance P antagonist is ([D-Pro ⁹ [spiro-γ-lactam] Leu ¹⁰ , Trp ¹¹ ] fizaremin- (1-11) (“GR82334”). The invention acting on the NK ₁ receptor. Other suitable antagonists for use in 1-Imino-2- (2-methoxy-phenyl) ethyl) -7,7-diphenyl-4-perhydroisoindolone (3aR, 7aR) ("RP67580" ) And 2S, 3S-cis-3- (2-methoxybenzylamino) -2-benzhydrylquinuclidine ("CP96,345"). Suitable concentrations for these agents are shown in Table 10.

2. Neurokinin ₂ receptor subtype antagonist Neurokinin A is a peptide that co-localizes with substance P in sensory neurons, which also promotes inflammation and pain. Neurokinin A activates the specific neurokinin receptor referred to as NK _2. Edmonds-Alt, S.M. Et al., “A Potential and Selective Non-Peptide Antigenist of the Neurokinin A (NK ₂ ) Receptor”, Life Sci. 50: PL101 (1992). In the urinary tract, TK such, not only the urethra and urinary tract of a human, a potent spasm raw materials acting through only the NK ₂ receptor in the human bladder. Maggi, C.I. A. Gen. Pharmacol. 22: 1-24 (1991). Thus, the desired drug contained surgical solution for use in urological procedures, in order to reduce spasm, comprising an antagonist against NK ₂ receptor. Examples of suitable NK ₂ antagonists include ((S) -N-methyl-N- [4- (4-acetylamino-4-phenylpiperidino) -2- (3,4-dichlorophenyl) butyl] benzamide. ("(±) -SR48968"); Met-Asp-Trp-Phe-Dap-Leu ("Men10,627"); and cyc (Gln-Trp-Phe-Gly-Leu-Met) ("L659,877") Suitable concentrations of these agents are shown in Table 11.

G. CGRP receptor antagonist Calcitonin gene-related peptide (CGRP) is also a peptide that co-localizes with substance P in sensory neurons, which acts as a vasodilator and enhances the action of substance P. Brain, S.M. D. "Inflammatory Oedema Induced by Synthesis Between Calcinin Gene-Related Peptide (CGRP) and Mediators of Increased Viscosity Permeability", Br. J Pharmacol. 99: 202 (1985). An example of a suitable CGRP receptor antagonist is α-CGRP- (8-37) (a truncated form of CGRP). This polypeptide inhibits activation of the CGRP receptor. Suitable concentrations for this drug are shown in Table 12.

H. Interleukin receptor antagonists Interleukins are a family of peptides classified as cytokines that are produced by leukocytes and other cells in response to inflammatory mediators. Interleukins (ILs) can be potent sensory hypersensitivity factors peripherally. Ferriera, S .; H. Et al., “Interleukin-1β as a Potentative Agent Antigenized by a Tripeptide Analogue”, Nature 334: 698 (1988). One example of a suitable IL-1β receptor antagonist is Lys-D-Pro-Thr, which is a truncated form of IL-1β. This tripeptide inhibits the activation of the IL-1β receptor. Suitable concentrations for this drug are shown in Table 13.

I. Inhibitors of enzymes active in the synthetic pathway for arachidonic acid metabolites Production of arachidonic acid by phospholipase inhibitor phospholipase A ₂ (PLA ₂ ) occurs in a series of reactions that produce a number of inflammatory mediators known as eicosanoids. There are numerous stages throughout this pathway that can be inhibited, thereby reducing the production of these inflammatory mediators. Examples of inhibition at these various stages are given below.

Inhibition of the enzyme PLA ₂ isoform inhibits the release of arachidonic acid from the cell membrane, thus inhibiting the production of prostaglandins and leukotrienes, resulting in reduced inflammation and pain. Glaser, K.M. B. Et al., “Regulation of Phospholipase A2 Enzymes: Selective Inhibitors and Ther Pharmaceutical Pharmaceutical”, Adv. Pharmacol. 32:31 (1995). One example of a suitable PLA ₂ isoform inhibitor is manoalide. Suitable concentrations for this drug are shown in Table 14. Phospholipase C _gamma also inhibition (PLC _gamma) isoforms also result in the production of reduced of prostanoids and leukotrienes, thus, resulting in decreased pain and inflammation. An example of a PLC _gamma isoform inhibitor is 1- [6-((17β-3-methoxyestradi-1,3,5 (10) -trien-17-yl) amino) hexyl] -1H-pyrrole-2,5- Dione.

2. Cyclooxygenase inhibitors A variety of non-steroidal anti-inflammatory drugs (NSAIDs) are widely used as anti-inflammatory, antipyretic, antithrombotic and analgesic agents. Lewis, R.A. A. “Prostaglandins and Leukotrienes”, Textbook of Rheumatology, 3rd edition (Kelly W. N. et al.), 258 (1989). Molecular targets for these drugs are type I and type II cyclooxygenases (COX-1 and COX-2). These enzymes are also known as prostaglandin H synthase (PGHS) -1 (constitutive) and prostaglandin H synthase (PGHS) -2 (inducible), prostaglandins and thrombos It catalyzes the conversion of arachidonic acid to prostaglandin H, an intermediate in the biosynthesis of xanes. COX-2 enzymes have been identified in endothelial cells, macrophages and fibroblasts. This enzyme is induced by IL-1 and endotoxin, and its expression is upregulated at the site of inflammation. Both the constitutive activity of COX-1 and the induced activity of COX-2 result in the synthesis of prostaglandins that contribute to pain and inflammation.

  NSAIDs currently on the market (diclofenac, naproxen, indomethacin, ibuprofen, etc.) are generally non-selective inhibitors of both isoforms of COX, but the ratio varies from compound to compound, but for COX-1, It may exhibit greater selectivity than COX-2. The use of COX-1 and COX-2 inhibitors to block the formation of prostaglandins is more than an attempt to block the interaction of natural ligands with the seven described subtypes of prostanoid receptors. Also represents a good therapeutic approach. The reported antagonists of the eicosanoid receptors (EP-1, EP-2 and EP-3) are extremely rare and only specific high affinity antagonists of the thromboxane A2 receptor have been reported. Wallace, J.M. And Cirino, G .; , Trends in Pharm. Sci. 15: 405-406 (1994).

  Oral, intravenous or intramuscular use of cyclooxygenase inhibitors is contraindicated in patients with ulcer disease, gastritis or kidney damage. In the United States, the only available form of this class of drugs is the ketorolac (Toradol ™) available from Syntex Pharmaceuticals, which has traditionally been used intramuscularly or intravenously in post-operative patients. However, again, it is contraindicated in the above categories of patients. The use of ketorolac or any other cyclooxygenase inhibitor in solution at a substantially lower dosage than currently used during the perioperative period allows the use of this drug in patients who are otherwise contraindicated. obtain. Adding a cyclooxygenase inhibitor to the solution of the present invention adds a different mechanism for inhibiting the development of pain and inflammation during arthroscopy or other therapeutic or diagnostic procedures.

  Preferred cyclooxygenase inhibitors for use in the present invention are ketorolac and indomethacin. Of these two drugs, indomethacin is less preferred because a relatively large dosage is required. The therapeutic and preferred concentrations used in the solution are shown in Table 15.

3. Inhibition of lipoxygenase inhibitors enzyme lipoxygenase, production of leukotrienes, such as leukotriene B _4, which is known to be an important mediator of inflammation and pain is inhibited. Lewis, R.A. A. “Prostaglandins and Leukotrienes”, Textbook of Rheumatology, 3rd edition (Kelly W. N. et al.), Page 258 (1989). An example of a 5-lipoxygenase antagonist is 2,3,5-trimethyl-6- (12-hydroxy-5,10-dodecadiynyl) -1,4-benzoquinone (“AA861”), for which a suitable concentration is Table 16 shows.

J. et al. Prostanoid receptor antagonists Specific prostanoids produced as metabolites of arachidonic acid mediate their inflammatory effects through activation of prostanoid receptors. Examples of various classes of specific prostanoid antagonists include eicosanoid EP-1 receptor subtype antagonists, eicosanoid EP-4 receptor subtype antagonists, and thromboxane receptor subtype antagonists. A preferred prostaglandin E ₂ receptor antagonist is 8-chlorodibenzo [b, f] [1,4] oxazepine-10 (11H) -carboxylic acid 2-acetylhydrazide (“SC19220”). A suitable thromboxane receptor subtype antagonist is [15- [1α, 2β (5Z), 3β, 4α] -7- [3- [2- (phenylamino) carbonyl] hydrazino] methyl] -7-oxobicyclo-2. , 2,1] -hept-2-yl] -5-heptanoic acid (“SQ29548”). Suitable concentrations for these agents are shown in Table 17.

K. Leukotriene receptor antagonists leukotrienes _(LTB 4, LTC ₄ and LTD ₄₎ are enzymatically generated has important biological properties, the product of 5-lipoxygenase pathway of arachidonic acid metabolism. Leukotrienes are implicated in a number of pathological conditions including inflammation. Specific antagonists are currently sought by many pharmaceutical companies for potential therapeutic intervention in these pathologies. Halushka, P.A. V. Et al., Annu. Rev. Pharmacol. Toxicol. 29: 213-239 (1989); Ford-Hutchinson, A .; Crit. Rev. Immunol. 10: 1-12 (1990). LTB ₄ receptors have been found in certain immune cells including eosinophils and neutrophils. Binding of LTB ₄ to these receptors results in the release of chemotactic and lysosomal enzymes, thereby contributing to the inflammatory process. This signaling process associated with activation of the LTB ₄ receptor involves G protein-mediated stimulation of phosphatidylinositol (PI) metabolism and an increase in intracellular calcium (see FIG. 2).

An example of a suitable leukotriene _{B 4} receptor antagonist is SC (+) - (S) -7- (3- (2- ( cyclopropylmethyl) -3-methoxy-4 - [(methylamino) carbonyl] phenoxy (propoxy) -3,4-dihydro-8-propyl-2H-1-benzopyran-2-propanoic acid ("SC 53228") Concentrations for this drug suitable for the practice of the present invention are shown in Table 18. Other suitable leukotriene B ₄ receptor antagonists include [3-[-2 (7-chloro-2-quinolinyl) ethenyl] phenyl] [[3- (dimethylamino-3-oxopropyl) thio] methyl] thiopropane. Acid ("MK0571"), and drugs LY66,071 and ICI 20,3219. D ₄ also acts as a receptor subtype antagonists.

L. Opioid Receptor Agonists Activation of opioid receptors provides a nociceptive effect and therefore agonists for these receptors are desirable. Opioid receptors include the opioid receptor subtypes of μ, δ and κ. The μ-receptor is present at the end of sensory neurons in the periphery, and activation of this receptor inhibits sensory neuron activity. Basbaum, A.M. I. Et al., "Opiate Analgesia: How Central is a Peripheral Target?" Engl. J. et al. Med. 325: 1168 (1991). δ- and κ-receptors are present at the efferent terminal of the sympathetic nerve, which inhibit the release of prostaglandins, thereby inhibiting pain and inflammation. Taiwo, Y .; O. Et al., “Kappa- and Delta-Opioids Block Symmetrically Dependent Hypergesia”, J. et al. Neurosci. 11: 928 (1991). These opioid receptor subtypes are members of the G protein coupled receptor superfamily. Thus, all opioid receptor agonists interact and initiate signaling through their cognate G protein-coupled receptors (see FIGS. 3 and 7). Examples of suitable μ-receptor agonists include fentanyl and Try-D-Ala-Gly- [N-MePhe] —NH (CH ₂ ) —OH (“DAMGO”). An example of a suitable δ-receptor agonist is [D-Pen ² , D-Pen ⁵ ] enkephalin (“DPDPE”). An example of a suitable kappa-receptor agonist is (trans) -3,4-dichloro-N-methyl-N- [2- (1-prolidinyl) cyclohexyl] benzeneacetamide ([U50,488]). Suitable concentrations for each of these agents are shown in Table 19.

M.M. Antagonists and agonists extracellular ATP purine receptors act as signaling molecules through the interaction of the P ₂ purinoreceptor. One of the major classes of purine receptors is the P _2X purine receptor, which is a ligand-gated ion channel with a unique ion channel that is permeable to Na ⁺ , K ⁺ and Ca ²⁺ . The P _2X purine receptor described in sensory neurons is important for primary afferent neurotransmission and nociception. ATP is known to depolarize sensory neurons, and ATP released from damaged cells stimulates P _2X purine receptors, thereby depolarizing nociceptive nerve fiber terminals. It plays a role in the activation of nociceptors. P2X ₃ receptors have a very limited distribution (Chen, C.C other, Nature 377:. 428~431 (1995 )). This is because P2X ₃ receptors are selectively expressed in sensory C-fibers nerves leading to the spinal cord, also, many of these C-fibers are known to have receptors for painful stimuli . Therefore, very limited localization of expression for P2X ₃ receptor subunits, these subtypes, and good target for analgesia effect (see FIGS. 3 and 7).

Suitable antagonists of the P _2X / ATP purine receptor used in the present invention include, by way of example, suramin and pyridoxyl phosphate-6-azophenyl-2,4-disulfonic acid (“PPADS”). Suitable concentrations for these agents are shown in Table 20.

P _2Y receptor (G protein-coupled receptor) agonists are known to cause smooth muscle relaxation by increasing inositol triphosphate (IP ₃ ) levels with subsequent increases in intracellular calcium. An example of a P _2Y receptor agonist is 2-me-S-ATP.

N. Adenosine triphosphate (ATP) sensitive potassium channel openers A variety of ATP sensitive potassium channels have been found in numerous tissues including blood vessels and non-vascular smooth muscle and brain. Studies using radiolabeled ligands have confirmed its presence. Opening of these channels causes potassium (K ⁺ ) efflux and the cell membrane is hyperpolarized (see FIG. 2). This hyperpolarization induces a decrease in intracellular free calcium by inhibition of voltage-gated calcium (Ca ²⁺ ) and receptor-operated Ca ²⁺ channels. These combined actions cause cells (eg, smooth muscle cells) to become relaxed or, in the case of vascular smooth muscle cells, which are more resistant to activation, cause vascular relaxation. Be made. Various K ⁺ channel openers (KCO) have been characterized as having potent antihypertensive activity in vivo and vasorelaxant activity in vitro (see FIG. 4). K ⁺ channel openers (KCOs) have also been shown to interfere with stimulation-coupled secretion and are thought to act on pre-junctional neuronal receptors, thus releasing neurostimulation and inflammatory mediators Inhibits various effects of. Quast, U .; Et al., “Cellular Pharmacology of Potassium Channel Operators in Viscous Smooth Muscle”, Cardiovasc. Res. 28: 805-810 (1994).

_A synergistic interaction between an endothelin (ET _A ) antagonist and an ATP-sensitive potassium channel opener (KCO) is expected in achieving vascular relaxation or smooth muscle relaxation. The rationale for dual use is based on the fact that these drugs have different molecular mechanisms of action in promoting smooth muscle relaxation and prevention of vasospasm. First of intracellular calcium elevation in smooth muscle cells induced by ET _A receptor subsequently, activation of voltage-gated channels, and to start the entry of extracellular calcium which is required for contraction. ET antagonists of _A receptors will block the action of this receptor mediated specifically, but increased calcium initiated by activation of other G-protein coupled receptor in muscle cells do not inhibit.

Potassium channel opener drugs such as pinacidil open these channels, causing K ⁺ influx and cell membrane hyperpolarization. This hyperpolarization acts to reduce contractions mediated by other receptors by the following mechanisms: (1) Hyperpolarization is by reducing the likelihood of opening L-type or T-type calcium channels. Inhibition of voltage-dependent Ca ²⁺ channels induces a decrease in intracellular free calcium; (2) Hyperpolarization is due to agonist induction from intracellular sources by inhibition of inositol triphosphate (IP ₃ ) formation. (Receptor-operated channel) inhibits Ca ²⁺ release; and (3) Hyperpolarization reduces the efficiency of calcium as an activator of contractile proteins. As a result, the combined action of these two classes of drugs causes the target cells to become relaxed or 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 ′-[1,1-dimethyl- [2,2,3 , ^3- 3 H] propyl] -N "- (3- pyridinyl) guanidine (" P1075 "); and N- cyano -N '- (2-nitro-carboxyethyl) -3-pyridine-carboximidamide mono methanesulfonate Naruto ("KRN2391") is included. The concentrations for these drugs are shown in Table 21.

III. Anticonvulsants A. Multifunctional Agents Some of the pain / anti-inflammatory agents described above are also useful for inhibiting vasoconstriction or smooth muscle spasm. As such, these agents also serve as anticonvulsants and are therefore beneficially used in vascular and urological applications. Anti-inflammatory / pain-preventing agents that also serve as anticonvulsants include serotonin receptor antagonists (especially serotonin ₂ antagonists), tachykinin receptor antagonists, and ATP sensitive potassium channel openers.

B. Nitric oxide donors Nitric oxide donors can be included in the solutions of the present invention, especially because of their anticonvulsant activity. Nitric oxide (NO) plays a very important role as a molecular mediator of many physiological processes, including vasodilation and regulation of normal vascular tone. In endothelial cells, an enzyme known as NO synthase (NOS) catalyzes the conversion of L-arginine to NO, which acts as a diffusible second messenger in adjacent smooth muscle cells. Mediates the response (see FIG. 8). NO is continuously formed and released by vascular endothelial cells under basal conditions, inhibits contraction, controls basal coronary tone, various agonists (such as acetylcholine) and Produced in the endothelium in response to other endothelium-dependent vasodilators. Thus, regulation of NO synthase activity and the resulting NO levels are important molecular targets that control vascular tone (see FIG. 8). Muramatsu, K .; Other, Coron. Artery Dis. 5: 815-820 (1994).

A synergistic interaction between the NO donor and the ATP-sensitive potassium channel opener (KCO) is expected to achieve vascular relaxation or smooth muscle relaxation. The rationale for dual use is based on the fact that these drugs have different molecular mechanisms of action in promoting smooth muscle relaxation and prevention of vasospasm. Evidence has been obtained from cultured coronary artery smooth muscle cells that vasopressin, angiotensin II and endothelin vasoconstrictors all inhibit _KATP currents by inhibition of protein kinase A. It has also been reported that _KATP current in bladder smooth muscle is inhibited by muscarinic agonists. The action of NO in mediating smooth muscle relaxation occurs through an independent molecular pathway (described above) with protein kinase G (see FIG. 8). This suggests that the combination of these two classes of drugs has greater efficacy in relaxing smooth muscle than using only one class of drugs alone.

  Suitable nitric oxide donors for the practice of the present invention include nitroglycerin, sodium nitroprusside, drugs FK409, FR144420, 3-morpholinoside nonimine or linsidomine chlorohydrate (“SIN-1”), and S-nitroso-N-acetylpenicillamine (“SNAP”) is included. The concentrations for these drugs are shown in Table 22.

C. Endothelin receptor antagonist Endothelin is a 21 amino acid peptide that is one of the most potent vasoconstrictors known. Three different human endothelin peptides (these, ET-1, are termed ET-2 and ET-3) have been described, these are at least two receptor sub-called ET _A receptors and ET _B receptors It mediates its physiological effects through types. Smooth muscle of the heart and blood vessels contains primarily ET _A receptors, the subtype is related to contraction in these tissues. Furthermore, ET _A receptors is 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. Accordingly, antagonists against ET _A receptors, and should be therapeutically beneficial in perioperative inhibition of vasospasm of the coronary arteries and may be useful in the inhibition of smooth muscle contraction in urological applications. Miller, R.M. C. Et al., Trends in Pharmacol. Sci. 14: 54-60 (1993).

  Suitable endothelin receptor antagonists include cyclo (D-Asp-Pro-D-Val-Leu-D-Trp) (“BQ123”); (N, N-hexamethylene) -carbamoyl-Leu-D-Trp- ( (CHO) -D-Trp-OH ("BQ610"); (R) 2-([R-2-[(s) -2-([1-hexahydro-1H-azepinyl] carbonyl] amino-4-methylpenta Noyl] amino-3- (3 [1-methyl-1H-indolyl]) propionylamino-3 (2-pyridyl) propionic acid ("FR139317"); cyclo (D-Asp-Pro-D-Ile-Leu-D -Trp) ("JKC301"); cyclo (D-Ser-Pro-D-Val-Leu-D-Trp) ("JK302"); 5- (di Tilamino) -N- (3,4-dimethyl-5-isoxazolyl) -1-naphthalenesulfonamide (“BMS182874”); and N- [1-formyl-N- [N-[(hexahydro-1H-azepine-1 -Yl) carbonyl] -L-leucyl] -D-tryptophyl] -D-tryptophan ("BQ610") Concentrations for three representatives of these agents are shown in Table 23.

D. CA ²⁺ Channel Antagonists Calcium channel antagonists are a distinct group of drugs that interfere with the transmembrane flux of calcium ions required for activation of cellular responses that mediate neuroinflammation. Calcium entry into platelets and leukocytes is an important event that mediates the activation of responses in these cells. Furthermore, the role of bradykinin and neurokinin receptors (NK ₁ and NK ₂ ) in mediating neuroinflammatory signaling pathways includes an increase in intracellular calcium, thus activation of calcium channels in the plasma membrane. Brought about. In many tissues, calcium channel antagonists (eg, nifedipine) can reduce the release of arachidonic acid, prostaglandins and leukotrienes induced by various stimuli. Moncada, S .; Flower, R .; And Vane, J .; , Goodman's and Gilman's Pharmaceutical Basis of Therapeutics (7th edition), MacMillan Public. Inc. 660-5 (1995).

  Calcium channel antagonists also interfere with the transmembrane flux of calcium ions required by vascular smooth muscle for contraction. This action is the rationale for the use of calcium channel antagonists during the perioperative period during treatment, in which case the goal is to relieve vasospasm and reduce smooth muscle relaxation. Is to promote. Dihydropyridine compounds, including nisoldipine, act as specific inhibitors (antagonists) of the voltage-dependent gating of L-type subtypes of calcium channels. Systemic administration of the calcium channel antagonist nifedipine during cardiac surgery has been previously utilized to prevent or minimize coronary vasospasm. Seitelberger, R.A. Other, Circulation 83: 460-468 (1991).

Calcium channel antagonists are one of the anticonvulsants useful in the present invention and show a synergistic effect when combined with other drugs of the present invention. Calcium (Ca ²⁺ ) channel antagonists and nitric oxide (NO) donors interact in achieving vasorelaxation or smooth muscle relaxation, ie in inhibiting convulsive activity. The rationale for dual use is that these two classes of drugs have different molecular mechanisms of action and when used alone may not be completely effective in achieving relaxation, and Based on the fact that the validity period can vary. In fact, there are numerous studies showing that calcium channel antagonists alone cannot achieve complete relaxation of vascular muscles pre-contracted with receptor agonists.

  The effect of nisoldipine on internal thoracic artery (IMA) convulsions, when used alone and in combination with nitroglycerin, is greatly positive in preventing contraction due to the combination of these two drugs. It was shown that a synergistic effect was produced (Liu et al., 1994). These studies are the scientific basis for combining calcium channel antagonists and nitric oxide (NO) donors for effective vasospasm prevention and smooth muscle relaxation. Various examples of systemic administration of nitroglycerin and nifedipine during cardiac surgery to prevent and treat myocardial ischemia or coronary vasospasm have been reported (Cohen et al., 1983; Seitelberger et al., 1991).

Calcium channel antagonists also show a synergistic effect with endothelin receptor subtype A (ET _A ) antagonists. Yanagisawa and coworkers observed that dihydropyridine antagonists blocked the effects of ET-1 (endogenous agonists at ET _A receptor in coronary arterial smooth muscle), thus, ET-1 is voltage-sensitive calcium channels It was speculated to be an endogenous agonist. Sustained period of intracellular calcium elevation in smooth muscle cells induced by activation of ET _A receptors requests extracellular calcium, also been found to be at least partially blocked by nicardipine. Therefore, the inclusion of calcium channel antagonists, when combined in a surgical solution, it is expected that synergistically enhance the effect of ET _A antagonists.

Calcium channel antagonists and ATP-sensitive potassium channel openers similarly show a synergistic effect. ATP-sensitive potassium channels (K _ATP ) couple the cellular membrane potential to the cellular metabolic state by sensitivity to adenosine nucleotides. K _ATP channels are inhibited by intracellular ATP, but are stimulated by intracellular nucleotide diphosphates. The activity of these channels is controlled by electrochemical driving forces on potassium and intracellular signals (eg, ATP or G protein), but not gated by the membrane potential itself. The _KATP channel hyperpolarizes the membrane, thus allowing the resting potential of the cell to be controlled by potassium and intracellular signals. ATP-sensitive potassium current has been found in skeletal muscle, brain, and smooth muscles other than heart and heart. Studies with radiolabeled ligands have confirmed the presence of ATP-sensitive potassium channels that are receptor targets for potassium channel opener drugs such as pinacidil. Opening of these channels causes potassium efflux and hyperpolarizes the cell membrane. This hyperpolarization induces a decrease in intracellular free calcium by inhibiting voltage-dependent Ca ²⁺ channels by reducing (1) the possibility of opening L-type or T-type calcium channels, and (2) inositol. Inhibition of triphosphate (IP ₃ ) formation suppresses agonist-induced (receptor-actuated channel) Ca ²⁺ release from intracellular sources, and (3) calcium as an activator of contractile proteins Reduce efficiency. The combined action of these two classes of drugs (ATP-sensitive potassium channel openers and calcium channel antagonists) causes the target cells to become relaxed or more resistant to activation.

Finally, calcium channel antagonists and tachykinin antagonists and bradykinin antagonists show a synergistic effect in mediating neuroinflammation. The role of neurokinin receptors in mediating neuroinflammation has been elucidated. The signaling pathways of neurokinin ₁ (NK ₁ ) and neurokinin ₂ (NK ₂ ) receptors (which are members of the G protein-coupled superfamily) include an increase in intracellular calcium, and thus the plasma membrane Activation of calcium channels in. Similarly, activation of the bradykinin ₂ (BK ₂ ) receptor is coupled to an increase in intracellular calcium. Thus, calcium channel antagonists interfere with a common mechanism involving elevated intracellular calcium, some of which enter via L-type channels. This is the basis for the synergistic interaction between the calcium channel antagonist and the neurokinin receptor and bradykinin ₂ receptor.

  Suitable calcium channel antagonists for the practice of the present invention include nisoldipine, nifedipine, nimodipine, rasidipine, isradipine and amlodipine. Suitable concentrations for these agents are shown in Table 24.

IV. Anti-restenosis Agents The solutions of the present invention utilized for cardiovascular and general vascular procedures are also sometimes used for restenosis, especially for angioplasty, rotational atherectomy and other interventional vascular use. An inhibitor may be included. The following drugs are suitable for inclusion in the cardiovascular irrigation solutions and general vascular irrigation solutions when restenosis limitation is indicated. The following anti-restenosis agents are preferably combined with anticonvulsants, and also more preferably with pain / anti-inflammatory agents in the solutions of the present invention.

A. Antiplatelet agents At the site of arterial injury, platelets adhere to collagen and fibrinogen through specific cell surface receptors and are then activated by several independent mediators. Various agonists can activate platelets, and such agonists include collagen, ADP, thromboxane A2, epinephrine and thrombin. Collagen and thrombin are used as primary activators at the site of vascular injury, while ADP and thromboxane A2 act to attract additional platelets into the growing platelet plug. Activated platelets degranulate, releasing chemoattractants and other factors used as vasoconstrictors, thus promoting vasospasm and platelet accumulation. Thus, the antiplatelet agent can be an antagonist obtained from any of the agonist-receptor targets described above.

  Since platelets play such an important role in the coagulation cascade, oral antiplatelet agents are routinely administered to patients undergoing vascular treatment. Indeed, because of this diversity of activators and the observation that only one antiplatelet agent is ineffective, some researchers have found that mixed treatment protocols are not effective. We conclude that it is necessary. Recently, Willerson and co-workers reported the intravenous use of three mixed drugs: ridogrel (antagonist of thromboxane A2), ketanserin (serotonin antagonist) and clopidogrel (ADP antagonist). They found that the combination of the three antagonists inhibited several associated platelet functions and reduced neointimal proliferation in a canine coronary angioplasty model (JACC Abstracts, 1995 2). Moon). It remains unclear which method is most successful for treating coronary thrombosis. One possibility is to include antiplatelet and antithrombotic agents in the cardiovascular and general vascular solutions of the present invention.

1. Thrombin inhibitors and thrombin receptor antagonists Thrombin plays a central role in vascular injury formation and is thought to be a major mediator of thrombus formation. Thus, thrombus formation at the site of vascular injury during and after PTCA (percutaneous transluminal coronary angioplasty) or other vascular procedures is central to acute reocclusion and chronic restenosis. This process can be interrupted by the application of direct antithrombin agents including hirudin and its synthetic peptide analogs and thrombin receptor antagonist peptides (Harker et al., Am. J. Cardiol. 75: 12B (1995)). Thrombin is also a powerful growth factor that initiates smooth muscle cell proliferation at the site of vascular injury. Thrombin also plays a role in regulating the action of other growth factors such as PDGF (platelet-derived growth factor), and thrombin inhibitors are after vascular injury induced by balloon angioplasty. It has been shown to reduce PDGF mRNA expression.

  Hirudin is a prototypical direct antithrombin drug because it binds to the catalytic site and substrate recognition site (exosite) of thrombin. Animal studies using baboons have shown that this proliferative response can be reduced by 80% using recombinant hirudin (Ciba-Geigy). Hirulog (Biogen) is a dodecapeptide modeled after hirudin and binds to the active site of thrombin via the Phe-Pro-Arg linker molecule. Large-scale clinical trials of hirudin and hirulog are underway to examine their efficacy in reducing vascular injury after PTCA, and so far phase II data for these inhibitors is positive Both drugs are considered suitable in the solutions of the present invention. Preliminary results of a study in 1,200 patients using repeated angiographic evaluations at 6 months to detect restenosis showed that ischemic events when using hirudin versus heparin Showed excellent short-term suppression. The advantage of this method is that no significant bleeding complications have been reported. Local hilllog therapy with sustained release has been found to reduce early thrombosis rather than neointimal thickening after arterial stenting in pigs. Muller, D.M. Et al., “Sustained-Release Local Hirallog Therapeutics Decrease Early Thrombosis But Neo-Thickening After Artificial Staging”, Am. Heart J.H. 133 (2): 211-218 (1996). In this study, hill logs were released from impregnated polymer placed around the artery.

  Other active antithrombin agents in testing that are theoretically assumed to be suitable for the present invention include argatroban (Texas Biotechnology) and ephegatran (Lilly).

2. ADP receptor antagonist (purine receptor antagonist)
Ticlopidine is an analog of ADP and inhibits both thromboxane and ADP-induced platelet aggregation. Ticlopidine is thought to block the interaction between ADP and its receptor, thereby inhibiting signaling by this G protein coupled receptor on the surface of the platelet membrane. Preliminary studies have shown that ticlopidine is more effective than aspirin in combination with dipyridamole. However, the clinical use of ticlopidine is limited because it causes neutropenia. Clopidogrel, an analog of ticlopidine, is thought to have fewer adverse side effects than ticlopidine and is currently being studied to prevent ischemic events. It is theoretically envisioned that these agents may be suitable for use in the solutions of the present invention.

3. Thromboxane Inhibitors and Thromboxane Receptor Antagonists Drugs currently utilized for conventional methods of treating thrombosis rely on aspirin, heparin and plasminogen activators. Aspirin irreversibly acetylates cyclooxygenase and inhibits the synthesis of thromboxane A2 and prostacyclin. Although the data support the benefits of aspirin for PTCA, it is believed that the fundamental efficacy of aspirin is only partial or moderate. This may be due to platelet activation by a thromboxane A2-independent pathway that is not blocked by aspirin-induced acetylation of cyclooxygenase. Platelet aggregation and thrombosis can occur regardless of aspirin treatment. Aspirin in combination with dipyridamole has also been shown to reduce the occurrence of acute complications during PTCA but not the occurrence of restenosis.

  Two thromboxane receptor antagonists appear to be more effective than aspirin and are considered suitable for use in the solutions and methods of the invention. Ticlopidine inhibits both thromboxane and ADP-induced platelet aggregation. Lidogrel (R68060) is a mixed form of thromboxane B2 synthase inhibitor and thromboxane-prostaglandin endoperoxide receptor blocker. Lidogrel has been compared to salicylate therapy in an open pilot study of patients receiving PTCA and administered in combination with heparin. Timemans, C.I. “Ridogrel in the Setting of Percutaneous Transluminal Coronary Angioplasty”, Am. J. et al. Cardiol. 68: 463-466 (1991). Treatment consisted of administering a 300 mg slow intravenous injection just prior to the start of PTCA treatment and continued by oral administration 12 hours later using a 300 mg dose twice daily. From this study, ridogrel was found to be largely successful because early acute reocclusion did not occur in 30 patients. Bleeding complications occurred in a significant number (34%) of patients, which appears to be a complicating factor that requires special attention. In this study, ridogrel was identified as a potent long-lasting inhibitor of thromboxane B2 synthase.

B. Inhibitors of cell adhesion molecules Selectin Inhibitors Selectin inhibitors block the interaction of a particular selectin with its cognate ligand or receptor. Representative examples of selectin targets on which these inhibitors act include, but are not limited to, E-selectin receptor and P-selectin receptor. Upjohn Co. Has a licensed right to monoclonal antibodies developed by Cytel Corps that inhibit the activity of P-selectin. The product CY1748 is in preclinical development and a potential indication is restenosis.

2. Integrin inhibitors The platelet glycoprotein IIb / IIIa complex is present on the surface of resting platelets as well as activated platelets. This complex appears to undergo a conversion upon platelet activation that allows the complex to be used as a binding site for fibrinogen and other adhesion proteins. A very promising new antiplatelet agent is directed at this integrin cell surface receptor, which represents the last common pathway for platelet aggregation.

  Several types of drugs fit into the class of GPIIb / IIIa integrin antagonists. The monoclonal antibody c7E3 (CentoRx; Centocor, Malvern, PA) has been intensively studied to date in a PTCA study of 3,000 patients. c7E3 is a chimeric human / mouse hybrid. A 0.25 mg / kg bolus of c7E3 followed by an intravenous infusion of 10 μg / min over 12 hours resulted in over 80% blockade of the GPIIb / IIIa receptor during the duration of the infusion. This correlated with greater than 80% inhibition of platelet aggregation. This antibody was co-administered with heparin and an increased risk of bleeding was observed. Further information comes from EPIC trials that have shown significant reductions in primary endpoint, mortality synthesis, the occurrence of non-fatal myocardial infarction, and the need for coronary revascularization, which information has long-term benefits Suggested. Tcheng, Am. Heart J.H. 130: 673-678 (1995). A Phase IV study (EPILOG) designed to investigate safety and efficacy issues with c7E3 Fab is planned or ongoing. This monoclonal antibody can also be classified as a platelet membrane glycoprotein receptor antagonist directed against the glycoprotein IIb / IIIa receptor.

  The platelet glycoprotein IIb / IIIa receptor blocker Integrelin is a cyclic heptapeptide that is very specific for this molecular target. In contrast to antibodies, Integrelin has a short biological half-life (about 10 minutes). The safety and efficacy of Integrelin was first evaluated in a Phase II Impact test. Intravenous infusion of Integrelin at either 4 or 12 hours at 1.0 μg / kg / min was utilized (Topol, E., Am. J. Cardiol, 27B-33B (1995)). Integrelin was given in combination with other drugs (heparin, aspirin) and was shown to exhibit strong antiplatelet aggregation (> 80%). A phase III study of 4,000 patients (IMPACT II study) showed that Integrelin significantly reduced ischemic events in patients who underwent Rotablator atherectomy (JACC Abstracts, 1996). . Suitable concentrations for these drugs (c7E3 and Integrelin) used in the present invention are shown below.

  Two peptidomimetics, MK-383 (Merck) and RO4483 (Hoffmann-LaRoche), are also being studied in phase II clinical trials. Since these are both small molecules, they have a short half-life and large potency. However, they also appear to be less specific and thus interact with other related integrins. It is theoretically envisioned that these peptidomimetics may also be suitable for use in the present invention.

C. Anti-chemotactic drugs Anti-chemotactic drugs interfere with the chemotaxis of inflammatory cells. Representative examples of anti-chemotactic targets on which these agents act include F-Met-Leu-Phe receptor, IL-8 receptor, MCP-1 receptor and MIP-1-α / RANTES receptor. However, it is not limited to these. Drugs included in this class of drugs are in the early stages of development. However, it is theoretically envisioned that they may be suitable for use in the present invention.

D. Interleukin receptor antagonist An interleukin receptor antagonist is an agent that blocks the interaction of a particular interleukin with its cognate ligand or receptor. Specific receptor antagonists for any of a number of interleukin receptors are in the early stages of development. An exception to this is the naturally occurring entity of the secreted form of the IL-1 receptor, which is called IL-1 antagonist protein (IL-1AP). It has been theoretically assumed that this antagonist has been shown to bind to IL-1 and inhibit the biological action of IL-1, and thus may be suitable for the practice of the present invention.

E. Intracellular signal transduction inhibitor Protein Kinase Inhibitor A. Protein Kinase C (PKC) Inhibitors Protein kinase C (PKC) plays a pivotal role in cell-surface signaling for many physiological processes. Various PKC isoenzymes are used as downstream targets resulting from initial activation of G protein coupled receptors (eg, serotonin, endothelin, etc.) or downstream targets resulting from initial activation of growth factor receptors (eg, PDGF etc.). Both of these receptor classes play an important role in mediating vasospasm and restenosis after coronary balloon angioplasty procedures.

Molecular cloning analysis has revealed that PKC exists as a large family of at least 8 subspecies (isoenzymes). These isoenzymes differ substantially in structure and mechanism for linking receptor activation to changes in the proliferation response of specific cells. Specific isoenzyme expression has been found in a wide variety of cell types including platelets, neutrophils, myeloid cells and smooth muscle cells. Thus, inhibitors of PKC can affect signaling pathways in several cell types as long as the inhibitor does not exhibit isoenzyme specificity. Thus, inhibitors of PKC can be expected to be effective in blocking the smooth muscle cell proliferative response, and also anti-inflammatory in blocking neutrophil activation and subsequent adhesion May have an effect. Several inhibitors have been described and the first report shows an IC ₅₀ of 50 μM for the inhibitory activity of calphostin C. G-6203 (also known as Go6976) is a new potent PKC inhibitor with great selectivity for certain PKC isoforms with IC ₅₀ values in the range of 2 μM to 10 μM . The concentration of these drugs and another drug (GF109203X, also known as Go6850 or Bisindoylmaleimide I (available from Warner-Lambert)) considered suitable for use in the present invention is Shown below.

B. Protein tyrosine kinase inhibitors Although there is tremendous diversity among the many members of the receptor tyrosine kinase (RTK) family, the signal transduction mechanisms used by these receptors have many common features with each other. Biochemical and molecular genetic studies have shown that ligand binding to the extracellular domain of RTK rapidly activates the intrinsic tyrosine kinase catalytic activity of the intracellular domain (see FIG. 5). ) Increased activity results in tyrosine-specific phosphorylation of numerous intracellular substrates that contain common sequence motifs. As a result, this results in a number of “downstream” signaling molecules that regulate phospholipid metabolism, arachidonic acid metabolism, protein phosphorylation (which involves mechanisms other than protein kinases), calcium mobilization and transcriptional activation, and Activation of a series of intracellular pathways is triggered (see Figure 2). The growth factor-dependent tyrosine kinase activity of the RTK cytoplasmic domain is the primary mechanism for generating intracellular signals that lead to cell proliferation. Thus, various inhibitors have the potential to block this signaling and thereby prevent the proliferative response (see FIG. 5).

Platelet derived growth factor (PDGF) receptors are attracting considerable attention as targets for inhibition in the cardiovascular field because they are thought to play a significant role in both atherosclerosis and restenosis. Release of PDGF by platelets on the damaged surface of the endothelium in the blood vessels results in stimulation of PDGF receptors in vascular smooth muscle cells. As described above, this initiates a series of intracellular events that result in increased proliferation and neointimal thickening. Inhibitors of PDGF kinase activity are expected to prevent proliferation and increase the likelihood of success after cardiovascular and general vascular procedures. Some of the related tyrphostin compounds have the potential as specific inhibitors of PDGF receptor tyrosine kinase activity because they have little effect on other protein kinases and other signaling systems ( The in vitro IC ₅₀ is in the range of 0.5 μM to 1.0 μM). To date, only a few of the many tyrphostin compounds are commercially available. Suitable concentrations for these agents when used in the present invention are shown below. Staurosporine has also been reported to show potent inhibitory effects on several protein tyrosine kinases of the src subfamily. Suitable concentrations for this drug when used in the present invention are shown below.

2. Inhibitors of intracellular protein tyrosine phosphatases Non-transmembrane protein tyrosine phosphatases (PTPases) containing the src homology ₂ (SH2) domain are known and are designated by the nomenclature as SH-PTP1 and SH-PTP2. . SH-PTP1 is also known as PTP1C, HCP or SHP. SH-PTP2 is also known as PTP1D or PTP2C. Similarly, SH-PTP1 is expressed at high levels in all lineages and all differentiation stages of hematopoietic cells, and the SH-PTP1 gene has been identified as being associated with the motheaten (me) mouse phenotype. This is the basis for predicting the action of an inhibitor that blocks its interaction with its cellular substrate. Stimulation of neutrophils by chemotactic peptides is known to result in activation of tyrosine kinases that mediate neutrophil responses (Cui et al., J. Immunol. (1994)). PTPase activity also regulates agonist-induced activity by reversing the action of tyrosine kinases activated in the early stages of cell stimulation. Agents that can stimulate PTPase activity may have potential therapeutic uses as anti-inflammatory mediators.

  These same PTPases have also been shown to modulate the activity of certain RTKs. They appear to balance the actions of activated receptor kinases and may therefore represent important drug targets. In vitro experiments have shown that PTPase injection blocks insulin-stimulated phosphorylation of tyrosyl residues in endogenous proteins. Thus, activators of PTPase activity can be used to reverse the activation of PDGF receptor action in restenosis and are therefore considered useful in the solutions of the present invention. PTPase linked to the receptor also functions as an extracellular ligand similar to the extracellular ligand of the cell adhesion molecule. The functional consequences of ligand binding to the extracellular domain have not yet been elucidated, but it is reasonable to speculate that binding will help regulate intracellular phosphatase activity (Fashena and Zinn). , Current Biology, 5: 1367-1369 (1995)). Such action can block adhesion mediated by other cell surface adhesion molecules (NCAM) and provide an anti-inflammatory effect. Drugs for these applications have not yet been developed.

3. Inhibitors of the SH2 domain (SRC homology ₂ domain) The SH2 domain (which was first identified in the src subfamily of protein tyrosine kinases (PTKs)) is a non-catalytic protein sequence that is a variety of signaling proteins. Consisting of about 100 amino acids conserved between (Cohen et al., 1995). The SH2 domain functions as a phosphotyrosine binding module, thereby mediating a very important protein-protein association in the intracellular signaling pathway (Pawson, Nature, 573-580, 1995). In particular, the role of the SH2 domain is clearly demonstrated as very important for receptor tyrosine kinase (RTK) -mediated signal transduction, such as in the case of platelet derived growth factor (PDGF) receptors. The phosphotyrosine-containing site in autophosphorylated RTKs serves as a binding site for SH2-protein, thereby mediating activation of biochemical signaling pathways (see FIG. 2) (Carpenter, G., FASEB J. 6: 3283-3289 (1992); Sierke, S. and Koland, J. Biochem. 32: 10102-10108 (1993)). The SH2 domain is involved in coupling activated growth factor receptors to cellular responses including changes in gene expression and ultimately cell proliferation (see FIG. 5). Thus, inhibitors that selectively block the action of specific RTK activation expressed on the surface of vascular smooth muscle cells are effective in blocking the proliferation and restenosis process after PTCA or other vascular treatments It is predicted that One RTK target that is currently of interest is the PDGF receptor.

At least 20 cytosolic proteins that contain SH2 domains and function in intracellular signaling have been identified. The distribution of SH2 domains is not limited to a particular protein family, but is found in several classes of proteins, protein kinases, lipid kinases, protein phosphatases, phospholipases, Ras regulatory proteins and some transcription factors. Many of the SH2-containing proteins have known enzymatic activity, while other SH2-containing proteins (Grb2 and Crk) are “linkers” between cell surface receptors and “downstream” effector molecules. And “adapter” (Marengere, L. et al., Nature 369: 502-505 (1994)). Examples of SH2 domain-containing proteins with enzymatic activity that are activated in signal transduction include, but are not limited to, the src subfamily protein tyrosine kinases (src (pp60 ^c-src ), abl, lck, fyn, fgr, etc.) Phospholipase Cγ (PLCγ), phosphatidylinositol 3-kinase (PI-3-kinase), p21-rasGTPase activating protein (GAP), and SH2-containing protein tyrosine phosphatase (SH-PTPase) (Songyang et al., Cell 72). : 767-778 (1993)). Because of the central role these various SH2-proteins occupy in transmitting signals from activated cell surface receptors into a series of additional molecular interactions that ultimately define the cellular response. In addition, inhibitors that block the binding of specific SH2 proteins are desirable as agents for a variety of potential therapeutic applications.

  Also, many immune / inflammatory response modulations are mediated through receptors that signal through non-receptor tyrosine kinases containing SH2 domains. Activation of T cells via the antigen-specific T cell receptor (TCR) initiates a signaling cascade that results in secretion of lymphokines and T cell proliferation. One of the earliest biochemical responses after TCR activation is an increase in tyrosine kinase activity. In particular, neutrophil activation is controlled in part by the response of cell surface immunoglobulin G receptors. Activation of these receptors mediates activation of unidentified tyrosine kinases known to have SH2 domains. Further evidence indicates that several kinases of the src family (lck, blk, fyn) are involved in signal transduction pathways leading from cytokine receptors and integrin receptors, thus integrating stimuli received from several independent receptor structures It has been shown that it can be used to Thus, specific SH2 domain inhibitors have the potential to block many neutrophil functions and serve as anti-inflammatory mediators.

  Efforts to develop drugs targeted to the SH2 domain are currently being made at the biochemical in vitro and cellular levels. If such efforts are successful, it is theoretically assumed that the resulting drug will be useful in the practice of the present invention.

4). Calcium Channel Antagonists Calcium channel antagonists have been previously described in connection with convulsion inhibitory function and can also be used as anti-restenosis agents in the cardiovascular and general vascular solutions of the present invention. Activation of growth factor receptors such as PDGF is known to result in an increase in intracellular calcium (see FIG. 2). Studies at the cellular level have shown that the action of calcium channel antagonists is effective in inhibiting mitogenesis of vascular smooth muscle cells.

F. Synergistic interactions resulting from therapeutic combinations of restenosis-preventing agents and other agents used in cardiovascular and general vascular solutions after PTCA or other cardiovascular or general vascular therapeutic treatment Given the complexity of the disease process associated with restenosis, and the diversity of molecular targets involved, blocking or inhibiting only one molecular target is sufficient to prevent vasospasm and restenosis. It is not expected to result (see Figure 2). In fact, numerous animal studies targeting different individual molecular receptors and / or enzymes have proved ineffective in animal models or have provided efficacy for both pathologies in clinical trials to date. Absent. (Free, M. et al., “An Intensive Poly-pharmaceutical Approach to the Prevention of Retention of The Mevacor, Ace Inhibitor, Colchicine (BIG-MAC) Pilot C. Serruys, P., et al., “PARK: the Post Angioplasty Resistensis Ketserin Trial”, J. Am. Coll. Of Cardiol. 21: 322A (1993)). Thus, therapeutic combinations of drugs that act on different molecular targets and are delivered locally appear to be necessary for clinical effectiveness in therapeutic efforts against vasospasm and restenosis. As discussed below, the rationale for this synergistic molecular targeted therapy is the stimulation that vascular smooth muscle cells in the vascular wall are exposed to cells during PTCA or other vascular interventional procedures. Derived from recent developments in understanding and understanding the underlying biochemical mechanisms that integrate the stimuli.

1. “Crosstalk” and convergence in the major signaling pathways The molecular switches responsible for cell signaling are traditionally divided into two distinct and distinct signaling pathways, each of which is a specific set. It contains a different set of protein families that act as transducing agents for the extracellular stimuli of and mediates different cellular responses. By one such pathway, signals from neurotransmitters and hormones are transduced via a G protein coupled receptor (GPCR) to produce a contractile response using intracellular targets of the trimeric G protein and Ca ^2+. (See FIG. 2). These stimuli and their respective receptors mediate smooth muscle contraction and vasospasm can be induced in the context of PTCA or other therapeutic or diagnostic procedures of cardiovascular or general vascularity. Examples of signaling molecules involved in mediating convulsions through the GPCR pathway include 5-HT and endothelin, which contain antagonists to them that act through their respective G protein-coupled receptors .

  Another major pathway converts signals from growth factors (eg, PDGF, etc.) into the regulation of cell growth and differentiation via tyrosine kinases, adapter proteins and Ras proteins (FIGS. 2 and 5). checking). This pathway can also be activated during PTCA or other cardiovascular or general vascular procedures that cause vascular smooth muscle cell proliferation with a high incidence. An example of a restenosis drug target is the PDGF receptor.

Signals transmitted from neurotransmitters and hormones stimulate either of the two classes of receptors (G protein-coupled receptors composed of 7-helical transmembrane regions, or ligand-gated ion channels). “Downstream” signals from both types of receptors converge when controlling the concentration of cytoplasmic Ca ²⁺ that initiates contraction in smooth muscle cells (see FIG. 2). Each GPCR transmembrane receptor activates a specific class of trimeric G proteins, including G _q , G _i or many other G proteins. The G _α subunit and / or G _βγ subunit activates phospholipase C _β , resulting in activation of protein kinase C (PKC) and increased levels of cytoplasmic calcium due to the release of calcium from intracellular stores.

Growth factor signaling (eg, PDGF mediated signaling) converges upon regulation of cell growth. This pathway, receptor tyrosine kinases and "downstream" enzymes is dependent on phosphorylation of tyrosine residues in the (with respect to the tyrosine kinase phospholipase C _gamma discussed above). Activation of the PDGF receptor also results in PKC stimulation and intracellular calcium elevation (a common step shared by various GPCRs) (see FIG. 2). It is now recognized that ligand-independent "crosstalk" can transactivate the tyrosine kinase receptor pathway in response to GPCR stimulation. In recent studies, Shc (adapter protein in the tyrosine kinase / Ras pathway) has been identified as an important mediator protein that relays the message from the GPCR pathway to the tyrosine kinase pathway (see FIG. 2). Lev et al., Nature 376: 737 (1995)). The activation of Shc is calcium dependent. Thus, a combination of selective inhibitors that block transactivation of a common signaling pathway that results in the proliferation of vascular smooth muscle cells would prevent convulsions and restenosis after PTCA or other cardiovascular or general vascular procedures. Acts synergistically to prevent. Specific examples are briefly described below.

2. Synergistic interaction between PKC inhibitor and calcium channel antagonist In this case, the synergistic interaction between PKC inhibitor and calcium channel antagonist in achieving vascular relaxation and growth inhibition is Occurs due to “crosstalk” with the tyrosine kinase signaling pathway (see FIG. 2). The rationale for dual use is based on the fact that these drugs have different molecular mechanisms of action. As described above, stimulation of GPCRs results in activation of protein kinase C and increased levels of cytoplasmic calcium by the release of calcium from the intracellular reservoir. Calcium activated PKC is the central control point in transmitting extracellular responses. “Crosstalk” from the GPCR-stimulated pathway via PKC can cause mitogenesis of vascular smooth muscle cells, and thus calcium channel antagonists can prevent convulsions by inhibiting the activation of Shc. It has a dual effect of directly blocking and preventing proliferation activation. Conversely, PKC inhibitors act on parts of the pathway that lead to contraction.

3. PKC inhibitor, synergistic effects 5-HT ₂ receptor family 5-HT ₂ antagonists and ET _{A antagonists,} includes three members, named _{5-HT 2A, 5-HT} 2B and _{5-HT 2C,} these All have the common property of being coupled to phosphotidylinositol turnover and increased intracellular calcium (Hoyer et al., 1988; Hartig et al., 1989). The distribution of these receptors includes vascular smooth muscle and platelets, and because of their localization, these 5-HT receptors are important in mediating convulsions, thrombosis and restenosis. is there. Sustained period of intracellular calcium elevation in smooth muscle cells induced by activation of ET _A receptors requires extracellular calcium, also been found to be at least partially blocked by nicardipine. Since 5-HT ₂ activation of both receptors and ET _A receptors is mediated by calcium, it is synergistically the effect of an antagonist to both of these receptors when combined in a surgical solution containing PKC inhibitor It is expected to increase (see FIGS. 2 and 4).

4). Synergistic Effect of Protein Tyrosine Kinase Inhibitor and Calcium Channel Antagonist The mitogenic effect of PDGF (or basic fibroblast growth factor or insulin-like growth factor-1) is a receptor that has intrinsic protein tyrosine kinase activity. Is mediated through. There are many substrates for PDGF phosphorylation, resulting in activation and ultimately proliferation of mitogen-activated protein kinase (MAPK) (see FIG. 5). Endothelin receptors, 5-HT receptors and thrombin receptors are members of the G protein-coupled superfamily that initiate signal transduction pathways that include an increase in intracellular calcium, resulting in activation of calcium channels in the plasma membrane. Thus, calcium channel antagonists interfere with the common mechanism used by these GPCRs. Recently, activation of certain GPCRs, including endothelin and bradykinin, has been shown to result in a rapid increase in tyrosine phosphorylation of a number of intracellular proteins. Some of the phosphorylated proteins are comparable to proteins known to be necessary for mitogenic stimulation. The speed of the process was such that changes could be detected in seconds. The acted target probably plays a role in mitogenesis. These tyrosine phosphorylation events were not blocked by selective PKC inhibitors or were clearly not mediated by increased intracellular calcium. Therefore, it is necessary to block both independent signaling divisions because two independent pathways (GPCR pathway and tyrosine phosphorylation pathway) can promote vascular smooth muscle cells to proliferative state. This is the basis for the synergistic interaction between calcium channel antagonists and tyrosine kinase inhibitors in surgical solutions. Since the action of protein tyrosine kinase inhibitors in preventing the proliferation of vascular smooth muscle cells occurs via an independent molecular mechanism (described above) from pathways involving calcium and protein kinase C, these two classes of drugs The combination (calcium channel antagonist and protein tyrosine kinase inhibitor) is expected to be more effective at inhibiting convulsions and restenosis than when using only one class of drugs alone.

5. Synergistic effects of protein tyrosine kinase inhibitors and thrombin receptor antagonists Thrombin mediates its action through 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 a number of pathways: activation of phospholipase (PLC) by G protein and activation of tyrosine kinases. Activation of tyrosine kinase activity is also essential for mitogenesis of vascular smooth muscle cells. Experimentally, inhibition with specific tyrosine kinase inhibitors is effective in preventing thrombin-induced mitosis, but what is the effect on the PLC pathway when monitored by measurement of intracellular calcium? (Weiss and Nucitelli, J. Biol. Chem. 267: 5608-5613 (1992)). Since the action of protein tyrosine kinase inhibitors in preventing the proliferation of vascular smooth muscle cells occurs via a molecular mechanism independent from the pathway involving calcium and protein kinase C (described above), protein tyrosine kinase inhibitors and thrombin Receptor antagonist combinations are expected to be more effective at inhibiting platelet aggregation, convulsions and restenosis than using either class of agents alone.

G. Cyclooxygenase-2 (COX-2) inhibitors A variety of non-steroidal anti-inflammatory drugs (NSAIDs) are widely used as anti-inflammatory, antipyretic, antithrombotic and analgesic agents (Lewis, RA, “Prostaglandins”). and Leukotrienes ", Textbook of Rheumatology, 3rd edition (Kelly W. et al., 258 (1989)). The molecular target for these drugs is the first enzyme in the prostaglandin synthesis pathway (which is called prostaglandin endoperoxide synthase), or fatty acid cyclooxygenase. Currently, it is understood that there are two forms of cyclooxygenase, which are termed cyclooxygenase-1 or type 1 (COX-1) and cyclooxygenase-2 or type 2 (COX-2). These isoenzymes are also known as prostaglandin H synthase (PGHS) -1 and PGHS-2, respectively. Both enzymes catalyze the conversion of arachidonic acid to labile intermediates (PGG ₂ and PGH ₂ ) that are intermediates in the biosynthesis of prostaglandins and thromboxanes. COX-1 is present in platelets and endothelial cells and exhibits constitutive activity. COX-2 has been identified in endothelial cells, macrophages and fibroblasts, including synovial cells following treatment (induction) with cytokines.

  COX-2 isoenzymes are induced in the context of inflammation by cytokines and inflammatory mediators such as IL-1, TNF-α and endotoxins, and their expression is upregulated at the site of inflammation. A concomitant increase in the activity of COX-2 over basal COX-1 activity that occurs simultaneously with its upregulation results in the synthesis of prostaglandins that contribute to pain and inflammation. Because COX-2 is usually only expressed in inflamed tissues or after exposure to mediators of inflammation, selective inhibitors are undesirable side effects associated with the use of non-selective NSAID drugs (eg, Anti-inflammatory activity without concomitant effects on constitutively expressed COX-1 activity present in platelets and other cell types, which is believed to be responsible for clotting time, bleeding, kidney and gastrointestinal side effects) Can be shown.

The two COX isoenzymes have been shown to be pharmacologically distinct and therefore it is possible to develop isoenzyme specific (selective) cyclooxygenase inhibitors that are useful for anti-inflammatory treatments It has become. A variety of biochemical, cellular and animal assays have been developed to assess the relative selectivity of inhibitors for the COX-1 and COX-2 isoforms. These assays include measuring prostaglandin E2 production in microsomes prepared from various cell types, and bioassay systems that use intact human cells. For any given drug, a compound that is a selective inhibitor for COX-2 has been identified, despite experimental variations in the degree of selectivity observed between assay systems and biological sources. Yes. In general, the criteria for defining selectivity is the ratio of the COX-1 / COX-2 inhibition constant (or COX-2 / COX-1) obtained for a given biochemical or cellular assay system. It is. This selectivity ratio provides different absolutes for inhibition of enzyme activity obtained between microsomal and cellular assay systems (eg, platelets and macrophages, cell lines that stably express recombinant human COX isoenzyme). IC ₅₀ values are described.

  Many of the conventional NSAIDs currently on the market (naproxen, indomethacin, ibuprofen) are generally non-selective inhibitors of both isoforms of COX, but the ratio varies from compound to compound, although COX-1 May exhibit greater selectivity than COX-2. The use of COX-2 inhibitors to block the formation of prostaglandins includes eight descriptions of endogenous prostanoid ligands (eg, PGE2) produced by COX-2 at the site of inflammation and prostanoid receptors. This is a preferred treatment strategy rather than trying to prevent interaction with any of the subtypes. This method is currently available because there are no selective and potent antagonists for all of the prostanoid receptors (EP-1, EP-2, EP-3, EP-4, DP, FP, IP and TP). Cannot be implemented.

In a study by Riendeau and co-workers, the selectivity of more than 45 NSAIDs and selective COX-2 inhibitors indicates that microsomes are sensitive microsomes for inhibition of human COX-1 based on prostaglandin E2 production. Assay and platelet assay (Can. J. Physiol. Pharmacol. 75: 1088-95 (1997)). In this study, among the compounds that were reported to be selective for COX-2 over COX-1, the rank order of efficacy was DuP697> SC-58451, Celecoxib> Nimesulide = Meloxicam = Piroxicam = NS -398 = RS-57067>SC-57666>SC-58125>Froslide>etodolac>L-745,337> DFU-T-614, IC ₅₀ values ranged from 7 μM to 17 μM. A good correlation is that of IC ₅₀ values for inhibition of microsomal COX-1, inhibition of TXB ₂ production by Ca ²⁺ ionophore-stimulated platelets, and prostaglandin E2 production by CHO stably expressing human COX-1. Obtained between both inhibitions. The microsomal assay is more sensitive to inhibition than the cell-based assay, and the detection of the inhibitory effect on COX-1 for all NSAIDs and selective COX-2 inhibitors examined was limited to arachidonic acid. Enabled with the identification of their efficacy under the terms of availability made.

  From molecular and cellular mechanisms of action revealed for selective COX-2 inhibitors such as celecoxib, and also from animal studies, these compounds can be directly irrigated into tissues or joints It is expected to exhibit anti-inflammatory effects when applied intraoperatively. In particular, it is expected to be an effective drug delivered by an irrigation solution during an arthroscopic procedure, urological procedure, cardiovascular procedure or systemic surgical procedure (during treatment). For example, a selective COX-2 inhibitor can be replaced by ketorolac (a relatively non-selective cyclooxygenase inhibitor) in Example IV, Example V and Example VI. Selective COX-2 inhibitors may be used alone or with other small molecule drugs, peptides, proteins, recombinant chimeric proteins, against the joint space, urogenital tract, cardiovascular system or any body cavity It can be delivered in combination with antibodies or gene therapy vectors (viral and non-viral). For example, a selective COX-2 inhibitor may exert its effect on any cell associated with the joint fluid space and structures including the joint. In this case, such cells are involved in the normal functioning of the joint or are present due to pathological conditions. These cells and structures include both type A fibroblasts and type B macrophages synovial cells; articular cartilage components such as chondrocytes; bone related cells, including periosteal cells , Osteoblasts, osteoclasts; immunological components such as lymphocytes, mast cells, monocytes, inflammatory cells including eosinophils, etc .; and other cells such as fibroblasts; As well as combinations of the above cell types.

  Selective COX-2 inhibitors are useful for the introduction and administration of drugs to targeted tissues or joints and can be delivered in formulations that enhance the delivery, uptake, stability or pharmacokinetics of inhibitor drugs . Formulations can include, but are not limited to, administration using microparticles, microspheres or nanoparticles composed of lipids, proteins, carbohydrates, synthetic organic or inorganic compounds. Examples of formulated molecules include lipids that can form liposomes or other aligned lipid structures, cationic lipids, hydrophilic polymers, polycations (eg, protamine, spermidine, and polylysine), substances specific to certain cell types Peptides or synthetic ligands and antibodies that can be targeted to, gels, sustained release matrices, soluble and insoluble particles, and formulation elements not shown, but are not limited to these.

  In the present invention, local delivery of a selective COX-2 inhibitor drug using a irrigation solution that contains the drug present at a low concentration and allows the drug to be delivered directly to the affected tissue or joint. be written. Drug-containing irrigation solutions are used during the perioperative period during surgical procedures. Other conventional methods used for drug delivery include higher concentrations of COX-2 drug (and that will be administered to achieve significant therapeutic concentrations in the targeted tissue or joint). Systemic administration (e.g., intramuscular, intravenous, subcutaneous, oral) is required to force larger total doses. Systemic administration also results in high concentrations in tissues that are not targeted tissues, which is undesirable and, depending on the dose, can lead to adverse side effects (eg, bleeding, ulceration). These systemic delivery methods also expose the drug to two-pass metabolism and rapid degradation, thereby limiting the duration of effective therapeutic concentrations. The drug in the irrigation solution is administered directly to the desired tissue, which means that selective COX- at a lower therapeutically effective concentration and lower total effective dose than by other routes of administration. 2 Provides significant advantages by allowing delivery of inhibitor drugs. Furthermore, the use of a COX-2 inhibitor in solution at a substantially lower dosage than currently used during the perioperative period may allow the use of this drug in patients who are otherwise contraindicated.

  Suitable compounds for the present invention are shown in the table below.

V. Application Methods The solutions of the present invention have applications for a variety of surgical / interventional procedures, including surgical techniques, diagnostic techniques, and therapeutic techniques. Irrigation solutions are applied during arthroscopic surgery of anatomical joints, urological procedures, cardiovascular and general vascular diagnostic and therapeutic procedures, and perioperatively for systemic surgery. As used herein, the term “perioperative” encompasses treatment, before and during treatment, during and after treatment, and before, during and after treatment. Preferably, the solution is applied before and / or after treatment as well as during treatment. Such treatments conventionally utilize a variety of physiological irrigation fluids (eg, normal saline or lactated Ringer's) that are applied to the surgical site by techniques well known to those skilled in the art. Applied. In the method of the present invention, the pain prevention / anti-inflammatory / anticonvulsant / restenosis prevention irrigation solution of the present invention is used instead of the conventionally applied irrigation solution. The irrigation solution is used to prevent pain and inflammation, convulsions and restenosis, before the start of the treatment, preferably before the tissue trauma and continuously throughout the duration of the treatment. Applied to the site. The term “irrigation” as used throughout this specification is intended to mean flowing the wound structure or anatomical structure with a flow of liquid. The term “application” refers to irrigation and other methods of locally delivering a solution of the invention (eg, a surgical site in a gelable form of a solution where the gelled solution then remains at that site throughout the treatment period. Etc.) are intended to be included. The term “continuously” as used throughout this specification refers to the situation where there is repeated frequent irrigation of the wound site with sufficient frequency to maintain a predetermined therapeutic local concentration of applied drug. It is also intended to include applications where there may be intermittent interruptions in the flow of irrigation fluid forced by manipulation techniques.

  The concentration indicated for each of the drugs in the solution of the present invention is the concentration in the absence of metabolic transformation of the drug that is locally delivered to the surgical site to achieve a predetermined level of efficacy at the surgical site. It will be appreciated that the drug concentration in a given solution may need to be adjusted to compensate for local dilution upon delivery. For example, for cardiovascular applications, the average human coronary blood flow rate is 80 cc / min and the average delivery rate for the solution is 5 cc / min with the local delivery catheter (ie, 16 to 1 blood flow to solution Assuming a delivery ratio), the drug concentration in the solution is required to be 16 times greater than the desired in vivo drug concentration. The concentration of the solution is not adjusted to compensate for metabolic conversion or dilution due to systemic distribution, in contrast to oral, intravenous, subcutaneous or intramuscular application. Because these situations are avoided by local delivery.

  Non-limiting examples of arthroscopic techniques in which the solution of the present invention can be used include partial meniscectomy and ligament reconstruction in the knee, shoulder acromioplasty, rotator cuff debridement, elbow Includes synovectomy and wrist and ankle arthroscopy. The irrigation solution is continuously supplied to the joint during surgery at a flow rate sufficient to expand the joint capsule, remove surgical debris, and allow unobstructed intra-articular visualization.

Suitable irrigation solutions for suppressing pain and edema during such arthroscopic techniques are provided herein in Example I below. For arthroscopy, the solution preferably includes the following combinations, and preferably all or any of the following: serotonin ₂ receptor antagonist, serotonin ₃ receptor antagonist, histamine ₁ receptor antagonist, 1A, 1B, Serotonin receptor agonists, bradykinin ₁ receptor antagonists, bradykinin ₂ receptor antagonists, and cyclooxygenase inhibitors that act on 1D, 1F and / or 1E receptors.

In the solutions of the present invention, very low doses of these pain and inflammation inhibitors are utilized for local application of the drug directly to the surgical site during the treatment period. For example, less than 0.05 mg amitriptyline (a suitable “dual” receptor antagonist of serotonin ₂ and histamine ₁ ) provides the desired effective local tissue concentration that inhibits 5-HT ₂ and H ₁ receptors. Is needed for 1 liter of irrigation solution. This dosage is very low relative to 10-25 mg of oral amitriptyline, which is the usual starting dose for this drug. This same rationale is to reduce convulsions associated with urological, cardiovascular and general vascular procedures, and to inhibit restenosis associated with cardiovascular and general vascular procedures. This is true for anticonvulsants and anti-restenosis agents utilized in the solutions of the present invention. For example, less than 0.2 mg nisoldipine (a preferred calcium channel antagonist) irrigation solution 1 to provide the desired effective local tissue concentration that inhibits voltage-dependent gating of L-type subtype calcium channels Required for liters. This dose is very low compared to a single oral dose of nisoldipine that is 20 mg to 40 mg.

  In each of the surgical solutions of the present invention, the drug is included at a lower concentration than that required using conventional methods of drug administration to achieve the desired effect, and at lower doses. Delivered locally. Equivalent therapeutic effects cannot be obtained by delivering similarly dosed drugs by other routes of drug administration (ie, intravenous, subcutaneous, intramuscular or oral routes). This is because systemically administered drugs undergo first-pass metabolism and two-pass metabolism.

For example, using a rat model of arthroscopy, we examine the ability of amitriptyline (5-HT ₂ antagonist) to inhibit 5-HT-induced plasma extravasation in the rat knee according to the present invention. It was. This study (described in more detail in Example XII below) compares the therapeutic dosing of amitriptyline delivered locally (ie, intra-articularly) with amitriptyline delivered intravenously in the knee. It was. The results revealed that intra-articular administration of amitriptyline required a total dosage level of about 1/200 of that required by the intravenous route to achieve the same therapeutic effect. Given that only a small percentage of the drug delivered into the joint is absorbed by local synovial tissue, the difference in plasma drug levels between these two routes of administration is at the overall amitriptyline dosage level. Much bigger than the difference.

  Implementation of the present invention should be distinguished from conventional intra-articular injections of opiates and / or local anesthetics when an arthroscopic procedure or “open” joint (eg, knee, shoulder, etc.) procedure is completed . The solution of the present invention is used for continued infusion during the entire surgical procedure to provide prophylactic inhibition of pain and inflammation. In contrast, the large concentrations (0.5% to 2% solution) required to achieve therapeutic efficacy using a constant infusion of a local anesthetic such as lidocaine results in serious systemic toxicity.

  When the treatment of the present invention is complete, as an alternative or supplement to opiates, inject higher concentrations of the same pain and inflammation inhibitors used in the irrigation solution at the surgical site, or otherwise apply It may be desirable to do so.

The solutions of the present invention also provide cardiovascular and general vascular prostheses to potentially reduce vessel wall spasm, platelet aggregation, vascular smooth muscle cell proliferation, and nociceptor activation caused by vessel growth. Applied in diagnostic and therapeutic treatments. Reference herein to arterial treatment is intended to encompass treatment of venous grafts harvested and placed in the arterial system. Suitable solutions for such techniques are disclosed herein in Example II below. The cardiovascular solution and the general vascular solution preferably comprise any combination of the following, and preferably all of the following: 5-HT ₂ receptor antagonist (Saxena, PR et al., “Cardiovascular Effects of Serotonin Inhibition Agents” and Antagonists ", J. Cardiovasc. Pharmacol. 15 (Suppl. 7): S17-S34 (1990); Douglas, 1985); activation of these receptors in sympathetic neurons and C-fiber nociceptive neurons in the vascular wall. 5-HT ₃ receptor antagonist to block, such activation has been shown to cause bradycardia and tachycardia (Saxena et al., 1990) Bradykinin ₁ receptor antagonists; and prevents the production of prostaglandins at the tissue injury sites and thereby, cyclooxygenase inhibitors for reducing pain and inflammation. Also, cardiovascular and general vascular solutions also preferably contain a serotonin _1B (which is also known as serotonin _1Dβ ) antagonist. This is because serotonin has been shown to cause significant vasospasm by activation of the serotonin _1B receptor in humans. Kaumann, A.M. J. et al. Et al., “Variable Participation of 5-HT1-Like Receptors and 5-HT2 Receptors in Serotonin-Induced Contrast of Human Isolated Coronaries 53” (Circle 114). This excitatory action of serotonin _1B receptor in the vessel wall causes vasoconstriction, but in contrast to the previously discussed inhibitory action of serotonin _1B receptor in neurons. The cardiovascular and general vascular solutions of the present invention are also preferably disclosed herein that reduce the occurrence and severity of post-procedure restenosis resulting from, for example, angioplasty or rotational atherectomy One or more of the anti-restenosis agents to be included.

The solutions of the present invention are also useful for reducing pain and inflammation associated with urological procedures such as transurethral prostatectomy and similar urological procedures. References herein to application of a solution to the urinary tract or urological structure are intended to encompass application to the urinary tract itself, the bladder and prostate, and related structures. Various studies have shown that serotonin, histamine and bradykinin cause inflammation in lower urinary tract tissue. Schwartz, M.M. 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). Suitable irrigation solutions for urological treatment are disclosed herein below in Example III. This solution preferably comprises the following combinations, and preferably all of the following: histamine ₁ receptor antagonists for inhibiting histamine-induced pain and inflammation; of these receptors in peripheral C-fiber nociceptive neurons 5-HT ₃ receptor antagonists to block activation; bradykinin ₁ antagonists; bradykinin ₂ antagonists; and cyclooxygenase inhibitors to reduce pain / inflammation caused by prostaglandins at the site of tissue injury. Preferably, anticonvulsants are also included to prevent convulsions in the urethra and bladder wall.

  Some of the solutions of the present invention can also suitably include a gelling agent to produce a dilute gel. Such a gellable solution can be applied, for example, in the urinary tract or in the arterial wall to deliver a continuous thin local predetermined concentration of drug.

  The solution of the present invention can also be used perioperatively to inhibit pain and inflammation in surgical wounds, and similarly to reduce pain and inflammation associated with burns. Burns result in the release of significant amounts of biogenic amines, such amines not only cause pain and inflammation, but also lead to severe plasma extravasation (fluid loss), often with severe burns, It is an element related to life and death. Holliman, C.I. J. et al. “The Effect of Ketanserin, a Specific Serotonin Antagonist, on Burn Shock Parameters in a Porcine Burn Model”, J. et al. Trauma 23: 867-871 (1983). The solution disclosed in Example I for arthroscopy is also suitably applied to wounds or burns to control pain and inflammation and for surgical procedures such as arthroscopy Can be done. Alternatively, the solution of the solution of Example I can be included in a paste base or salve base for application to a burn or wound.

VI. Examples Below are shown some formulations according to the present invention that are suitable for certain surgical procedures, followed by a summary of three clinical studies utilizing the agents of the present invention.

A. Example I
Irrigation solutions for arthroscopy The following compositions are suitable for use in irrigating anatomical joints during arthroscopic procedures. As with the remaining solutions described in the subsequent examples, each drug is solubilized in a carrier fluid containing a physiological electrolyte (eg, normal saline or lactated Ringer's solution). The

B. Example II
Irrigation solutions for cardiovascular and general vascular therapeutic and diagnostic procedures The following drugs and concentration ranges in solution in physiological carrier fluids irrigate surgical sites during cardiovascular and general vascular procedures Suitable for use in

C. Example III
Irrigation solutions for urological procedures The following drugs and concentration ranges in solution in a physiological carrier fluid are suitable for use in irrigating surgical sites during urological procedures.

D. Example IV
Irrigation Solution for Arthroscopy, Burns, General Surgical Wounds and Oral / Dental Application The following composition is anatomical irrigation during arthroscopy and oral / dental procedures, and burns and general Preferred for use in the management of surgical wounds. While the solution shown in Example I is suitable for use with the present invention, the following solutions are even more preferred as greater efficacy is expected.

E. Example V
Alternative Irrigation Solutions for Cardiovascular and General Vascular Therapeutic and Diagnostic Treatments The following drugs and concentration ranges in solution in physiological carrier fluids are the surgical site during cardiovascular and general vascular procedures. Is preferred for use in irrigating. Again, this solution is preferred compared to the solution shown in Example II above because of its greater potency.

F. Example VI
Alternative Irrigation Solutions for Urological Treatments The following drugs and concentration ranges in solution in physiological carrier fluids are preferred for use in irrigating the surgical site during urological treatments. This solution is believed to have a greater potency than the solution shown in Example III above.

G. Example VII
Cardiovascular and general vascular restenosis prevention irrigation solutions The following drugs and concentration ranges in solution in physiological carrier fluids are for use in irrigation during cardiovascular and general vascular therapeutic and diagnostic procedures Is preferred. The drug in this preferred solution is also the cardiovascular of Example II and Example V above or the Example VIII described below for the preferred anticonvulsant, anti-restenosis, and anti-pain / anti-inflammatory solutions. It can be added to the irrigation solution and general vascular irrigation solution at the same concentration.

H. Example VIII
Alternative irrigation solutions for cardiovascular and general vascular therapeutic and diagnostic procedures Further preferred solutions for use in cardiovascular and general vascular therapeutic and diagnostic procedures are nitric oxide (NO Donor) The formulation described above in Example V, except that SIN-1 is replaced by two agents, FK409 (NOR-3) and FR144420 (NOR-4), at the concentrations indicated below. Is blended in the same way.

I. Example IX
Alternative irrigation solutions for arthroscopy, burns, general surgical wounds and oral / dental applications Alternatives for use in arthroscopy, burns, general surgical wounds and irrigation of oral / dental applications A preferred solution is formulated the same as in Example IV described above using the following substitutions, deletions and additions at the concentrations indicated below.
1) Amitriptyline is replaced by mepyramine as an H ₁ antagonist;
2) the kallikrein inhibitor aprotinin is deleted;
3) The bradykinin ₁ antagonist [leu ⁹ ] [des-Arg ¹⁰ ] calidene is added;
4) The bradykinin ₂ antagonist HOE140 is added; and 5) the μ-opioid agonist fentanyl is added.

J. et al. Example X
Alternative irrigation solutions for urological procedures Alternative preferred solutions for use in irrigation during urological procedures are described above, with the following substitutions, deletions and additions at the concentrations indicated below: Formulated as in Example VI.
1) SIN-1 is
a) FK409 (NOR-3), and b) FR144420 (NOR-4)
Is replaced as a NO donor by a combination of the two drugs:
2) the kallikrein inhibitor aprotinin is deleted;
3) The bradykinin ₁ antagonist [leu ⁹ ] [des-Arg ¹⁰ ] Cariden is added; and 4) the bradykinin ₂ antagonist HOE 140 is added.

K. Example XI
Effects of histamine / serotonin receptor blockade on normal iliac artery balloon dilatation and its response in New Zealand white rabbits. First, a new in vivo model for the study of arterial tone was used. The time course of arterial size change before and after balloon angioplasty is described below. Second, the role of the combined histamine and serotonin in controlling arterial tone in this situation is then to selectively connect the histamine receptor and serotonin receptor blocker to the artery before and after angioplasty injury. Studied by injecting.

1. Design Considerations This study describes the time course of changes in arterial lumen size in one artery group and the effect of histamine / serotonin receptor blockade on these changes in another similar artery group Was intended to be evaluated. To facilitate comparison of the two different groups, both groups were treated in the same manner except for the contents of the injections performed at the time of the experiment. In control animals (arteries), the infusion was normal saline (vehicle for test solution). Treated arteries with histamine / serotonin receptor blockade received saline containing receptor antagonist at the same rate as control animals in the same part of the protocol. Specifically, the test solutions were all in normal saline (a) a serotonin ₃ antagonist (metoclopramide) at a concentration of 16.0 μM; (b) a serotonin ₂ antagonist (trazodone at a concentration of 1.6 μM). ); And (c) a histamine antagonist (promethazine) at a concentration of 1.0 μM. The drug concentration in the test solution is 16 over the drug concentration delivered at the surgical site due to the 16 to 1 flow rate ratio between the iliac artery (80 cc / min) and the solution delivery catheter (5 cc / min). It was twice as big. The study was conducted in a prospective, randomized, blinded fashion. Assignment to a specific group is random, and the investigator has completed an angiographic analysis for the contents of the infusion solution (saline alone or saline containing a histamine / serotonin receptor antagonist alone) I wasn't told until.

2. Animal Protocol This protocol was approved by the Animal Use Committee of the Seattle Military Medical Center. The facility is fully accredited by the American Institute for Laboratory Animal Care. The iliac arteries of 3-4 kg male New Zealand white rabbits fed on normal rabbit food were examined. The animals are sedated using intravenous administration of xylazine (5 mg / kg) and ketamine (35 mg / kg) dosed to produce an effect, an incision is made at the ventral midline of the neck, and the carotid artery is Isolated. The artery was ligated distally, an arteriotomy was performed, and a 5 French sheath was introduced into the descending aorta. Baseline blood pressure and heart rate are recorded, and then angiograms of distal and bilateral iliac arteries use manual injection of 76% (Squibb Diagnostics, Princeton, NJ) of iopamidol into the descending aorta Was recorded on 35 mm motion picture film (frame speed, 15 per second). For each angiogram, the calibration object was placed in the X-ray field of view to allow correction for the magnification when the diameter measurement was made. A 2.5 French infusion catheter (Advanced Cardiovascular Systems, Santa Clara, Calif.) Was placed through the carotid sheath and placed 1 cm to 2 cm above the aortic bifurcation. Infusion of test solution (either saline alone or saline containing histamine / serotonin receptor antagonist) was started at a rate of 5 cc / min and continued for 15 minutes. Five minutes after injection, a second angiogram is performed using the previously described technique, followed by a 2.5 mm balloon angioplasty catheter (the Lightning, Cordis Corp., Miami, FL) was rapidly advanced to the left iliac artery and then to the right iliac artery under fluoroscopic guidance. In each iliac artery, a balloon catheter was carefully placed between the proximal and distal deep femoral bifurcations using osseous viewing points and the balloon was inflated to a pressure of 12 atmospheres for 30 seconds. . The balloon catheter was inflated using a thin solution of radiographic contrast agent so that the diameter of the inflated balloon could be recorded on the motion picture film. The angioplasty catheter was quickly removed and another angiogram was recorded on motion picture film on average 8 minutes after the start of infusion. The infusion continued until 15 minutes and another angiogram (4th) was performed. Thereafter, the injection was stopped (75 cc of the solution was injected in total) and the injection catheter was removed. At 30 minutes (15 minutes after stopping the infusion), the last angiogram was recorded as before. Blood pressure and heart rate were recorded immediately before the angiogram at 15 and 30 minutes. After the last angiogram, the animals were euthanized with an overdose of intravenously administered anesthetic and the iliac artery was removed and immersed and fixed in formalin for histological analysis.

3. Angiographic analysis Angiograms were recorded on 35 mm motion picture film at a frame rate of 15 per second. For analysis, angiograms were projected from a Vangard projector at a distance of 5.5 feet. The diameter of the iliac artery at a pre-determined location relative to the balloon angioplasty site was recorded based on the portable caliper measurement after correction for magnification by measuring the calibration object. Measurements were taken at baseline (before test solution injection was started), 5 minutes after injection, immediately after balloon angioplasty (average 8 minutes after test solution was started), 15 minutes (infusion Performed immediately before stopping) and 30 minutes (15 minutes after stopping the injection). Diameter measurements were made at three locations in each iliac artery: proximal to the balloon dilation site, balloon dilation site, and just distal to the balloon dilation site.

The diameter measurement was then converted to an area measurement by the following formula:
Area = (Pi) (diameter ² ) / 4
For the calculation of vasoconstriction, the value at baseline is used to represent the maximum area of the artery,
% Vasoconstriction = {(area in reference state−area at subsequent time) / area in reference state} × 100
As calculated.

4). Statistical methods All values are expressed as mean ± 1 [standard error of the mean]. The time course of vasomotor response in control arteries was assessed using a one-way analysis of variance with correction for repeated measurements. Post hoc comparison of data between specific time points was performed using Scheffe's test. Once the time at which significant vasoconstriction has occurred is determined in the control artery, the control artery and the artery treated with the histamine / serotonin receptor antagonist are analyzed using multiple analysis of variance for treatment groups identified as independent variables, Comparisons were made at those time points when significant vasoconstriction occurred in the control artery. To compensate for the absence of any a priori defined hypothesis, a p-value less than 0.01 was considered significant. Statistics were performed using Statistica for Windows® (version 4.5) (Statsoft, Tulsa, OK).

5. Results The time course of changes in arterial diameter before and after balloon angioplasty in normal arteries given saline infusion was evaluated in 16 arteries obtained from 8 animals. (Table 30). Three segments of each artery were examined: a proximal segment just upstream of the balloon dilation segment, a balloon dilation segment, and a distal segment just downstream of the balloon dilation segment. A similar pattern of arterial size was revealed in the proximal and distal segments. That is, in each, a significant change in arterial diameter was observed when all time points were compared (proximal segment, p = 0.0002, and distal segment, p <0.001, ANOVA) . Post hoc tests showed that the diameter at the time immediately after angioplasty was significantly smaller in each of these segments than the diameter at baseline or 30 minutes. On the other hand, the arterial diameter in each segment at the 5 min, 15 min and 30 min time points was similar to the baseline diameter. The balloon dilation segment showed less change in arterial size than the proximal and distal segments. The baseline diameter of this segment was 1.82 ± 0.05 mm; the nominal inflation diameter of the balloon used for angioplasty was 2.5 mm, and the actual measured inflation diameter of the balloon Was 2.20 ± 0.03 mm (p <0.0001, relative to the baseline diameter of the balloon-treated segment). Thus, the inflated balloon caused the balloon expansion segment to expand in the circumferential direction. However, there was only a slight increase in lumen diameter from baseline to 30 minutes (1.82 ± 0.05 mm to 1.94 ± 0.07 mm, post-test p = NS).

  The lumen diameter of the artery was used to calculate the lumen area, which was then used to measure percent vasoconstriction, 5 minutes, immediately after angioplasty, 15 minutes and 30 minutes data It was calculated by comparing against the measured value in the state. The proximal segment and distal segment data expressed as percent vasoconstriction is shown in FIG. 9; the change in vasoconstriction over time is significant (p = 0.0008 in the proximal segment; In the distal segment, p = 0.0001, ANOVA). Post hoc testing confirmed that the vasoconstriction immediately after angioplasty was significantly different from the vasoconstriction present at the 30 minute time point (P <0.001 in both segments). In the distal segment, the vasoconstriction immediately after angioplasty was also significantly less than the vasoconstriction at the 5 minute time point (p <0.01); any other differences in the comparison between time points were posterior It was not significant by the test.

  Lumen changes in the control arteries can be summarized as follows: 1) Vasoconstriction with a loss of about 30% of lumen area in baseline conditions occurs immediately after balloon dilation, proximal and distal of the balloon dilation segment. It occurs in the distal arterial segment. Smaller amounts of vasoconstriction tend to occur in the proximal and distal segments before dilation and at the 15 minute time point (approximately 7 minutes after dilation), but also at the 30 minute time point (after dilation) By about 22 minutes), the tendency towards vasodilation has replaced previous vasoconstriction; 2) there is only a minor change in lumen size in the balloon dilation segment, and Despite the use of a balloon with an inflated diameter significantly greater than that present in this segment at baseline, no significant increase in the lumen diameter of the dilated segment was observed. These findings led to the conclusion that any effect of postulated histamine / serotonin treatment could only be detected in the proximal and distal segments at the time when vasoconstriction was present.

  Histamine / serotonin receptor blocking solution was injected into 16 arteries (8 animals). Angiographic data could be obtained at all time points in 12 arteries. Heart rate and systolic blood pressure measurements could be obtained in some animals (Table 31). There was no difference in heart rate or systolic blood pressure when the two groups of animals were compared at specific times. Animals treated with histamine / serotonin tend to decrease in systolic blood pressure (−14 ± 5 mmHg, p = 0.04) and decrease in heart rate (−26 ± 10, p = 0. 05). In control animals, there was no change in heart rate or systolic blood pressure throughout the duration of the experiment.

  Proximal and distal segments of arteries treated with histamine / serotonin were compared to control arteries using percent vasoconstriction measurements. FIG. 10A shows the effect of histamine / serotonin infusion on proximal segment vasoconstriction compared to vasoconstriction present in control arteries. When findings in the two treatment groups were compared at baseline, immediately after angioplasty and at 15 minutes, histamine / serotonin infusion resulted in significantly less vasoconstriction compared to control saline infusion (P = 0.003, two-way ANOVA). A comparison of the two treatment groups in the distal segment is illustrated in FIG. 10B. Despite the observed difference in mean diameter measurements in the distal segment, blood vessels treated with solution were treated with saline at baseline, immediately after angioplasty and at 15 minutes. Although it showed less vasoconstriction than control vessels, this pattern did not achieve statistical significance (p = 0.32, two-way ANOVA). The lack of statistical significance can be attributed to smaller than expected vasoconstriction in control vessels.

L. Example XII
Amitriptyline inhibition of knee joint plasma extravasation induced by 5-hydroxytryptamine-comparison of intra-articular versus intravenous routes The following study shows _two of the 5-HT ₂ receptor antagonist amitriptyline in a rat knee synovial model of inflammation Initiated to compare the two routes of administration: 1) continued intra-articular injection vs. 2) intravenous injection. The ability of amitriptyline to inhibit joint plasma extravasation induced by 5-HT was demonstrated by comparing both the efficacy and total drug dose of amitriptyline delivered by each route.

1. Animals Approval from the institutional animal care committee of the University of California (San Francisco) was obtained for these studies. Male Sprague-Dawley rats (Bantin and Kingman, Fremont, Calif.) Weighing 300-450 g were used in these studies. Rats were housed under controlled lighting conditions (lighting from 6 am to 6 pm) with free access to food and water.

2. Plasma extravasation Rats were anesthetized with sodium pentobarbital (65 mg / kg), after which the rats were injected with tail vein injection (2.5 ml / kg) of Evans blue dye used as a marker for plasma extravasation. 50 mg / kg by volume) was given. The knee joint capsule was exposed by incising the overlying skin and a 30 gauge needle was inserted into the joint and used for fluid injection. The infusion rate (250 μl / min) was controlled by a Sage Instruments Syringe pump (Model 341B, Orion Research Inc., Boston, Mass.). A 25 gauge needle was also inserted into the joint space and the perfusate was removed at 250 μl / min controlled by a Sage Instruments Syringe pump (model 351).

  Rats were randomly assigned to three groups: 1) rats receiving only intra-articular (IA) 5-HT (1 μM), 2) amitriptyline (0.01 mg / kg to 1.0 mg / kg) by intravenous (IV) administration. rats receiving HT 5-HT (1 mM), and 3) amitriptyline (1 μM to 100 μM dose) by intra-articular (IA) administration, followed by IA 5-HT (1 μM) ) Rats receiving + IA amitriptyline. In all groups, the baseline level of plasma extravasation is 0.9% saline perfused into the joint and 3 perfusate samples over a 15 minute period (one every 5 minutes). ) Was collected at the time of disclosure of each experiment. The first group then received 5-HT IA for a total of 25 minutes. Perfusate samples were collected every 5 minutes for a total of 25 minutes. Samples were then analyzed for the concentration of Evans Blue dye by spectrophotometric measurement of absorbance at 620 nm, which is directly proportional to that concentration (Carr and Wilhelm, 1964). In the IV amitriptyline group, the drug was administered at the time of tail vein injection of Evans Blue dye. The knee joint was then perfused with physiological saline for 15 minutes (baseline), followed by 25 minutes of perfusion with 5-HT (1 μM). Perfusate samples were collected every 5 minutes for a total of 25 minutes. Samples were then analyzed using a spectrophotometer. In the IA amitriptyline group, amitriptyline was perfused into the joint for 10 minutes after 15 minutes of saline perfusion, after which amitriptyline was perfused in combination with 5-HT for an additional 25 minutes. Perfusate samples were collected every 5 minutes and analyzed as described above.

  Some rat knees have physical damage to the knee joint, and inflow and outflow mismatch (the presence of blood in the perfusate and high plasma extravasation level at baseline or improper needle placement Because it can be detected by the enormous number of knee joints).

A. Plasma Extravasation with 5-HT Induction Plasma extravasation at baseline was measured in all knee joints examined (total n = 22). Plasma extravasation levels at baseline were low with an average value of 0.022 ± 0.003 absorbance units (mean value ± standard error of mean value) at 620 nm. The extravasation level in this reference state is shown as a broken line in FIGS.

  Perfused rat knee joint 5-HT (1 μM) produces a time-dependent increase in plasma extravasation above baseline levels. At 25 minutes of perfusion within the 5-HT joint, the maximum level of plasma extravasation was achieved by 15 minutes and continued until perfusion was stopped at 25 minutes (data not shown). Therefore, the reported 5-HT induced plasma extravasation levels are the mean values at the 15 min, 20 min and 25 min time points during each experimental period. The plasma extravasation by 5-HT induction averaged 0.192 ± 0.011, which was about 8 times stimulation above baseline. This data is illustrated in FIGS. 11 and 12, corresponding to the “0” dose of IV amitriptyline and the “0” concentration of IA amitriptyline, respectively.

B. Effect of intravenous amitriptyline on 5-HT-induced plasma extravasation. Amitriptyline administered by tail vein injection resulted in a dose-dependent decrease in 5-HT-induced plasma extravasation, as shown in FIG. It was. The IC ₅₀ for IV amitriptyline inhibition of 5-HT-induced plasma extravasation is about 0.025 mg / kg. Plasma extravasation due to 5-HT induction is completely inhibited by a dose of 1 mg / kg IV amitriptyline, with plasma extravasation averaging 0.034 ± 0.010.

C. Effect of intra-articular amitriptyline on 5-HT-induced plasma extravasation. Amitriptyline administered intra-articularly alone at increasing concentrations does not affect plasma extravasation levels relative to baseline conditions, and plasma extravasation Averaged 0.018 ± 0.002 (data not shown). Amitriptyline co-administered with 5-HT at increasing concentrations resulted in a concentration-dependent decrease in 5-HT-induced plasma extravasation, as shown in FIG. Plasma extravasation by 5-HT induction in the presence of 3 μM IA amitriptyline is not significantly different from the plasma extravasation caused by 5-HT alone, however, 30 μM of co-perfused with 5-HT Amitriptyline caused more than 50% inhibition, whereas 100 μM amitriptyline caused complete inhibition of 5-HT-induced plasma extravasation. The IC ₅₀ for IA amitriptyline inhibition of 5-HT-induced plasma extravasation is about 20 μM.

The main finding of this study is that 5-HT (1 μM) perfused intra-articularly in the rat knee joint results in stimulation of plasma extravasation that is approximately 8-fold above baseline levels. Either intravenous or intra-articular administration of the HT ₂ receptor antagonist amitriptyline can inhibit 5-HT-induced plasma extravasation. However, the total dosage of amitriptyline administered is dramatically different between these two drug delivery methods. The IC ₅₀ for IV amitriptyline inhibition of 5-HT-induced plasma extravasation is 0.025 mg / kg, ie 7.5 × 10 ⁻³ mg in 300 g adult rats. The IC ₅₀ for IA amitriptyline inhibition of 5-HT-induced plasma extravasation is about 20 μM. Since 1 ml of this solution was delivered every 5 minutes for a total of 35 minutes during the experimental period, the total dosage perfused into the knee was 4.4 × 10 ⁻⁵ mg total perfused into the knee. The dosage was 7 ml. This IA amitriptyline dose is approximately 1/200 of the IV amitriptyline dose. Furthermore, it is possible that only a small percentage of the IA perfused drug is absorbed systemically, thereby creating an even greater difference in the total delivered dose of drug.

  Since 5-HT plays an important role in surgical pain and inflammation, as discussed above, 5-HT antagonists such as amitriptyline are beneficial when used during the perioperative period. possible. A recent study attempted to clarify the effect of oral amitriptyline on pain after orthopedic surgery (Kerrick et al., 1993). Oral doses as low as 50 mg produced undesirable central nervous system side effects such as “reduced satisfaction”. Kerrick et al. Also showed that oral amitriptyline produced a greater pain scale score (P <0.05) than placebo in patients after surgery. It is unclear whether this was due to the overall discomfort caused by oral amitriptyline. In contrast, the intra-articular route of administration allows for extremely low concentrations of the drug to be delivered locally to the inflammatory site and can provide maximum benefit with minimal side effects.

M.M. Example XIII
Effects of cardiovascular and general vascular solutions on vasospasm induced by rotational atherectomy in rabbit arteries Tested Solution In this study, an irrigation solution consisting of the drug shown in Example V above was used with the following exceptions. Nitroprusside was replaced by SIN-1 as the nitric oxide donor and nicardipine was replaced by nisoldipine as the Ca ²⁺ channel antagonist.

The concentration of nitroprusside was selected based on its previously defined pharmacological activity (EC ₅₀ ). The concentration of the other drugs in this test solution was determined based on the drug's binding constant for their cognate receptor. In addition, all concentrations were adjusted based on a blood flow rate of 80 cc per minute in the rabbit's distal aorta and a flow rate of 5 cc per minute in the solution delivery catheter. Three components were mixed into 1 cc or less of DMSO, after which these components and the remaining three components were mixed to their final concentrations in normal saline. A control solution consisting of normal saline was used. Test solution or control solution was infused at a rate of 5 cc / min for 20 minutes. A brief interruption in the infusion was necessary when blood pressure measurements were taken. Thus, each animal was given approximately 95 cc of solution for a 20 minute treatment period.

2. Animal Protocol This protocol has been approved by the Animal Use Committee of the Seattle Military Medical Center, which is accredited by the American Institute for Laboratory Animal Care and Certification. The iliac arteries of 3-4 kg male New Zealand white rabbits bred for 3-4 weeks on a 2% cholesterol rabbit diet were examined. The animals are sedated using intravenous administration of xylazine (5 mg / kg) and ketamine (35 mg / kg) dosed to produce an effect, an incision is made at the ventral midline of the neck, and the carotid artery is Isolated. The artery was ligated distally, an arteriotomy was performed, and a 5 French sheath was introduced into the descending aorta and placed at the level of the renal artery. Blood pressure and heart rate at baseline were recorded. Angiograms of the distal aorta and bilateral iliac arteries were recorded on 35 mm motion picture film using a manual injection of 76% iopamidol (Squibb Diagnostics, Princeton, NJ) into the descending aorta (frame speed, 1 sec. 15).

  For each angiogram, the calibration object was placed in the X-ray field of view to allow correction for the magnification when the diameter measurement was made. Infusion of either the test solution or saline control was initiated from the lateral arm of the 5 French sheath (delivered to the distal aorta) at a rate of 5 cc / min and continued for 20 minutes. Five minutes after injection, a second angiogram was performed using previously described techniques. A 1.25 mm or 1.50 mm rotating atherectomy bar (Heart Technology / Boston Scientific Inc.) was then advanced into the iliac artery. A rotating atherectomy bar was advanced three times over the guidewire in each of the iliac arteries at a rotational speed of 150,000 RPM to 200,000 RPM. In each iliac artery, a rotating atherectomy bar was advanced from the distal aorta to the middle portion of the iliac artery between the first and second deep femoral branches. The rotating atherectomy bar was quickly removed and another angiogram was recorded on motion picture film on average 8 minutes after the start of infusion.

  The infusion was continued until 20 minutes and another angiogram (4th) was performed. Thereafter, the injection was stopped. A total of about 95 cc of control or test solution was injected. At 30 minutes (15 minutes after stopping the infusion), the last angiogram was recorded as before. Blood pressure and heart rate were recorded immediately before the angiogram at 15 and 30 minutes. After the last angiogram, the animals were euthanized with an overdose of anesthetic administered intravenously.

3. Angiographic analysis Angiograms were recorded on 35 mm motion picture film at a frame rate of 15 per second. Angiograms were examined in random order without knowing the treatment evaluation. For analysis, angiograms were projected from a Vangard projector at a distance of 5.5 feet. The entire angiogram for each animal was examined to identify the anatomy of the iliac artery and to identify the largest convulsive site in the iliac artery. A map of the iliac anatomy was prepared to help consistently identify the site for measurement. Measurements were first made on angiograms 15 minutes after rotational atherectomy and then on the remaining angiograms obtained from that animal in random order. Measurements were made using an electronic portable caliper (Brown & Sharp Inc., N. Kingston, RI). The iliac artery diameter was measured at three locations: proximal to the first deep femoral bifurcation of the iliac artery; at the site of the most severe convulsions (this is the first and second in all cases) And occurred at the distal site (near or distal to the origin of the second deep femoral artery bifurcation of the iliac artery). Measurements were made at baseline (before test solution injection was started), 5 minutes after injection, immediately after rotational atherectomy (average 8 minutes after test solution was started), and injection stopped Immediately after 20 minutes (this was 15 minutes after the start of the rotational atherectomy) and 15 minutes after the stop of the injection (30 minutes after the start of the rotational atherectomy). The calibration object was measured in each angiogram.

The diameter measurement was then converted to an area measurement by the following formula:
Area = (Pi) (diameter ² ) / 4
For the calculation of vasoconstriction, the value at baseline is used to represent the maximum area of the artery,
% Vasoconstriction = {(area in reference state−area at subsequent time) / area in reference state} × 100
As calculated.

4). Statistical methods All values are expressed as mean ± 1 [standard error of the mean]. The time course of vasomotor response in control arteries was assessed using a one-way analysis of variance with correction for repeated measurements. Post hoc comparison of data between specific time points was performed using Scheffe's test. Arteries treated with the test solution were compared to arteries treated with saline using a multiple analysis of variance (MANOVA) at specified locations in the iliac arteries and at specified times. . To compensate for the absence of any a priori defined hypothesis, a p-value less than 0.01 was considered significant. Statistics were performed using Statistica for Windows® (version 4.5) (Statsoft, Tulsa, OK).

5. Results Eight arteries in 4 animals received saline solution and 13 arteries in 7 animals received test solution. In each artery, despite the solution tested, rotational atherectomy was performed by passing a rotating bar from the distal aorta to the middle part of the iliac artery. Therefore, the proximal iliac artery segment and the segment referred to as the maximum vasoconstriction site were exposed to the rotating bar. The guide wire for the rotating atherectomy catheter passed through the distal segment, but the rotating bar of the rotating atherectomy catheter itself did not enter the distal segment.

  Table 32 summarizes the iliac artery diameters at the saline treated arteries in the three designated segments. In the proximal segment, there was no significant change in arterial diameter over the course of the experiment (p = 0.88, ANOVA). In the middle iliac artery at the site of maximum vasoconstriction, a large decrease in diameter was observed, with the greatest decrease occurring at 15 minutes after rotational atherectomy (p <0.0001, at all 5 time points). ANOVA to compare measured values of). The diameter of the distal segment did not change significantly over the course of the experiment (p = 0.19, ANOVA comparing all time points). However, a trend towards a decrease in diameter was observed immediately after rotational atherectomy and at 15 minutes after rotational atherectomy.

  The diameter of the iliac artery treated with the test solution is shown in Table 33. Angiograms are not recorded at 3 of these arteries at 5 minutes after the start of the infusion, and angiographic data are taken from 2 arteries (1 animal) at 30 minutes after rotational atherectomy. Excluded from. This is because the animal received a 15-minute vascularization diagram with air embolism that caused hemodynamic anxiety. ANOVA statistics were not applied to this data because the number of observations at the five time points was not constant. Nonetheless, it is clear that the magnitude of the change in diameter measurements within the segment in the treated artery with the test solution throughout the course of the experiment is less than that observed in the artery treated with saline. .

Due to the different number of observations at various time points, ANOVA was not performed to reveal statistical similarities / differences in diameter at specific segments.

  The primary endpoint for the study was a comparison of vasoconstriction in arteries treated with saline and arteries treated with the test solution. Vasoconstriction was based on the arterial lumen area obtained from arterial diameter measurements. The area values at 5 minutes, immediately after and after the rotational atherectomy, were compared to the area values at baseline, and the relative area changes were calculated. The result is referred to as “vasoconstriction” if the lumen area is smaller than in the baseline state at a later time point, and the lumen area is compared to the baseline state at a later time point. If it became larger in, it was referred to as “vasodilation” (Tables 27 and 28). In order to facilitate statistical analysis using as many observations as possible in both treatment groups, arterial data treated with test solution and arterial treated with saline were used for rotational atherectomy. Comparisons were made immediately and at 15 minutes after rotational atherectomy.

  In the proximal segment (FIG. 13), there is no substantial change in lumen area with either procedure immediately after rotational atherectomy, but by 15 minutes after rotational atherectomy. Some vasodilation was observed in this segment. The test solution did not change the outcome of rotational atherectomy in this segment compared to saline treatment. However, in the middle vessel at the site of maximum vasoconstriction (FIG. 14), the test solution significantly attenuated the vasoconstriction caused by rotational atherectomy in saline-treated arteries (p = 0.0004). , MANOVA corrected for repeated measurements). In the distal segment (FIG. 15), there was little vasoconstriction in saline treated arteries, and the test solution did not significantly alter the response to rotational atherectomy.

  The hemodynamic responses in the saline treated artery and the artery treated with the test solution are summarized in Table 36. When compared to animals treated with saline, animals treated with the test solution sustained substantial hypotension and significant tachycardia during the period of solution infusion. By 15 minutes after completion of the infusion (or 30 minutes after rotational atherectomy), animals treated with the test solution show a partial, but not complete, recovery of blood pressure towards baseline. It was.

6). Summary of research Rotational atherectomy in hypercholesterolemic New Zealand white rabbits causes significant vasospasm in the middle portion of the iliac artery exposed to the rotating bar. This vasospasm is most apparent 15 minutes after the rotational atherectomy procedure and has almost completely disappeared without pharmacological intervention by 30 minutes after the rotational atherectomy.

  2. Under the rotational atherectomy procedure investigated in this protocol, the test solution treatment according to the present invention almost completely inhibited the vasospasm seen after the intermediate iliac artery was exposed to the rotating bar. Yes.

  3. Treatment with a test solution of the present invention given the concentrations of the components used in this protocol causes severe hypotension upon infusion of the solution. A weakening of vasospasm after rotational atherectomy with test solution occurred in the presence of severe hypotension.

  While preferred embodiments of the invention have been illustrated and described, it will be appreciated that various changes to the disclosed solutions and methods may be made in the embodiments of the invention without departing from the spirit and scope of the invention. The For example, according to the disclosure contained herein, alternative pain inhibitors and anti-inflammatory and anticonvulsants and anti-restenosis agents that can enhance or replace the disclosed agents in accordance with the disclosure contained herein. Can be discovered. Accordingly, it is intended that the scope of patents granted be limited only by the definition of the appended claims.

The present invention will now be described in more detail, by way of example, with reference to the accompanying drawings.
FIG. 1 provides a schematic diagram of a general vascular cell showing the molecular target and flow of signaling information that causes contraction, secretion and / or proliferation. The integration of external signals through receptors, ion channels and other membrane proteins is common for platelets, neutrophils, endothelial cells and smooth muscle cells. Representative examples of molecular targets are included for the major group of molecules that are therapeutic targets for drugs contained in the solutions of the present invention. FIG. 2 provides a detailed diagram of the signaling pathway illustrating “crosstalk” between the G protein coupled receptor (GPCR) pathway and the receptor tyrosine kinase (RTK) pathway in vascular smooth muscle cells. Only representative proteins in each pathway are shown to simplify the flow of information. Activation of GPCRs results in increased intracellular calcium and increased protein kinase C (PKC) activity, followed by smooth muscle contraction or convulsions. In addition, “crosstalk” to the RTK signaling pathway results from activation of PYK2 (a newly discovered protein tyrosine kinase) and PTK-X (an unidentified protein tyrosine kinase), which initiates proliferation. It is done. Conversely, activation of RTKs directly initiates proliferation, while “crosstalk” to the GPCR pathway occurs at the level of PKC activity and calcium levels. LGR represents a ligand-dependent receptor and MAPK represents a mitogen activated protein kinase. These interactions define the basis for synergistic interactions between molecular targets that mediate convulsions and restenosis. The GPCR signaling pathway also mediates signaling (FIGS. 3 and 7) that results in pain transmission in other cell types (eg, neurons). FIG. 3 provides a diagram of the G protein coupled receptor (GPCR) pathway. Specific molecular sites of action for several drugs in the preferred arthroscopic solutions of the present invention have been identified. FIG. 4 provides a diagram of the G protein coupled receptor (GPCR) pathway, including signaling proteins involved in “crosstalk” with the growth factor receptor signaling pathway. Specific molecular sites of action for several drugs in the preferred cardiovascular / general vascular solution of the present invention have been identified (see also FIG. 5). FIG. 5 provides a diagram of a growth factor receptor signaling pathway including signaling proteins involved in “crosstalk” with the G protein coupled receptor signaling pathway. Specific molecular sites of action for several drugs in the preferred cardiovascular / general vascular solution of the present invention have been identified (see also FIG. 4). FIG. 6 provides a diagram of the G protein coupled receptor pathway, including signaling proteins involved in “crosstalk” with the growth factor receptor signaling pathway. Specific molecular sites of action for several drugs in preferred urological solutions have been identified. FIG. 7 provides a diagram of the G protein coupled receptor pathway. Specific molecular sites of action for several drugs in the preferred general surgical wound solution of the present invention have been identified. FIG. 8 provides a diagram of the mechanism of action of nitric oxide (NO) donor drugs and NO that cause relaxation of vascular smooth muscle. Physiologically, certain hormones and transmitters can activate certain forms of NO synthase in endothelial cells by elevated intracellular calcium that results in increased synthesis of NO. NO donors can generate NO extracellularly or can be metabolized to NO inside smooth muscle cells. Extracellular NO can diffuse into endothelial cells or can directly enter smooth muscle cells. The main target of NO is soluble guanylate cyclase (GC), which leads to activation of cGMP-dependent protein kinase (PKG) and subsequent excretion of calcium from smooth muscle cells by a membrane pump. NO also hyperpolarizes cells by opening potassium channels that in turn cause closure of voltage-sensitive calcium channels. Thus, a synergistic interaction of calcium channel antagonist, potassium channel opener, and NO donor is evident from the above signaling pathway. FIG. 9, FIG. 10A and FIG. 10B show examples of the effect of injection with histamine antagonist and serotonin antagonist used in the solution of the present invention on vasoconstriction during balloon angioplasty. For the animal studies described in VII, a graph of the ratio of vasoconstriction over time in the control artery, the proximal segment of the subject artery and the distal segment of the subject artery is provided, respectively. FIG. 9, FIG. 10A and FIG. 10B show examples of the effect of injection with histamine antagonist and serotonin antagonist used in the solution of the present invention on vasoconstriction during balloon angioplasty. For the animal studies described in VII, a graph of the ratio of vasoconstriction over time in the control artery, the proximal segment of the subject artery and the distal segment of the subject artery is provided, respectively. FIG. 9, FIG. 10A and FIG. 10B show examples of the effect of injection with histamine antagonist and serotonin antagonist used in the solution of the present invention on vasoconstriction during balloon angioplasty. For the animal studies described in VII, a graph of the ratio of vasoconstriction over time in the control artery, the proximal segment of the subject artery and the distal segment of the subject artery is provided, respectively. FIGS. 11 and 12 provide an illustration of plasma extravasation versus dosage of amitriptyline used in the solutions of the present invention and delivered intravenously and intra-articularly to the knee joint, respectively. In this case, extravasation has been induced by the introduction of 5-hydroxytryptamine in the animal studies described in Example VIII herein. FIGS. 11 and 12 provide diagrams of plasma extravasation versus dosage of amitriptyline used in the solutions of the present invention and delivered intravenously and intra-articularly to the knee joint, respectively. In this case, extravasation has been induced by the introduction of 5-hydroxytryptamine in the animal studies described in Example VIII herein. FIGS. 13, 14 and 15 are treated with physiological saline immediately after rotation atherectomy and at 15 minutes after rotation atherectomy in the animal study of Example XIII described herein. Proximal segment (FIG. 13), intermediate segment (FIG. 14) and distal segment (FIG. 15) of an artery (N = 4) or an artery (N = 7) treated with a solution formulated according to the present invention FIG. 4 provides a mean ± 1 [standard error of mean] mean vasoconstriction (negative value) or vasodilation (positive value) FIGS. 13, 14 and 15 are treated with physiological saline immediately after rotation atherectomy and at 15 minutes after rotation atherectomy in the animal study of Example XIII described herein. Proximal segment (FIG. 13), intermediate segment (FIG. 14) and distal segment (FIG. 15) of an artery (N = 4) or an artery (N = 7) treated with a solution formulated according to the present invention FIG. 2 provides a diagram of mean ± 1 [standard error of mean] of vasoconstriction (negative value) or vasodilation (positive value). FIGS. 13, 14 and 15 are treated with physiological saline immediately after rotation atherectomy and at 15 minutes after rotation atherectomy in the animal study of Example XIII described herein. Proximal segment (FIG. 13), intermediate segment (FIG. 14) and distal segment (FIG. 15) of an artery (N = 4) or an artery (N = 7) treated with a solution formulated according to the present invention FIG. 2 provides a diagram of mean ± 1 [standard error of mean] of vasoconstriction (negative value) or vasodilation (positive value).

Claims (19)

  Use of the composition in manufacturing a medicament for inhibiting tumor cell adhesion, pain and inflammation in a wound during a surgical procedure by applying the composition locally to the surgical site perioperatively Wherein the composition comprises a therapeutically effective amount of at least one tumor cell adhesion inhibitor and at least one pain / inflammatory inhibitor in solution in a liquid carrier, wherein the agent is selected to act on different molecular targets. Wherein the at least one tumor cell anti-adhesive agent is selected to be locally deliverable to the surgical site to provide a therapeutic anti-adhesive effect without metabolic transformation.   Manufacture pharmaceuticals to inhibit tumor cell attachment and transplantation during surgical procedures in the body cavity of patients in need of such treatment by applying the composition locally to the body cavity in the perioperative period Use of the composition in doing so, wherein the composition comprises a therapeutically effective amount of at least one tumor cell adhesion inhibitor and at least one pain / inflammation inhibitor in solution in a liquid carrier, the medicament comprising different molecular targets At least one tumor cell adhesion preventive agent against the body cavity to provide a therapeutic tumor cell adhesion prevention effect and a tumor cell transplantation prevention effect without metabolic transformation Use, selected to be locally deliverable.   Use of the composition in manufacturing a medicament for inhibiting tumor cell adhesion in a wound site during a surgical procedure by applying the composition locally to the surgical site perioperatively, The composition comprises a therapeutically effective amount of a first tumor cell adhesion inhibitor and a second tumor cell adhesion inhibitor in solution in a liquid carrier, wherein the agents are selected to act on different molecular targets, Use, wherein at least one of the agents is selected such that it can be delivered locally to the surgical site to provide a therapeutic anti-adhesion effect without metabolic transformation.   Manufacture pharmaceuticals to inhibit tumor cell attachment and transplantation during surgical procedures in the body cavity of patients in need of such treatment by applying the composition locally to the body cavity in the perioperative period Use of the composition in a method comprising the steps of containing a therapeutically effective amount of a first tumor cell adhesion inhibitor and a second tumor cell adhesion inhibitor in solution in a liquid carrier, wherein the drug comprises different molecules Selected to act on the target, and at least one of the agents is local to the body cavity to provide a therapeutic tumor cell adhesion prevention and tumor cell transplantation prevention effect without metabolic transformation Use, selected to be deliverable.   5. Use according to any one of claims 1 to 4, wherein the perioperative application of the solution comprises application during treatment together with application before or after treatment of the solution.   6. Use according to any one of claims 1 to 5, wherein perioperative application of the solution includes application before, during and after treatment of the solution.   7. Use according to any one of the preceding claims comprising continuously applying the solution to a surgical site or body cavity.   8. Use according to any one of claims 1 to 7, wherein the liquid carrier is a liquid irrigation carrier and the solution is delivered by irrigation of a surgical site or body cavity.   Use according to any one of claims 1 to 7, wherein the liquid carrier comprises a sustained release carrier.   8. Use according to any one of claims 1 to 7, wherein the liquid carrier comprises a gel.   Use according to any one of claims 1 to 10, wherein the anti-adhesive agent is selected from the group consisting of a CD44 receptor antagonist, an integrin receptor antagonist and a selectin receptor antagonist.   11. Use according to any one of claims 1 to 10, wherein at least one of the anti-adhesive agents comprises an inhibitor of matrix metalloproteinase.   Use according to claim 1 or 2, further comprising a plurality of pain / inflammatory inhibitors.   Use according to claim 3 or 4, further comprising at least one pain / inflammatory inhibitor. The pain / inflammation inhibitor is a serotonin receptor antagonist, serotonin receptor agonist, histamine receptor antagonist, bradykinin receptor antagonist, kallikrein inhibitor, tachykinin receptor antagonist (including neurokinin ₁ receptor subtype antagonist and neurokinin ₂ receptor subtype antagonist), calcitonin Gene-related peptide receptor antagonist, interleukin receptor antagonist, phospholipase inhibitor (including PLA ₂ isoform inhibitor and PLC _γ isoform inhibitor), cyclooxygenase inhibitor, lipooxygenase inhibitor, prostanoid receptor antagonist ( Including eicosanoid EP-1 receptor subtype antagonists and eicosanoid EP-4 receptor subtype antagonists and thromboxane receptor subtype antagonists), leukotriene receptor antagonists (including leukotriene B ₄ receptor subtype antagonists and leukotriene D ₄ receptor subtype antagonists) Opioid receptor agonists (including μ-opioid receptor subtype agonists, δ-opioid receptor subtype agonists and κ-opioid receptor subtype agonists), purine receptor agonists and purine receptor antagonists (P _2Y receptor agonists and P _2X receptor antagonists) Including) 15. Use according to any one of claims 1, 2, 13 or 14, selected from the group consisting of ATP sensitive potassium channel openers. The pain / inflammation inhibitor is a serotonin receptor antagonist, serotonin receptor agonist, histamine receptor antagonist, bradykinin receptor antagonist, kallikrein inhibitor, tachykinin receptor antagonist (including neurokinin ₁ receptor subtype antagonist and neurokinin ₂ receptor subtype antagonist), calcitonin Gene-related peptide receptor antagonists, phospholipase inhibitors (including PLA ₂ isoform inhibitors and PLC _γ isoform inhibitors), lipooxygenase inhibitors, prostanoid receptor antagonists (eicosanoid EP-1 receptor subtype antagonists and eicosanoids) EP-4 containing receptor subtype antagonists and thromboxane receptor subtype antagonists), including leukotriene receptor antagonists (leukotriene B ₄ receptor subtype antagonists and leukotriene D ₄ receptor subtype antagonists), opioid receptor agonists (.mu.-opioid receptor sub Type agonists, including δ-opioid receptor subtype agonists and κ-opioid receptor subtype agonists), purine receptor agonists and purine receptor antagonists (including P _2Y receptor agonists and P _2X receptor antagonists), and ATP-sensitive potassium channel openers Claims 1, 2 selected from the group consisting of , 13 or 14.   Each of the agents in the applied solution is sufficient to provide a predetermined level of inhibitory effect at the surgical site when applied topically, and the same level at the surgical site when applied systemically 17. Use according to any one of claims 1 to 16, comprising a concentration or dosage that results in a plasma concentration that is less than that required to provide an inhibitory effect.   17. Use according to any one of claims 1 to 16, wherein each of the drugs in the solution is locally delivered at a concentration of less than 100,000 nanomolar.   17. Use according to any one of claims 1 to 16, wherein each of the agents in the solution is locally delivered at a concentration of less than 10,000 nanomolar.

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