CN1694717A - Irrigation solution and method for inhibition of tumor cell adhesion, pain and inflammation - Google Patents

Abstract

A method and solution for perioperatively inhibiting tumor cell adhesion and a variety of pain and inflammation processes at wounds from general surgical procedures including oral/dental procedures. The solution preferably includes at least one anti-tumor cell adhesion agent and multiple pain and inflammation inhibitory agents at dilute concentration in a physiologic carrier, such as saline or lactated Ringer's solution. The solution is applied by continuous irrigation of a wound during a surgical procedure for preemptive inhibition of pain and while avoiding undesirable side effects associated with oral, intramuscular, subcutaneous or intravenous application of larger doses of the agents. One preferred solution to inhibit tumor cell adhesion, pain and inflammation includes at least one anti-tumor cell adhesion agent, a serotonin2 antagonist, a serotonin3 antagonist, a histamine antagonist, a serotonin agonist, a cyclooxygenase inhibitor, a neurokininl antagonist, a neurokinin2 antagonist, a purinoceptor antagonist, an ATP-sensitive potassium channel opener, a calcium channel antagonist, a bradykininl antagonist, a bradykinin2 antagonist and a mu -opioid agonist.

Description

Irrigating solutions and methods for inhibiting tumor cell adhesion, pain and inflammation

field of invention

The present invention relates to the use of perioperative local delivery of combinations of therapeutic agents to inhibit tumor cell adhesion and/or invasion and/or local tumor cell metastasis while simultaneously treating pain and/or inflammation and/or smooth muscle spasm and /or restenosis method.

Background of the invention

Endoscopy is a surgical procedure in which a camera connected to a distal light source and video monitor is inserted into a body cavity (e.g., joint, peritoneal cavity, bladder, etc.) through small incisions in the overlying skin and body wall. , thorax, etc.). With similar incisions, surgical instruments can be placed into body cavities and their application guided by arthroscopic imaging. As endoscopists improve, more and more procedures that once required "open" surgical techniques can now be performed endoscopically. These procedures include, for example, appendectomy, cholecystectomy, and cardiac surgery. The widening range of surgical indications and the development of small-diameter endoscopes have made pediatric endoscopy routine.

Throughout each endoscopic procedure, a physiological irrigation solution (eg, saline or lactated Ringer's solution) is used to continuously irrigate the joint, dilate the body cavity, and remove surgical debris to obtain clear contrast. US Patent No. 4,504,493 to Marshall discloses an equimolar solution of glycerol in water as a nonconductive 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 in the treatment of burns and any surgical wounds. In each case, physiological fluids were used to irrigate the wound or body cavity or passage. Conventional physiological flushes neither inhibit tumor cell adhesion and/or invasion and/or local tumor cell metastasis, nor produce analgesic, anti-inflammatory, antispasmodic and antirestenotic effects.

Cell adhesion molecules play an important role in cell-cell interactions and interactions between cells and extracellular matrix components. Although these adhesion molecules are required for a variety of biological processes and regulate intracellular signaling events, these molecules are particularly important in the earliest stages of tumor metastasis when tumor cell adhesion and/or invasion are required. Specific cell adhesion molecules and proteases have been implicated in the attachment and engraftment of dissociated tumor cells that occur in a wide variety of human malignancies, including breast, prostate, liver, ovarian and Metastatic period of bladder cancer. The various adhesion molecules that mediate adhesive interactions consist of cell surface receptor-ligand pairs. The cell surface receptors constitute a group of transmembrane proteins that can be classified as members of related biological families or superfamilies based on their homologous structures and shared functional features. Adhesion of cancer molecules at secondary sites is known to be regulated by several adhesion protein families including CD44 proteoglycans, integrins and selectins. The main function of these receptors is to mediate the binding of cells to specific structural proteins, including the extracellular matrix (ECM), and to recognize membrane-bound ligands that can mediate cell-cell adhesion. Proteases, such as metalloproteases (MMPs) and their natural inhibitors, tissue inhibitors of metalloproteinases (TIMPs), also promote cell adhesion and invasion.

Surgical trauma has been found to increase the frequency of tumor implantation at the site of surgical injury and wound healing. One consequence of surgical trauma to malignant tissue is the local spread of free tumor cells located at the surgical site. For example, patients with pancreatic cancer who undergo surgery often present with peritoneal spread and liver metastases. Adhesion molecules and receptors that mediate the attachment and implantation of dissociated tumor cells to surgical and wound healing sites are often present on a variety of other normal cell types. Surgical trauma also stimulates and induces the production and release of proteases by tumor cells, inflammatory cells, and extracellular matrix components. Therefore, a pharmacological approach aimed at interfering with the function of cell adhesion molecules to reduce tumor cell adhesion and inhibit protease production and activation is to deliver therapeutic agents only to tissues at risk of local tumor metastasis. Since the process of free tumor cells attaching to and invading the extracellular matrix or other cells during surgery is a dynamic process involving specific interactions that are temporally related to surgical trauma, the optimal time for drug intervention is surgery hour. Discovery of the role of multiple adhesion proteins, adhesion receptors and proteases in the extent of cancer metastasis leads to pharmaceutical compositions that block tumor cell attachment to extracellular matrix proteins and/or inhibit tumor cell invasion during surgical procedures developed.

Alleviating pain and suffering in postoperative patients is an area of particular interest in clinical medicine, especially as the number of outpatient procedures increases each year. The most widely used agents, cyclooxygenase inhibitors (eg ibuprofen) and opioids (eg morphine, fentanyl) have significant side effects including gastrointestinal irritation/bleeding and respiratory depression. The high incidence of opioid-related nausea and vomiting is a particularly unresolved problem in the postoperative period. Therapeutic agents aimed at treating postoperative pain while avoiding adverse side effects are not easy to develop because the molecular targets of these agents are widely distributed throughout the body and mediate various physiological effects. Although the clinical need for inhibition of pain and inflammation, as well as vasospasm, smooth muscle spasm, and restenosis is significant, methods to deliver effective doses of inhibitors of pain, inflammation, spasm, and restenosis while minimizing adverse systemic side effects Has not yet been developed. For example, conventional (ie, intravenous, oral, subcutaneous, or intramuscular) methods of administering therapeutic doses of opiates are often associated with significant adverse side effects, including severe respiratory depression, mood changes, mental clouding, extreme nausea, and vomiting.

Previous studies have demonstrated that endogenous agents such as serotonin (serotonin, sometimes referred to herein as "5-HT"), bradykinin, and histamine can cause pain and inflammation. "Serotonin-Bradykinin Potentiation in the Pain Receptors in Man (Serotonin-Bradykinin Potentiation in Human Pain Receptors)" by Sicuteri, F. et al. in Life Sci.4:309-316 (1965); Rosenthal, S.R., "Histamine as the Chemical Mediator for Cutaneous Pain" in J.Invest.Dermat.69:98-105 (1977); Richardson, B.P. et al. in Nature 316:126- "Identification of Serotonin M-Receptor Subtypes and their Specific Blockade by a New Class of Drugs" in 131(1985); Whalley , E.T. et al. in Naunyn-SchmiedebArch.Pharmacol, 36:652-57 (1987) in "The Effect of KininAgonists and Antagonists (kinin agonists and antagonists)"; Lang, E. et al. in J. "Chemo-Sensitivity of Fine Afferents from Rat Skin In Vitro" in Neurophysiol. 63:887-901 (1990).

For example, Richardson et al. (1985) demonstrated that 5-HT-induced pain applied to human blister substrates (new skin) could be inhibited by 5- _HT3 receptor antagonists. Similarly, pain induced by peripherally administered bradykinin can be blocked by bradykinin receptor antagonists. Sicuterl et al., 1965; Whalley et al., 1987; Dray, A. et al. "Bradykinin and Inflammatory Pain" in Trends Neurosci. 16:99-104 (1993). Vasodilation, pruritus, and pain induced by peripherally applied histamine can be inhibited by histamine receptor antagonists. Rosenthal, 1977; Douglas, WW "Histamine" in The Pharmacological Basis of Therapeutics (1985), pp. 605-638, published by MacMillan Publishing Company, New York, edited by Goodman, LS et al. and 5-Hydroxytryptamine (Serotonin) and their Antagonists (histamine and 5-hydroxytryptamine (serotonin) and their antagonists)"; Rumore, MM et al. "Analgesic Effects in LifeSci.36:403-416 (1985) ofAntihistaminics (Analgesic effect of antihistamines)". The combined application of the above three agonists has been shown to have a synergistic analgesic effect, producing a persistent and intense pain signal. Sicuteri et al., 1965; Richardson et al., 1985; Kessler, W., et al. "Excitation of Cutaneous Afferent Nerve Endings In Vitro by a Combination of Inflammatory Mediators and Conditioning Effect of Substance P (stimulatory effect of inflammatory mediator combination on in vitro skin afferent nerve endings and regulation of substance P)".

In the body, 5-HT is located in platelets and central neurons, histamine is present in mast cells, and bradykinin is produced from larger precursor molecules during tissue trauma, pH changes, and temperature changes. Since 5-HT can be released in large quantities by platelets at the site of tissue injury, the resulting plasma level is 20 times higher than the resting level (Ashton, J.H. et al. in "Serotonin as a Mediator of Cyclic FlowVariations in Circulation73:572-578 (1986) Stenosed Canine Coronary Arteries (Serotonin is a Mediator of Circulatory Flow Changes in Canine Constricted Coronary Arteries)"), therefore, endogenous 5-HT may play a role in producing postoperative pain, hyperalgesia, and inflammation. In fact, activated platelets have been shown to stimulate peripheral nociceptors in vitro. Ringkamp, M. et al. "Activated Human Platelets in Plasma Excite Nociceptors in Rat Skin, In Vitro" in Neurosci. Lett. 170:103-106 (1994) (Activated human platelets in plasma stimulated lesions in rat skin in vitro sex receptors)". Likewise, histamine and bradykinin are released into tissues during trauma. Kimura, E., et al. "Changes in Bradykinin Level in Coronary Sinus Blood After the Experimental Occlusion of a Coronary Artery" in Am Heart J.85:635-647 (1973) peptide levels)”; Douglas, 1985; Dray et al. (1993).

In addition, prostaglandins are also known to cause pain and inflammation. Cyclooxygenase inhibitors, such as ibuprofen, are commonly used in nonsurgical and postoperative settings to block prostaglandin production, thereby reducing prostaglandin-mediated pain and inflammation. See Flower, R.J., et al., "Analgesic- Antipyretics and Anti-Inflammatory Agents; Drugs Employed in the Treatment of Gout (analgesic-antipyretic drugs and anti-inflammatory drugs; drugs used in the treatment of gout)". Cyclooxygenase inhibitors, when used routinely, have been associated with certain adverse systemic side effects. For example, indomethacine or ketorolac are well known to have adverse gastrointestinal and renal side effects.

As mentioned above, 5-HT, histamine, bradykinin and prostaglandins cause pain and inflammation. Over the past 20 years, a variety of receptors through which these substances mediate their own effects on peripheral tissues have been known and/or discussed. Most studies have been performed in rats or other animal models. However, there are differences in pharmacology and receptor sequences between human and animal species. No studies have conclusively established the importance of 5-HT, bradykinin, or histamine in producing postoperative pain in humans.

Furthermore, antagonists of the aforementioned mediators have not been used so far in the treatment of postoperative pain. A class of drugs known as serotonin and norepinephrine uptake antagonists and including amitriptyline has been used orally to successfully relieve chronic pain conditions. But the mechanisms underlying chronic versus acute pain conditions are thought to be distinct. In fact, two studies of perioperative use of amitriptyline in acute pain settings have shown no pain relief from amitriptyline. "Desipramine Enhances Opiate Postoperative Ahalgesia" by Levine, J.D. et al. in Pain 27:45-49 (1986); Kerrick, J.M. et al. in Pain 52:325 "Low-Dose Amitriptyline as an Adjunct to Opioids for Postoperative Orthopedic Pain: a Placebo-Controlled TrialPeriod" in -30 (1993) ". In both studies, the drug was given orally. A second study indicated that oral amitriptyline actually produced a lower overall feeling of goodness in postoperative patients, possibly due to the drug's affinity for various amine receptors in the brain.

In addition to blocking the uptake of 5-HT and norepinephrine, amitriptyline is also a potent 5-HT receptor antagonist. Thus, the lack of efficacy in reducing postoperative pain in the above studies seems to contradict the notion of a role for endogenous 5-HT in acute pain. There are a number of reasons for the lack of acute pain reduction found with amitriptyline in the two studies described above. (1) The first study (Levine et al., 1986) administered amitriptyline one week before surgery until the night before surgery, while the second study (Kerrick et al., 1993) administered amitriptyline only after surgery For Lin. Therefore, the tissue at the surgical site was not present with amitriptyline during the actual period of tissue injury, which is also considered to be the period of 5-HT release. (2) It is known that amitriptyline is extensively metabolized by the liver. Orally administered, amitriptyline may not be present in high enough concentrations for long enough in surgical site tissues to inhibit the activity of postoperatively released 5-HT in the second study above. (3) Due to the existence of multiple inflammatory mediators, and studies have confirmed that there is a synergistic effect between these inflammatory mediators, blocking only one substance (5-HT) is not enough to inhibit inflammation in response to tissue damage.

Several studies have demonstrated that histamine ₁ (H ₁ ) receptor antagonists at very high concentrations (1%-3% solution - ie 10-30 mg/ml) act as local anesthetics for surgical procedures. This anesthetic effect is not thought to be mediated through _H1 receptors, but is due to a non-specific interaction of the antagonist with neuronal membrane sodium channels (similar to the effect of lidocaine). Topical application of histamine receptor antagonists has not currently been applied in the perioperative setting, just as certain side effects, such as sedation, are associated with the aforementioned high "narcotic" concentrations of histamine receptor antagonists.

Summary of the invention

One aspect of the present invention is directed to the prevention and treatment of tumor cell adhesion and/or invasion and/or local metastasis in patients undergoing surgical procedures. The present invention describes methods and pharmaceutical compositions based on combinations of several agents that inhibit the attachment and/or invasion and/or local metastasis of tumor cells during surgical procedures and at the same time inhibit the pain associated with such procedures and/or inflammation and/or smooth muscle spasm and/or restenosis. According to one aspect of the present invention, there is provided a method of reducing or preventing tumor cell adhesion and/or invasion and/or local metastasis, the method comprising directly applying a composition to the surgical site, the composition in a specific pharmaceutical The pharmaceutically effective carrier includes a combination of two or more metabolically active agents, at least one agent in the combination is an anti-tumor adhesion or anti-invasion or anti-local metastasis agent, and the specific pharmaceutically effective carrier is suitable for flushing liquid delivery. Agents with metabolic activity include, but are not limited to, all compounds that can act directly or indirectly to regulate or change the biological, biochemical or biophysical state of cells, including changes in cell membrane potential, ligand-receptor binding, cell Agents of the enzymatic activity or expression level of receptors, as well as enzyme inhibitors or activators, inhibitors of protein-protein interactions, RNA-protein interactions or DNA-protein interactions. For example, functional receptor antagonists may include monoclonal antibodies that competitively inhibit the receptor binding site, soluble receptors that reduce the concentration of free ligand required to activate the receptor, inhibitors of receptor signaling pathways, intracellular or Activators and inhibitors of extracellular enzymes, and agents that modulate transcription factor binding to DNA.

The present invention specifically provides a method of treating tumor cell adhesion and/or invasion and/or local metastasis by local and perioperative delivery of a combination of several agents for maximum therapeutic effect. The combined application of these agents overcomes the limitations of existing therapeutic methods relying on the use of a single substance to block the multifactorial tumor cell adhesion and invasion process, which is regulated by many pro-inflammatory cytokines and receptors. The regulation of these pro-inflammatory cytokines and receptors is related to the inflammatory response induced by surgical procedures. It has been reported that pro-inflammatory cytokines stimulate the expression of adhesion receptors and metalloproteinases present on both tumor and normal cells, thereby enhancing their adhesion and invasion properties. The combination of an anti-adhesion agent and an anti-inflammatory agent, for example, can result in an additive or synergistic effect of the pharmaceutical composition.

The present invention employs a method of combining several agents that act simultaneously on specific molecular targets that are associated with tumor cell adhesion and/or invasion and/or metastasis, and delivering these agents to the surgical site through surgical procedures , and/or inflammation and/or pain and/or smooth muscle spasm and/or restenosis. The combination of these agents can preemptively inhibit tumor cell adhesion and/or invasion throughout the surgical procedure, and can also preemptively inhibit pain and/or inflammation and/or smooth muscle spasm and/or restenosis. Inhibition of a single adhesion receptor-ligand interaction by systemic delivery is likely to elicit adverse effects because these molecules also function to deliver signals to normal cells. For example, CD44 receptor binding has a physiological role in the immune response of normal T cells and other hematopoietic cells, therefore, inhibition of CD44 binding may have adverse effects on the immune system.

The present invention specifically provides pharmaceutical compositions of several metabolically active agents based on the combination of at least two agents acting simultaneously on different molecular targets. At least one of the agents is an anti-tumor cell adhesion or anti-invasive or anti-local metastasis agent that directly reduces or prevents the attachment of tumor cells to the extracellular matrix or other cells (both normal and tumor cells). Agents suitable for inclusion in the above-mentioned pharmaceutical compositions should further have the following characteristics, that is, they need to have pharmacological selectivity and specificity, so as to limit the interaction of the agent with a single family (or class) of receptors or a single enzyme family (such as CD44 , integrins, protein tyrosine kinases, MMPs and MAP kinases). Particularly suitable agents interact specifically with one or more receptor subtypes (or isotypes) and exhibit specificity for that receptor family. Each suitable agent is linked to its molecular target by a specific molecular mechanism, one feature of which is a specific stoichiometry of the ligand-receptor (or inhibitor-enzyme) complex (typically 1 : 1), another characteristic lies in its equilibrium binding or kinetic constants.

At least one other agent belongs to the class of receptor antagonists, enzyme inhibitors or cytokines having analgesic, anti-inflammatory, antispasmodic and/or anti-restenotic activity. The optimal drug combination includes at least one agent selected from the class of anti-tumor cell adhesion and/or anti-invasion agents, such anti-tumor cell adhesion and/or anti-invasion agents include molecular targets that can inhibit and reduce cell adhesion receptors Receptor antagonists (eg, integrin receptor antagonists, CD44 receptor antagonists, and selectin receptor antagonists) or protease inhibitors of the activity or expression of

In addition, other suitable agents included in the optimal drug combination have the function of anti-tumor cell adhesion agents and inhibit cell signal transduction pathways, which are activated by cell adhesion receptors. Molecular targets comprising these cellular signaling pathways include membrane-associated, intracellular enzymes that convert and integrate signals generated by activated cell surface adhesion receptors. For example, such agents include inhibitors that inhibit enzymes belonging to specific families: protein tyrosine kinase, protein kinase C, and mitogen-activated protein kinase (MAPK). These enzyme inhibitors may interact with specific one or more members of the aforementioned kinase family through a specific mechanism characterized by a specific stoichiometric relationship (typically 1:1) of the enzyme-inhibitor complex and the equilibrium inhibition constant.

Despite extensive experimental studies, no effective therapy has previously been developed to prevent tumor cell adhesion and/or invasion during surgical procedures. It is an object of the present invention to provide a method of preemptively treating tumor cell adhesion and/or invasion and/or local metastasis that occurs during surgical procedures by administering an irrigation fluid that can continuously deliver a therapeutic agent. Existing delivery methods fail to provide a means to initially deliver agents directly to the surgical site and maintain drug levels at or above therapeutically effective concentrations throughout the surgical procedure. Since large volumes of irrigating fluid are used during certain surgical procedures (eg, laparoscopic surgery), having the irrigating fluid contain a specific agent provides a means of avoiding dilution of the agent and maintaining a therapeutic concentration level. By delivering the therapeutic agent locally in the irrigating fluid directly to the surgical site, the systemic delivery of therapeutic peptides, proteins and small molecules as potential anti-tumor cell adhesion/invasion and anti-metastatic agents can be reduced or eliminated challenges, including toxicity, drug stability, and poor pharmacokinetic profiles.

Multiple drug combinations can be delivered by topical and perioperative irrigation (ie, delivered intraoperatively only or not only intraoperatively, but also preoperatively and/or postoperatively). The invention also provides anti-tumor adhesion and/or anti-invasive and/or local metastatic agents that can be delivered in combination with one or more analgesic and/or anti-inflammatory and/or antispasmodic and/or anti-restenotic agents agent.

A significant advantage of the present invention resides in the rapid onset of pharmacological action. Direct, local delivery of anti-tumor adhesion agents initiated at the beginning of the surgical procedure and continued during the surgical procedure (i.e., perioperative period) may inhibit one of the earliest stages of metastasis, the initial adhesion process. Direct and constant delivery of therapeutically effective concentrations of anti-adhesion and/or invasion agents is believed to pre-empt the initial attachment of tumor cells to the extracellular matrix and other cells and render tumor cells unable to subsequently invade into tissues. The study found that the highest incidence of tumor implantation occurred when tumor cells were injected immediately after surgery, suggesting that this period is a critical time for therapeutic inhibition of the adhesion process.

The unique advantages of the delivery method and pharmaceutical composition of the present invention include: 1) The combined drug therapy targets the multifactorial causes of tumor cell adhesion/invasion/local metastasis, inflammation, pain, smooth muscle spasm and restenosis during surgical operations, 2) Local delivery of the drug combination can instantly obtain therapeutic concentrations of anti-tumor adhesion agents at the surgical site, and 3) continuous delivery of therapeutic agents in the flushing fluid can allow the surgical site to obtain therapeutically effective concentration ranges during surgical operations. Constant drug concentration, 4) local delivery reduces total drug dose and application frequency compared to systemic delivery, and 5) direct, local delivery to the surgical site so that it cannot be delivered systemically, but is drug active Novel agents such as certain peptides and antibodies can be utilized.

The present invention further provides a solution comprising a mixture of low concentrations of agents in a physiological electrolyte carrier fluid for targeted local inhibition of mediators of pain, inflammation, spasm and restenosis. The present invention also provides a method of perioperatively delivering an irrigant containing the above agents directly to the surgical site, wherein the irrigant acts locally at the receptor and enzyme level to preemptively inhibit pain, Inflammation, spasm and restenosis. By the local perioperative delivery method of the present invention, the desired therapeutic effect can be achieved with lower doses of agent than would be necessary for other delivery methods (ie, intravenous, intramuscular, subcutaneous, and oral). Analgesic/anti-inflammatory agents in the above solutions include agents selected from the following classes of receptor antagonists and agonists and enzyme activators and inhibitors, each of which inhibits pain and inflammation through different molecular mechanisms of action: (1) Serotonin receptor antagonists; (2) serotonin receptor agonists; (3) histamine receptor antagonists; (4) bradykinin receptor antagonists; (5) kallikrein inhibitors; (6) ) tachykinin receptor antagonists, including neurokinin ₁ and neurokinin ₂ receptor subtype antagonists; (7) calcitonin gene-related peptide (CGRP) receptor antagonists; (8) interleukin receptor antagonists (9) Inhibitors that inhibit enzymes that function in the synthetic pathway of arachidonic acid metabolites, including (a) phospholipase inhibitors, including PLA ₂ isoform inhibitors and PLC _γ isoform inhibitors , (b) cyclooxygenase inhibitors, and (c) lipoxygenase inhibitors; (10) prostanoid hormone receptor antagonists, including eicosanoid EP-1 and EP-4 receptor subclasses (11) Leukotriene receptor antagonists, including leukotriene _B4 receptor subtype antagonists and leukotriene _D4 receptor subtype antagonists; ( 12) Opioid receptor agonists, including μ-opioid, δ-opioid, and κ-opioid receptor subtype agonists; (13) Purinergic receptor agonists and antagonists, including P _2X receptor antagonists and _P2Y receptor agonists; and (14) adenosine triphosphate (ATP)-sensitive potassium channel openers. Each of the aforementioned agents acts as an anti-inflammatory and/or antinociceptive, ie, analgesic or analgesic agent. Suitable substances can be selected from these classes of compounds for a particular application.

Several preferred embodiments of the solutions of the invention also include antispasmodic agents for specific applications. For example, solutions for vascular procedures may contain antispasmodics alone or in combination with analgesic/anti-inflammatory agents to inhibit vasospasm, and solutions for urological procedures may also contain antispasmodics to inhibit Spasms of the urinary tract and bladder walls. For these applications, antispasmodic agents can be included in the solution required for the procedure. For example, the solution may contain analgesic/anti-inflammatory agents that also act as antispasmodic agents. Suitable anti-inflammatory/analgesic agents that may also act as antispasmodics include serotonin receptor antagonists, tachykinin receptor antagonists and ATP-sensitive potassium channel openers. Other agents that may be used in the above solutions include calcium channel antagonists, endothelin receptor antagonists and nitric oxide donors (enzyme activators), especially because of their antispasmodic properties.

A particularly preferred embodiment of the solution of the present invention suitable for use in cardiovascular and general vascular procedures includes an anti-restenotic agent, most preferably in combination with an antispasmodic agent. Suitable anti-restenotic agents include: (1) antiplatelet agents, including (a) thrombin inhibitors and receptor antagonists, (b) adenosine diphosphate (ADP) receptor antagonists (also known as purine receptor ₁ receptor antagonists), (c) thromboxane inhibitors and receptor antagonists, and (d) platelet membrane glycoprotein receptor antagonists; (2) inhibitors of cell adhesion molecules, including (a) selected Protein inhibitors and (b) integrin inhibitors; (3) antichemotactic agents; (4) interleukin receptor antagonists (also act as analgesic/anti-inflammatory agents); and (5) intracellular signaling inhibitors , including (a) protein kinase C (PKC) inhibitors and protein tyrosine kinase inhibitors, (b) regulators of intracellular protein tyrosine phosphatases; (c) src homology region ₂ (SH2) domain inhibitors, and (d) calcium channel antagonists. These agents help prevent restenosis of arteries undergoing angioplasty, atherectomy, or other cardiovascular or general vascular therapeutic or diagnostic procedures.

The present invention also provides a method of manufacturing a specific drug compounded as a dilute irrigation solution for continuous irrigation of surgical sites or wounds during surgical operations. The method entails dissolving at least one tumor cell adhesion, invasion and/or metastasis inhibitor and preferably multiple analgesic/anti-inflammatory agents in a physiological electrolyte carrier fluid, and for some applications, an antispasmodic agent and/or anti-restenosis agents, the concentration of each agent included is preferably not greater than 100,000 nanomolar, more preferably not greater than 10,000 nanomolar.

The method of the present invention may combine at least one inhibitor of tumor cell adhesion, invasion and/or metastasis with, preferably, dilutions of various receptor antagonists and agonists, and enzyme inhibitors and activators during therapeutic or diagnostic procedures Delivered directly to trauma or surgical sites to inhibit pain, inflammation, spasticity and restenosis. Since the active ingredient in the solution is applied continuously and directly locally to the surgical tissue, the drug can be administered at extremely low doses compared to the doses required to achieve therapeutic efficacy when the same drug is delivered orally, intramuscularly, subcutaneously or intravenously. Dosage is used efficiently. The term "topical" as used herein encompasses the application of a drug in and around a wound or other surgical site, but excludes oral, subcutaneous, intravenous and intramuscular administration. The term "continuous" as used herein encompasses uninterrupted application, application repeated at frequent intervals (e.g., repeated intravascular boluses at frequent intervals during a procedure), and application that can still be considered continuous except for several brief stops, such as Circumstances when other drugs or agents are introduced or equipment is manipulated to maintain a substantially constant pre-set drug concentration locally at the wound or surgical site.

The low-dose application of the agent has a three-fold advantage. The most important advantage is the absence of systemic side effects that usually limit the efficacy of these agents. Furthermore, in the solutions of the invention, the agent selected for a particular application is highly specific for the mediator of its action. This specificity was maintained by the low dose employed. Finally, the cost of the aforementioned active agents required per surgical procedure is low.

The advantages of local application of the above-mentioned agents through cavity irrigation or other liquid applications are as follows: (1) local application ensures a known concentration at the target site without considering the variability in metabolism, blood flow, etc. of different patients; 2) Due to the direct mode of delivery, therapeutic concentrations are obtained instantaneously, thereby improving dosing control; and (3) Direct topical application of the active agent to the wound or surgical site also substantially reduces the risk of the agent passing through extracellular processes. Degradation, such as primary and secondary metabolism, also occurs when the agent is administered orally, intravenously, subcutaneously or intramuscularly. This advantage applies especially to active agents that are peptides themselves and are rapidly metabolized. Thus, topical administration enables the availability of compounds or agents that would not otherwise be available for therapy. For example, certain agents of the following classes are peptides: bradykinin receptor antagonists; tachykinin receptor antagonists; opioid receptor agonists; CGRP receptor antagonists; and interleukin receptor antagonists . Local and continuous delivery of agents to trauma or surgical sites can minimize the degree of drug degradation or metabolism, and at the same time, it can also continuously replenish some of the agents that may be degraded, so as to ensure that a specific local therapeutic concentration is maintained throughout the surgical operation, This concentration is sufficient to maintain receptor occupancy.

According to the present invention, perioperative topical administration of the solutions of the present invention throughout the surgical procedure produces preemptive analgesic, anti-inflammatory, antispasmodic or antirestenotic effects. The term "perioperative" as used herein encompasses intraoperative use, preoperative and intraoperative use, intraoperative and postoperative use, and preoperative, intraoperative, and postoperative use. For optimal anti-inflammatory, analgesic (for some applications), antispasmodic (for some applications) and anti-restenotic (for some applications) effects, the solutions of the present invention are most preferably used in before, during and after surgery. By occupying target receptors or inactivating or activating anchored enzymes before significant surgical trauma begins locally, the agents in the solutions of the invention can modulate specific pathways to preemptively inhibit anchored pathological processes. According to the present invention, if the inflammatory mediators and processes are preemptively inhibited before they cause tissue damage, the effect is more robust than if the inhibition has already started to cause damage.

Inhibiting more than one mediator of tumor cell adhesion, invasion and/or metastasis, inflammation, spasm or restenosis by applying the solution containing multiple agents of the present invention, helps to significantly reduce cell adhesion, invasion and/or metastasis, inflammation, The degree of pain and spasm, and theoretically reduce the incidence of restenosis. The flushing solution of the present invention includes a combination of multiple drugs, and each solution can act on multiple receptors or enzymes. Thus, these agents are simultaneously effective against multiple pathological processes, including tumor cell adhesion, invasion and/or metastasis, pain and inflammation, vasospasm, smooth muscle spasm and restenosis. The actions of these agents are believed to be synergistic, wherein the combination of multiple receptor antagonists and inhibitory agonists of the invention increases potency disproportionately compared to the potency of a single agent. The synergistic effect of several agents of the present invention is discussed below by way of examples given while describing in detail several of these agents.

In addition to arthroscopic procedures, the solutions of the present invention may also be applied topically to any human body cavity or passage, surgical wounds, wounds (such as burns), or in any surgical/interventional procedure where irrigation may be performed. These procedures include, but are not limited to, urological procedures, cardiovascular and general vascular diagnostic and therapeutic procedures, endoscopic procedures, and oral, dental and periodontal procedures. The term "wound" as used hereinafter includes surgical trauma, surgical/intervention sites, wounds and burns unless otherwise indicated.

Perioperative application of the solution of the present invention should provide a clinically significant reduction in pain and inflammation at the surgical site compared to currently applied irrigants, thereby reducing the patient's postoperative need for analgesia (i.e., opiates) and appropriately enabling the patient to Mobilize the surgical site earlier. The use of the solution of the invention places no additional demands on the surgeon and operating room personnel compared to conventional irrigating solutions.

Brief description of the drawings

The invention will be described more particularly by way of example with reference to the accompanying drawings, in which:

Figure 1 provides a schematic diagram of common vascular cells showing the molecular targets and signaling information flow leading to contraction, secretion and/or proliferation. Platelets, neutrophils, endothelial cells, and smooth muscle cells all integrate exogenous signals through receptors, ion channels, and other membrane proteins. The figure includes representative examples of molecular targets of the major molecular populations that are therapeutically targeted by drugs contained in the solutions of the present invention.

Figure 2 provides a detailed signaling pathway diagram illustrating the "crosstalk" between the G-protein coupled receptor (GPCR) pathway and the receptor tyrosine kinase (RTK) pathway in vascular smooth muscle cells. Only representative proteins are labeled for each pathway to simplify information flow. Activation of GPCRs results in increased intracellular calcium and increased protein kinase C (PKC) activity, resulting in smooth muscle contraction or spasm. Furthermore, "crosstalk" to the RTK signaling pathway occurs through the activation of PYK2 (a newly discovered protein tyrosine kinase) and PTK-X (an undefined protein tyrosine kinase), triggering the proliferation. In contrast, activation of RTKs directly initiates proliferation, while "crosstalk" to the GPCR pathway occurs at the level of PKC activity and calcium levels. LGR refers to ligand-gated receptor and MAPK refers to mitogen-activated protein kinase. These interactions define the basis for a synergistic interplay between molecular targets mediating spasticity and restenosis. The GPCR signaling pathway also mediates signal transduction leading to pain transmission in other cell types such as neurons (Figures 3 and 7).

Figure 3 provides a schematic diagram of the G-protein coupled receptor (GPCR) pathway. The specific molecular sites of action of certain drugs in the solution used in the preferred arthroscopic examination of the present invention are marked in the figure.

Figure 4 provides a schematic diagram of the G-protein coupled receptor (GPCR) pathway including the signaling proteins responsible for "cross-talking" with the growth factor receptor signaling pathway. The specific molecular sites of action of certain drugs in the preferred cardiovascular and general vascular solutions of the present invention are indicated in the figure. (See also Figure 5)

Figure 5 provides a schematic diagram of the growth factor receptor signaling pathway including the signaling proteins responsible for "cross-talking" with the G-protein coupled receptor signaling pathway. The specific molecular sites of action of certain drugs in the preferred cardiovascular and general vascular solutions of the present invention are indicated in the figure. (See also Figure 4)

Figure 6 provides a schematic diagram of the G-protein coupled receptor pathway including the signaling proteins responsible for "cross-talking" with the growth factor receptor signaling pathway. The specific molecular sites of action of certain drugs in the preferred urologically used solutions of the present invention are indicated in the figure.

Figure 7 provides a schematic diagram of the G-protein coupled receptor pathway. The specific molecular sites of action of certain drugs in the preferred solution for general surgical wounds of the present invention are indicated in the figure.

Figure 8 provides a schematic representation of nitric oxide (NO) donor drugs and the mechanism of action of NO in causing vascular smooth muscle relaxation. Physiologically, certain hormones and transmitters can increase NO synthesis by increasing intracellular calcium content and activating a form of NO synthase in endothelial cells. NO donors can generate NO extracellularly or be metabolized to NO in smooth muscle cells. Extracellular NO can diffuse across endothelial cells or directly into smooth muscle cells. The main target of NO is soluble guanylate cyclase (GC), which activates cGMP-dependent protein kinase (PKG) and subsequently excretes calcium from smooth muscle cells by means of membrane pumps. NO also hyperpolarizes cells by opening potassium channels, which in turn leads to the closure of voltage-sensitive calcium channels. Thus, a synergistic interaction between calcium channel antagonists, potassium channel openers and NO donors is evident from the above signal transduction pathways.

Figures 9, 10A and 10B provide, for the animal study described in Example 7 herein, the relationship between vasoconstriction and Graph as a percentage of time demonstrating the effect of infusion of histamine and serotonin antagonists employed in solutions of the invention on constriction of blood vessels during balloon angioplasty.

Figures 11 and 12 provide graphs of the relationship between plasma extravasation and the dose of amitriptyline contained in the solution of the invention in the case of intravenous and intra-articular delivery of the solution of the invention to the knee joint respectively, wherein the knee Intra-articular extravasation was induced following the introduction of serotonin into the knee joint in the animal study described in Example VIII herein.

Figures 13, 14 and 15 present the immediate and 15 minute time points following atherectomy with saline (N=4) or solutions formulated according to the invention for the animal study described in Example 13 herein. (N=7) Mean vasoconstriction (negative values) or vasodilation (positive values) plots for the proximal (Fig. 13), middle (Fig. 14) and distal (Fig. 15) segments of the arteries, present ±1 standard error of the mean.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

According to the present invention, there is provided a method and solution for reducing or preventing tumor cell adhesion and/or invasion and/or local metastasis, the method comprising directly applying to the surgical site a composition in a suitable manner to achieve The delivered pharmaceutical effective carrier includes a combination of two or more metabolically active agents, at least one of which is an anti-tumor adhesion or anti-invasion or anti-local metastasis agent.

I. Inhibitors of Cell Adhesion and Invasion Molecules

A. Antagonists of Metalloproteinases and Tissue Inhibitors of Metalloproteinases (TIMPs)

Matrix metalloproteinases (MMPs) are a family of zinc-dependent neutral endopeptidases whose enzymatic activity can control the degradation of extracellular matrix (ECM). This activity is a key control factor in tumor adhesion, invasion, metastasis, growth and angiogenesis. Through cloning and sequencing, 17 members of this family have been identified in humans. These members are divided into several subgroups, namely collagenases, gelatinases, stromelysins and stromelysin-like MMPs, membrane-type MMPs and neo-MMPs.

MMPs have shared structural and functional features. All family members discovered so far have been determined to have at least three functional domains: a proregion that contains conserved cysteine residues and does not play a role in enzyme activation; between residues 106-119 and contains a conserved structure the catalytic region of the metal-binding site; and the highly conserved zinc-binding active site between amino acids 52-58.

Cells generally do not express MMPs constitutively, but are rapidly induced to express MMPs in response to exogenous signals, cytokines or growth factors and altered cell-matrix and cell-cell interactions. Exceptions to this rule are MMP-8 and MMP-9, which are stored in secretory granules of neutrophils and eosinophils, and MMP-7, which is stored in secretory epithelial cells.

The expression of MMPs is mainly regulated at the transcriptional level, and their proteolytic activity is regulated by the activation and inhibition of activity by zymogens. Extracellular signals, such as cytokines (IL-1, tumor necrosis factor, etc.) and other signals activate the AP-1 transcription factor complex composed of Jun and Fos protein members, which bind to AP-1 in cis Element binding activates the transcription of the corresponding MMP gene. Most MMPs are secreted as latent precursors (zymogens), except for MMP-11 and MT1-MMP, which are activated by Golgi-linked furin-like proteases before secretion, and all other MMPs are proteolytically activated in the extracellular space ,

The activity of MMPs in the extracellular space is regulated by a family of natural inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMPs). To date, four members of this family (TIMP-1, 2, 3, 4) have been identified and well characterized. These TIMPs have similar inhibitory activity against most MMPs, but differ in their interactions with proMMPs, solubility, regulation of expression, and tissue-specific expression. Unlike other TIMPs, TIMP-3 inhibits the activity of TNF-α-converting enzyme (TACB), 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 expressed constitutively, whereas the expression of TIMP-4 is restricted. The expression of TIMP-3 is positively regulated by mitogens, phorbol esters and TGF-β.

MMPs have been proven to promote tumor cell invasion and metastasis through at least three mechanisms. First, in a physiological sense, MMPs can regulate tumor adhesion. This process is necessary for cells to attach and move through the ECM. Cell transfection studies varying the ratio of MMP-2 to TIMP-2 have demonstrated variation in the adhesion phenotype of tumor cells. Second, the protease action of MMPs can degrade ECM macromolecules such as collagen, laminin, and proteoglycans, thereby allowing tumor cell invasion. Finally, MMPs can act on BCM components or other proteins to activate biological functionality. For example, after laminin-5 is degraded by MMP-2, a soluble chemoattractant fragment is generated.

The typical role of MMPs in degrading ECM components, especially the basement membrane, is thought to be critical for tumor cell invasion. On the experimental side, the necessity of protease action in tumor cell invasion has been confirmed by chemical invasion assays. Extensive expression of different MMPs occurs during aggressive primary tumors or their metastases. The expression level of MMP-1 is related to the poor prognosis of colon and esophageal cancer, while the expression of MMP-2 and MMP-3 is closely related to lymph node metastasis. Expressions of MMP-2, TIMP-2 and MT1-MMP are associated with poor prognosis in bladder cancer. MMP-7 knockout mice show reduced intestinal tumorigenesis, and MMP-2-deficient mice show reduced angiogenesis and tumor progression. The infiltration of inflammatory cells is also a prominent feature of many malignant tumors, and inflammatory cells can be the source of MMPs and contribute to the invasion of tumor cells. These inflammatory cells also produce cytokines that increase MMP expression in tumor and stromal cells, leading to further tumor cell invasion.

TIMPs also play a role in tumor cell invasion. TIMP1, 2, 3 and 4 have been found to have the ability to inhibit tumor growth by overexpressing them in several human and rodent models. In melanoma cells, overexpression of TIMP-2 inhibited the growth of transplanted tumors in the skin of SCID mice. This growth inhibition appears to be independent of angiogenesis but dependent on the collagen matrix. Currently, the prevailing notion of the role of TIMPs in cancer focuses on the inhibition of tumor invasion, metastasis, and regulation of ECM homeostasis.

Based on in vitro, in vivo, in situ and clinical studies, the inhibition of MMP activity can inhibit the adhesion, invasion and metastasis of tumor cells, and effectively inhibit the growth and metastasis of tumors. Preventing tumor growth can be achieved by inhibiting the expression or activity of MMPs at various stages in the MMPs synthesis pathway.

Inhibition of MMP transcription can be achieved by directly blocking signaling pathways that regulate MMP transcription, or by inhibiting transcription factors responsible for activating transcription of MMP genes. By inhibiting the signaling pathways involved in the activation of AP-1, MAPKs (ERK1, 2, SAPK/JNK or p38) can significantly inhibit the expression of MMPs and the invasion of malignant cells in vitro. Expression of dominant-negative transcription factors is required for effective in vivo gene therapy approaches and can be used to inhibit MMP production. For example, studies have found that the expression of dominant-negative c-Jun inhibits the induction of MMP1, 2, 3, 10 and 14, and also inhibits the invasion of keratinocytes in vitro. Retinoids, as inhibitors of AP-1 activity, can also be used in certain human cancers to prevent the expression of MMP genes.

Another approach to inhibit the expression of specific MMPs is the use of antisense RNAs. Antisense oligonucleotides to MMP-7 were found to inhibit the invasion of colon cancer cells in vitro and in vivo. Studies have found that MMP-9 antisense ribozyme expression constructs can inhibit the expression of MMP-9 and the invasion of rat bone cancer cells.

The most extensive area of research to inhibit tumor cell invasion by inhibiting MMPs is the development of compounds that block the activity of MMPs. Synthetic MMP inhibitors (MMPIs), such as hydroxamic acid inhibitors, are small peptide analogs of fibrillar collagen that specifically interact with zinc in the catalytic site of MMPs and inhibit the activity of MMPs. Several of these agents are currently in clinical trials for the treatment of aggressive tumors. The first class of agents developed were broad-spectrum MMPIs that could non-specifically inhibit multiple MMPs. Examples are batimastat (BB-94), which has poor oral bioavailability and needs to be administered topically, and marimastat (BB-2516), which can be taken orally. Preclinical studies have shown that these agents inhibit tumor invasion, metastasis, and growth. The second is to complete the development of selective agents for anchoring certain MMPs. Because peptide-based MMP inhibitors are costly to produce and often have low bioavailability, non-peptide inhibitors have been developed through rational design based on X-ray crystallographic information of MMP active sites. Examples of non-peptide inhibitors are CGS27023A (Novartis), AG3340 (Agouron) and the bifenbutyric acid derivative BAY12-9566 (Bayer). In addition, tetracyclines, which lack antimicrobial effects, have also been developed to block host-derived MMPs. Studies have found that this kind of chemically modified tetracyclines (CMTs) without antimicrobial effect can block the expression of MMP2, 3 and 8 through various mechanisms, and can also induce apoptosis of malignant cells. Recently, bisphosphonates were identified as MMP inhibitors, and aggrecanase-1 inhibitors, SE206, BB-16, and XS309 were found to have good potency. Finally, the ability of TIMPs to potently and specifically inhibit MMP activity has drawn interest in their applications. TIMP1, 2, 3 and 4 inhibit the invasion of malignant cells in vitro and in vivo.

One aspect of the invention provides for the direct administration of one or more MMP inhibitors in combination with at least one other analgesic and/or anti-inflammatory and/or antispasmodic and/or antirestenotic agent by means of an irrigation solution A method at a surgical site, wherein the combination of the above agents can effectively prevent or inhibit tumor cell adhesion, invasion and metastasis. Inhibitors and antagonists may act at the level of MMP transcription, translation, secretion, activation or degradation.

Anyone skilled in the art will understand that when selecting an MMP inhibitor as a therapeutic agent for a patient, the required dose of the drug to be administered should be determined in part for any particular treatment regimen and should be readily determinable.

The following table lists MMP inhibitors suitable for use in the present invention.

Table 1

Metalloprotease antagonists and TIMPs potion Therapeutic preferred concentration optimal concentration Batimastat Malimastat CGS27032AAG3340Bay12-9566CMTl minocycline SE206BB16XS309 antisense RNATIMPSl 0.1-10,0000.1-10,0001-100,0000.1-10,00010-1,000,0000.1-1000mg/kg300-500,00010-1,000,00010-1,000,000100-10,000,0000.5-5,0000,000.1-10 100100100010010,000100mg/kg300,00010,00010,000100,0005,0001,000

B. CD44 receptor antagonists and CD44 signaling inhibitors

CD44 is a transmembrane glycoprotein that includes a family of surface receptors consisting of a large number of variant isoforms. CD44 receptors are found in a wide variety of tissues, including the central nervous system, lung, epidermis, liver, colon, and pancreas. A related family of cell surface adhesion molecules are multifunctional proteins expressed in both normal and malignant tissues. CD44 is encoded by a single gene containing 20 exons, of which 10 exons (v1-v10) are variant exons inserted by alternative splicing. The ubiquitously expressed canonical isoforms of CD44 (CD44s) do not contain the sequence encoded by this variant exon. Expression of a number of CD44 variant isoforms (CD44v) with insertions of different combinations of exons (v1-v10) in the ectodomain is restricted to proliferating epithelial cells, activated lymphocytes and malignant cells.

Recent studies have confirmed that the CD44 protein is the main receptor for hyaluronate in the human body. Since CD44 is the major transmembrane receptor for glycosaminoglycans of the extracellular matrix, CD44 is also known as the hyaluronate receptor. It also binds with lower affinity to certain other extracellular matrix ligands (chondroitin sulfate, heparan sulfate, and fibronectin).

CD44 receptor-ligand interactions are required for many normal cellular processes, including cell adhesion (aggregation and migration), hyaluronate degradation, lymphocyte activation, lymphocyte homing to sites of inflammation, and release of cytokines. Other specialized functions include chondrogenesis and assembly of the pericellular matrix during wound healing. The interaction between the CD44 receptor and hyaluronic acid (HA) delivers important signals to normal and transformed CD44-bearing cells. Like many other cell surface receptors, cells expressing CD44 regulate expression of this receptor in response to ligand binding.

The CD44 molecule is a glycosylated molecule with a molecular weight of 85kD, and its N-terminal sequence is homologous to chondronexin. CD44 isoforms of different sizes have been described on a number of cell types. The change in size of the CD44 isoforms is due to differences in glycosylation and the addition of chondroitin sulfate molecules to CD44. The 85 kD CD44 molecule has been identified in serum, plasma and synovial fluid (lines 1-26-20). Alternative splicing produces the hematopoietic form of this molecule, namely CD44H, and other isoforms. Alternative splicing results in changes in protein glycosylation and provides a mechanism to regulate CD44-binding activity. By existing in several isoforms, and because of its broad cellular distribution and functional association with other adhesion molecules, the CD44 molecule is a multifunctional pro-inflammatory molecule involved in immune cell activation as well as metastasis of certain tumor cell types.

The predominant isoform found on human leukocytes is CD44H. Both hyaluronate and CD44 mAb bound to monocytes induced IL-1 release. On T cells, the linkage of hyaluronate and CD44 mAbs to CD44 had differential effects; CD44 mAbs increased T cell triggering, whereas hyaluronate inhibited T cell triggering. Finally, CD44 mAbs and polyclonal anti-CD44 serum were found to inhibit lymphocyte binding to sites of inflammation, such as endothelial venules in the synovium.

The physiological functions of CD44 suggest that it plays a role in the metastatic spread of tumors. Properties of CD44, such as hyaluronate-mediated adhesion, cellular mobility, and adhesion to lymphoid tissues, are among several properties required for invasive and metastatic tumor cells. In vivo experiments demonstrated that tumor cells transfected to overexpress specific CD44 isoforms displayed enhanced metastatic potential. Among the many isoforms of CD44, some are more effective than others in promoting tumor cell metastasis. Non-metastatic cell lines can be conferred with metastatic potential by transfection of CD44 variants, and high levels of CD44 are associated with several types of malignancies.

Numerous studies have reported that human tumors exhibit specific alterations in CD44 isoform expression, and studies have further found that the extent of clinical disease in a given tumor correlates with the expression pattern of CD44 isoforms. For example, studies have found that expression of a CD44 splice variant is required for metastasis in adenocarcinoma cells and that monoclonal antibodies (mAbs) directed against this specific CD44 variant can prevent metastasis. In addition, a wide range of epithelial and mesenchymal malignancies expressed high levels of CD44H and multiple CD44 variant isoforms. The report uses data specifically demonstrating that expression of CD44 variants is a shared feature of epithelial ovarian cancer.

Many studies have investigated the distribution pattern of CD44 in tumors. The study found that the presence of CD44 canonical and CD44-9v isoforms on the surface of gastric cancer cells was significantly associated with higher patient mortality and shorter survival time due to tumors. Intestinal-type gastric cancer predominantly expresses the CD44-6v isoform that renders these tumor cells metastatic. There is significant evidence that integrin receptors and receptors of different CD44 isoforms are altered following malignant transformation of the colonic mucosa into adenomas and invasive carcinomas, which correlates with their metastatic potential. Expression of the CD44-6v isoform is associated with poor prognosis in colorectal cancer due to the development of tumor metastasis and adhesion. These data suggest that CD44 plays an important role in tumor metastasis, and that various molecules that inhibit the interaction between the CD44 receptor and its natural ligands can be used in therapy.

Recently, studies have characterized at the cellular level the cytoplasmic signaling pathway of CD44 in activated endothelial cells, which involves tyrosine kinases. The HA-binding activity of CD44 is associated with cellular activation in certain cell types. Adding hyaluronic acid degradation products (8-10 disaccharides) to cells can induce continuous and rapid tyrosine phosphorylation of various proteins. The study also observed increased phosphorylation of the CD44 receptor. Pretreatment of cells with anti-CD44-receptor antibodies or nonselective inhibitors of tyrosine kinase activity inhibited the induced protein tyrosine phosphorylation and proliferative responses. In the membranes of treated cells, protein kinase C (PKC) activity was increased 2-3 fold. In addition, studies have found that PKC activation triggers a cytoplasmic kinase cascade that includes Raf-1 kinase, MAP kinase (MEK-1), and extracellular signal-regulated kinase (ERK-1). ERK is a dual-specificity kinase that controls the expression of proteins associated with tumorigenesis, tumor cell proliferation, and motility.

In summary, phosphorylation of the CD44 receptor results in increased tyrosine phosphorylation, which activates cytoplasmic cascades and early response genes, and enables cell proliferation. Therefore, an alternative indirect approach to inhibiting CD44 activation and signaling should utilize inhibitors of CD44-activated cell signaling pathways rather than directly employing CD44 receptor antagonists. Tyrosine kinase inhibitors, PKC inhibitors (such as calcinein) or ERK inhibitors can all be employed as agents that can block signaling proteins mediated by CD44 and provide new targets for therapeutic agents.

During tumor growth and metastasis, one of the key events is the interaction between tumor cells and the host tissue matrix, which is mediated by different combinations of adhesion receptors on different tumor cell types. Several lines of evidence suggest that the interaction between the CD44 receptor expressed on tumor cells and tissue matrix HA can promote the growth and invasion of certain tumors. Transformation from colonic mucosa to carcinoma is associated with overexpression of several CD44 alternative splice variants. The importance of the CD44-hyaluronate interaction in tumor development was clarified by disrupting the CD44-hyaluronan interaction with soluble recombinant CD44, which was found to inhibit CD44-transfected lymphoma and melanoma tumor formation. Demonstrated by topical administration of a mutant, hyaluronate-incapable CD44-Ig fusion protein that had no effect on subcutaneous melanoma growth in mice, along with control infusions of wild-type CD44-Ig that blocked tumor development Differentiation inhibitory effects of soluble wild-type and mutant CD44-Ig fusion proteins on melanoma growth in vivo.

The present invention provides a method of administering a CD44 inhibitor or a CD44 receptor antagonist in combination with at least one other analgesic and/or anti-inflammatory and/or antispasmodic and/or antirestenotic agent, the combination of which is effective To prevent or inhibit the adhesion and metastasis of tumor cells, and at the same time inhibit one or more other adverse processes associated with surgical procedures. Inhibitors and antagonists can act directly by physically linking and binding to cell surface receptors, preventing binding of activating ligands, or as indirect inhibitors by inhibiting unique molecules involved in CD44 signaling.

Direct receptor antagonists include monoclonal or polyclonal antibodies prepared against CD44 variants, or fragments thereof, which can be used as inhibitors of HA binding to CD44 and can be used as therapeutic agents. Applicable anti-CD44 monoclonal antibodies include mAbs that recognize epitopes present on all CD44 isoforms, or suitable mAbs that recognize only post-translationally modified forms of CD44. Isotype (variant) specific anti-CD44 mAbs are all described in US Patent No. 5,863,540 (F12, A1G3, A3D8), also including mAbs IM7 and BU52. Other monoclonal antibodies suitable for use in the present invention include anti-CD44 mAbs directed against different CD44 isoforms that arise as a result of alternative splicing of the ten alternative exons.

The invention discloses the use of recombinantly prepared chimeric soluble CD44 (CSCD44) protein, wherein the extracellular domain of CD44 receptor or part thereof is covalently linked to the constant region of IgG molecule, and the chimeric soluble CD44 (CSCD44) protein can be used For anti-tumor, anti-cell adhesion and anti-metastasis agents. In particular, a first example is the use of chimeric polypeptides (recombinant chimeras) containing the extracellular domain of the CD44 receptor extracellular polypeptide coupled to the CH2 and CH3 regions of a mouse IgGl heavy chain polypeptide. A second example is a chimeric fusion construct comprising the HA ligand binding region of the CD44 receptor and the Fc antibody portion raised against the CD44 receptor (termed Fc-fused soluble receptor). The molecular form of the active soluble CD44 receptor can be a monomer or a dimer. Methods to test the binding of soluble CD44 antibodies to variant isoforms of CD44 expressed on the surface of cells have been established.

A second approach to inhibit CD44-HA receptor-ligand binding is to employ size-specific hyaluronan oligosaccharides (HA oligomers) to suppress tumors by saturating the CD44 receptor with excess ligand form. One study found that injections of hyaluronic acid oligomers at a low concentration of 1 mg/ml inhibited melanoma growth. Thus, local delivery of the above-mentioned oligomers inhibits the interaction of CD44 with its natural substrates and provides a suitable method to control local tumor development.

Those skilled in the art will understand that when selecting specific anti-CD44 monoclonal antibody or CD44 peptide sequence as a therapeutic agent for a patient, the dose to be administered should be partially determined for any specific treatment plan, and the dose should be easily determined. Those skilled in the art will appreciate that the amount of peptide, protein or antibody administered for a particular treatment regimen may be a function of the particular CD44 region associated with the pathological clinical situation, or the particular cell type mediating metastasis.

The table below lists anti-adhesion and/or anti-invasion and/or anti-metastatic agents suitable for surgical use according to the invention, in combination with analgesic and/or anti-inflammatory and/or antispasmodic and/or anti-restenotic agents Delivery of CD44 receptor antagonists.

Table 2

CD44 receptor antagonist potion Treatment preferred concentration (μg/ml) The most preferred concentration (μg/ml) Anti-CD44 mAb IM7 Anti-CD44 mAb BU52 Anti-CD44 mAb F12 Anti-CD44 mAb A1G3 Anti-CD44 mAb A3D8 Chimeric rhCD44:Fc Anti-CD44 Fab Hyaluronic Acid Oligomer Soluble CD44 Receptor 0.1-10000.1-10000.1-10000.1-10000.1-10000.1-10000.1-10000.1-10001.0-10000 10010010010010010010010001000

C. Integrin Receptor Antagonists and Integrin Signaling Inhibitors

Integrins are a superfamily of cell surface receptors involved in cell-cell and cell-matrix adhesion. These receptors play an important role in regulating various cellular functions, such as cell differentiation and migration, maintenance of tissue architecture, blood clot formation and contraction programmed cell death. Furthermore, integrins are thought to be involved in cancer cell differentiation, tumor progression, and metastasis due to their important role in mediating the interaction between tumor cells and extracellular matrix and endothelial cells. Although changes in integrin expression vary by tumor type, integrin expression and function are altered in many malignant cells. Specific integrin receptor subtypes on tumor cells mediate attachment to a number of adhesion proteins that are components of the extracellular matrix, activated platelets, and endothelial cells at surgical wound sites.

Integrins are a family of heterodimeric, transmembrane αβ receptors expressed on a wide variety of cells. This receptor family includes at least 14 known α-subunits and 8 β-subunits, which are connected to each other to form a variety of subtype combinations, showing different ligand specificities. The α-subunit appears to be the key determinant of ligand specificity, with αv-containing integrins demonstrating specificity for vitronectin, α5-containing integrins for fibronectin, α3-containing integrins Integrins demonstrated specificity for collagen/laminin. A large number of integrin heterodimeric subunit combinations are found on a variety of tumor cells, promoting the interaction of these tumor cells with fibronectin, laminin, vitronectin, fibrinogen, collagen and thrombospondin combined. Vascular cell adhesion molecule-1 (VCAM-1) is an endothelial cell ligand for two leukocyte integrins, α4β1 and α4β7. Mucosal addressin cell adhesion molecule (MadCAM-1) is also considered a ligand for the α4β7 integrin.

Interactions between fibronectin and several integrin receptor subtypes to which it binds are of particular importance at several stages of tumor development and influence the course of malignant metastasis. Fibronectin is an extracellular glycoprotein composed of repeating units of amino acids. It is encoded by a single gene, with alternative splicing giving rise to multiple isoforms. Specific protein regions enable the molecule to interact with a variety of cells via integrin and non-integrin receptors. Fibronectin contains at least two separate cell adhesion domains with different receptor specificities. The cell adhesion region in the central part of fibronectin includes at least two minimum amino acid sequences, namely Arg-Gly-Asp (RGD) sequence and Pro-His-Ser-Arg-Asn (PHSRN) sequence. The α5β1 fibronectin-specific integrin binds to a centrally located RGD/PHSRN site. α4β1 integrin binds to the IIICS site. Peptide and antibody inhibitors of fibronectin and integrin function have been identified as potent inhibitors of tumor metastasis and potentially important agents for controlling tumor cell adhesion.

Integrin activation pathways mediated by intracellular kinases and adapter proteins are particularly important for integrin receptor-mediated anchorage dependence. Recently, signal transduction studies in multiple cell types confirmed that the binding of integrin receptors to extracellular matrix proteins rapidly generates intracellular signaling through enhanced tyrosine phosphorylation, which also identified the adhesion Focal Kinase (FAK) The role of protein tyrosine kinases (PTKs) in linking integrin receptors to intracellular signaling pathways. FAK is linked to several different cytoplasmic signaling proteins, such as Src-family PTKs and several SH2-domain proteins (Shc, Grb2 and PI3-kinase). This enables FAK to function in a network of integrin-stimulated signaling pathways, thereby activating targets such as ERK and the JNK/mitogen-activated protein kinase pathway. These signaling mechanisms are based on direct inhibitors of integrin receptor-mediated signaling by tyrosine kinase inhibitors at points in the signal transduction cascade proximal to the integrin receptor, serving as antiadhesive The therapeutic potential of supplements provides a theoretical basis.

Among the fibronectin ligands of integrin receptors, the peptide sequence motif (arginine-glycine-aspartate) responsible for cell attachment provides the basis for the development of therapeutic agents because the integrin Protein binding activity can be reproduced by short synthetic peptides containing the RGD sequence. It was found that peptides containing the RGD sequence inhibited the attachment of tumor cells in vitro. By cyclizing peptides with specific sequences flanking the RGD motif, and by synthesizing RGD mimetics, agents have been prepared that can specifically bind only one or a few RGD-directed integrins. A series of peptide analogs of fibronectin RGD sequence have been developed and synthesized, and their anti-metastatic effects in mice and inhibition of tumor cell invasion in vitro have also been verified.

Cell and animal studies have shown that anti-adhesion peptides and peptides help stop the spread of certain forms of cancer in the peritoneum. For example, the peptide integrin antagonists tyrosyl-isoleucyl-glycyl-seryl-arginine (YIGSR), acetyl-arginyl - Effect of glycyl-aspartyl-serinamide (Ac-RGDS-NH2) and arginyl-glycyl-aspartic acid (RGD) on adhesion and invasiveness of gastric cancer cell lines. It was reported that the expression of both α2β1 and α3β1 integrins was significantly increased on OCUM-2MD3 cells, and the ability of these cells to bind to the extracellular matrix was also significantly higher than that of control cells. Adhesion polypeptides YIGSR and RGD and two RGD derivatives significantly inhibited the adhesion and invasion of OCUM-2MD3 cells to mesothelial ECM in a cell-specific manner. Peritoneally diffused nude mice administered ip with YIGSR or RGD sequences survived longer than mice not treated with this treatment. Studies have found that therapies based on the interference of integrin-binding peptides with tumor cell attachment are effective anti-metastatic regimens in animal experiments, and that if cells were pre-exposed to fibrinogen, laminin, or RGDS-motif-containing peptides, It will also reduce the frequency of tumor implantation at surgical trauma sites.

These and other studies point to the use of synthetic fibronectin fragments, peptide analogs and mimetics containing RGD sequences, or antibodies to inhibit specific integrin receptor-ligand interactions, providing a useful method for developing clinically A method for potent subtype-specific integrin receptor antagonists. Studies have found that a number of the above-mentioned peptides, such as those disclosed in U.S. Patent No. 5,627,263, are more effective than the original RGDS peptides in inhibiting tumor cell adhesion and tumor metastasis, and these peptides are disclosed in the present invention for use in combination with other agents . In particular, the present invention provides the use of peptides and peptide analogs specific for fibronectin-binding integrins, especially _{α5β1 integrins} . In addition, the present invention also provides specific agents that inhibit binding to a portion of the β1 integrin heterodimer, namely the _α2 , _α6 and _αv integrin subunits.

Other classes of inhibitors are naturally occurring antiadhesive snake venom proteins containing the RGD sequence, including a family of small, highly homologous, cysteine-rich polypeptides purified from snake venom. Antiadhesive venoms competitively inhibit integrin-ligand interactions and have been shown to inhibit cell adhesion interactions in a variety of systems. The study found that specific snake venom-derived antiadhesive venom proteins were 2000-fold more effective than short, synthetic, linear peptides containing RGD in blocking fibrinogen-dependent platelet aggregation. The greater potency of the antiadhesin venom molecule stems from the amino acids surrounding the RGD sequence and the intrachain disulfide bonds that maintain the RGD sequence in the proper configuration. Specific anti-adhesion venom proteins block the adhesive function of integrin cell surface receptors and thereby inhibit integrin-dependent cellular responses in a variety of cell types and tissues. Through affinity cross-linking studies, it was found that akitoxin anti-adhesion venom specifically binds with high affinity to αVβ3, but not to α5β1 or other bulk integrins. The related anti-adhesion venom protein echistatin specifically inhibits the binding of ¹²⁵ I-labeled akitoxin to αVβ3, while the structurally unique anti-adhesion venom protein, antithrombotic peptide, has a 1000-fold lower affinity.

Recently, two anti-adhesion venom proteins, ussuristatin 1 (US-1) and 2 (US-2), isolated from the venom of the Chinese viper (Agkistrodon ussuriensis), were characterized as potent inhibitors of cell adhesion. Both polypeptides consist of 71 amino acids and are characterized by a highly similar sequence to other anti-adhesion venom proteins. US-1 has a typical Arg-Gly-Asp (RGD) sequence, which is responsible for blocking the binding of fibrinogen to its receptor. In US-2, the corresponding sequence is Lys-Gly-Asp (KGD). US-2 also inhibits platelet aggregation, but with approximately 10-fold higher _IC50s . US-1 also dose-dependently inhibited the adhesion of human melanoma cells to fibrinogen and fibronectin with IC ₅₀ =17-33 nM, while US-2 did not inhibit the adhesion of the cells to fibronectin.

Another heterodimeric anti-adhesive venom EC3 (Mr=14,762) isolated from the venom of Viper serrata is a potent antagonist of α4 integrin. Each subunit contains 67 residues and shows high homology to other anti-adhesion venom proteins. EC3 inhibits the adhesion of cells expressing α4β1 and α4β7 integrins to natural ligands such as vascular cell adhesion molecule 1 (VCAM-1) and mucosal addressin cell adhesion molecule 1 (MadCAM-1) with _IC50 = 6-30 nM, the adhesion of K562 cells (α5β1) to fibronectin, its IC ₅₀ =150 nM, and the adhesion of αIIbβ3 Chinese hamster ovary cells to fibrinogen, its IC ₅₀ =500 nM.

Examples of integrin antagonists suitable for use in the present invention are as follows. For each of the listed agents, the examples provide preferred and most preferred concentrations where the irrigating solutions contain the listed agents, which are expected to be therapeutically effective concentrations.

table 3

Treatment and preferred concentrations of integrin receptor antagonists compound Therapeutic preferred concentration (μM) The most preferred concentration (μM) RGDGRGDSPYIGSRGACRGDCLGAGRGDSPKGRGDTPSDGRGRGDSPASSKP ring (RGD-D-Phe-Val) trimethylsulfonyl (DRGDS) ₃ US-1 Agkistrodon toxin EC-3 5-500005-500005-500000.1-100000.1-10000.1-10000.1-10000.1-10000.1-10000.1-10000.03-30000.1-10000.1-1000 5000500050001001010101010103101

D. Selectin Receptor Antagonists and Selectin Signaling Inhibitors

Adhesion of malignant cells to vascular endothelial surfaces involves a family of adhesion receptors similar to those involved in the recruitment of inflammatory cells to tissue sites. Selectins are involved in a cascade of sequential molecular stages following endothelial cell (EC) activation. The selectin family consists of E-selectin (ELAM-1), P-selectin (GMP-140) and L-selectin (LECAM-1). Carbohydrate determinants often expressed on human cancer cells, sialic acid Lewis A (SLe ^a ) and sialic acid Lewis X ( ^SLex ), act as ligands for E-selectin, which is expressed in blood vessels on endothelial cells. These carbohydrate determinants are involved in the adhesion of cancer cells to the vascular endothelium, thereby promoting cancer metastasis. These selectins are inducible endothelial-expressed adhesion molecules involved in leukocyte recruitment. The initial adhesion mediated by these molecules triggers the activation of integrin molecules through the action of several cytokines. The degree of expression of cancer cell surface carbohydrate ligands correlates well with the frequency of metastasis. Monoclonal antibodies against SLe ^a or SLe ^x block the adhesion of tumor cells (leukemia, colon cancer, and histiocytic lymphoma cells) to ECs and platelets. Likewise, selectin expression is often upregulated on breast cancer endothelium.

P-selectin, also known as GMP-140 or PADGEM, is a membrane glycoprotein located in the secretory granules and endothelium of resting (unstimulated) platelets. When mediators activate these cells, P-selectin is rapidly redistributed to the plasma membrane. These selectins constitute a family of structurally and functionally related molecules. Structural motifs shared by these molecules include an N-terminal lectin-like region followed by an EGF-like region, a series of consensus repeats related to repeats in complement fixin, a transmembrane domain, and a short cytoplasmic tail. P-selectin is a receptor for neutrophils and monocytes when expressed on activated platelets and endothelium. This property facilitates rapid adhesion of leukocytes to the endothelium in areas of tissue injury and also facilitates platelet-leukocyte interactions at sites of inflammation and bleeding. Studies have also found that P-selectin can bind tumor cells in various tissue sections and bind to the cell surface of a large number of cancer-derived cell lines. By acting in conjunction with other adhesion molecules, P-selectin is involved in tumor metastasis and thus becomes a target for drugs that block adhesion receptor function.

A role for selectins in tumor metastasis has been demonstrated based on the adhesion of human colon cancer cell lines to selectins using an in vitro flow model. Recombinant forms of P-selectin and Chinese hamster ovary cells expressing P-selectin supported the attachment and rolling of KM12-L4 colon cancer cells, an effect that was abrogated by pretreatment of KM12-L4 cells with neuraminidase. KM12-L4 cells interact with P-selectin through a PSGL-1-independent adhesion pathway. E-selectin-IgG chimeras have also been found to support partially sialylation-dependent adhesion of colon cancer cells under fluid flow conditions. Thus, sialylation is partly involved in the 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 human colon cancer cells to human and mouse EC correlates with the metastatic potential of the cancer cells.

Recently, a study evaluated the role of E-selectin-sialic acid Lewis x ( ^SLex )/sialic acid Lewisa (SLe ^a ) interaction in mediating the relationship between two human colon cancer cell lines, HT-29 and COLO 201, and human umbilical cord. Role in endothelial cell (HUVEC) adhesion in vitro. Glycotopes were strongly expressed by colic pain cell lines, and it was determined that monoclonal antibodies to E-selectin inhibited the adhesion of HT-29 and COLO 201 cells to IL-1-stimulated HUVECs. Pre-incubation of cells with two different antibodies against ^SLex and the related Lewis epitopes, Le ^x and Le ^a , had no significant effect on adhesion. The three antibodies to Sle ^a differed in their ability to inhibit the adhesion of HT-29 and COLO 201 cells. Thus, the Sle ^a epitope is important for colon cancer cell adhesion primarily mediated by E-selectin.

Other studies have demonstrated that pretreatment of HUVECs with tumor necrosis factor-α (TNF-α) can enhance the binding of HUVECs to the COLO 205 cancer cell line. This increase in adhesion was concentration- and time-dependent, with tumor cell adhesion being maximal at 4 hours. This result is consistent with increased expression of the adhesion molecule E-selectin on the endothelium. Incubation of TNF-stimulated HUVECs with an anti-E-selectin mAb, BB11, prior to the addition of COLO 205 resulted in complete inhibition of tumor cell adhesion. These studies demonstrate the utility of E-selectin-specific antibodies in inhibiting tumor cell attachment to endothelial targets. Other studies found that pretreatment of cells with anti-P-selectin or anti-L-selectin monoclonal antibodies (ie, MAb PB 1.3 and MAbDREG-200), or oligosaccharides containing sialic acid Lewis ^x (Slex ^- OS) Can effectively inhibit cell adhesion.

Similar in vitro and in vivo studies using malignant SW1990 cells derived from human pancreas determined the role of selectins in cancer cell metastasis. SW1990 cells can also strongly express Sle ^a and SLe ^x antigens, CD44H and β1 integrin. These cells exhibit binding activity to IL-1-activated HUVECs and human peritoneal mesothelial cells. Adhesion leading to pain cell engraftment into endothelial cells was inhibited by treatment with antibodies to Sle ^a and to β1 integrin. In animal studies, treatment with antibodies against Sle ^a and β1 integrin, respectively, could inhibit the development of liver metastases in nude mice carrying SW1990 cells and prolong their survival.

Elucidation of the signaling properties of selectins reveals mechanisms common to other cell adhesion receptors. Studies using HT-29 human colon carcinoma cells that specifically adhere to E-selectin-IgG chimeras showed that, on the basis of adhesion to E-selectin, tyrosine in several proteins in HT-29 cell lysates The amount of acid phosphorylation increased. This effect was specific for adhesion to E-selectin, as the addition of E-selectin blocking mAbs significantly reduced tyrosine phosphorylation compared to E-selectin alone. Kinase assays showed that c-src activity was dose-dependent and markedly reduced on the basis of adhesion E-selectin, which was associated with increased phosphorylation of the negative regulator tyrosine Tyr 527. These studies identified signaling pathways involving E-selectin ligands and c-src on HT-29 cells.

Other studies have demonstrated that lymphocyte binding to P-selectin induces tyrosine phosphorylation of various proteins. Activation appears to occur upon initial contact with surface adhesion molecules. P-selectin effect varies over time, eg early response after 10 min and maximal effect at 30 min. Identified proteins include pp125 focal adhesion kinase (FAK) and paxillin. Treatment with the tyrosine kinase inhibitor genistein, or with the protein kinase C inhibitor staurosporine, reduced pp125 FAK phosphorylation. These signaling mechanisms provide a rationale for the therapeutic potential of tyrosine kinase inhibitors as anti-adhesion agents, based on their role as direct inhibitors of selectin receptor-mediated signaling. The rapid signaling properties of selectin receptors highlight the need to deliver therapeutic agents at the onset of surgical procedures to provide preemptive inhibitory effects.

The present invention provides a method of inhibiting the spread of metastatic tumor cells by administering to a patient a pharmaceutical composition comprising a therapeutically effective amount of an anti-E-selectin specific mAb or antibody fragment, and/or or anti-P-selectin-specific mAb or antibody fragment, and/or anti-L-selectin-specific mAb or antibody fragment, and/or anti-sialic acid Lewis ^a (Sle ^a ) mAb, in combination with one or more Anti-inflammatory and/or analgesic and/or antispasmodic and/or antirestenotic agent.

Studies have also found that heparin can inhibit the binding of selectin to its sugar ligand. Heparin (enoxaparin), which inhibits selectin interaction, is thought to reduce adhesion of selectin-associated tumor cells.

Examples of selectin antagonists suitable for use in the present invention are listed in the table below. The table provides, for each of the agents listed, the preferred and most preferred concentration of the agent in the irrigant solution which is considered to be a therapeutically effective concentration.

Table 4

Treatment and preferred concentrations of selectin receptor antagonists potion Treatment preferred concentration (μg/ml) The most preferred concentration (μg/ml) Anti-SLe ^x mAb PB1.3 Anti-SLe ^a mAb DREG-200Sle ^x -OS Anti-E-selectin mAbBB11 Anti-P-selectin IgG Anti-E-selectin IgG Enoxaparin 0.1-10000.1-10000.1-10000.1-100000.1-100001.0-100000.1-1000 100100100100010001000100

Given the defined molecular and cellular mechanisms of action for the classes of agents identified here, these agents are expected to exhibit anti-tumor cell adhesion, anti-invasive and/or anti-metastatic effects when administered perioperatively in the form of irrigation fluids. These agents are expected to function as effective drugs when delivered as irrigants during surgical procedures involving established or potentially malignant conditions. The method of delivery and composition contained in the irrigating solution are believed to be useful in procedures involving the peritoneum, abdomen, thorax, pleura, mediastinum, genitourinary, epidural, intrathecal, and joint cavity, including but not limited to indications Surgery for the oncological treatment of ovarian, gastric, pancreatic and colon cancer. Each metabolically active anti-tumor adhesion agent may be delivered in combination with one or more other analgesic and/or anti-inflammatory and/or antispasmodic and/or anti-restenotic agents. Suitable antineoplastic agents that can be included in the pharmaceutical composition of the present invention should further have the following characteristics, that is, they must have the ability to limit the interaction of the agent with a single family (or class) of receptors or a single family of enzymes (such as CD44, integrins, selectins). , protein tyrosine kinases, MMPs and MAP kinases) interaction pharmacological selectivity and specificity. Particularly suitable agents interact specifically with one or more receptor subtypes (or isotypes) while exhibiting specificity for that receptor family. Each suitable agent should be linked to its molecular target by a specific molecular mechanism characterized by a specific stoichiometric relationship (typically 1:1) of the ligand-receptor (or inhibitor-enzyme) complex. ), and another characteristic lies in its equilibrium binding or kinetic constants. These agents may include small molecule drugs, natural or synthetic peptides or peptoids, polypeptides, proteins, recombinant chimeric proteins, monoclonal or polyclonal antibodies, oligonucleotides, or gene therapy vectors (viral and non-viral).

For example, an agent such as an integrin receptor antagonist can act on specific cells, which are any cells connected to the fluid space of the peritoneal cavity and specific structures, including those involved in normal function or present due to pathological conditions cavity. These cells and structures include, but are not limited to: epithelial cells; mesothelial cells; inflammatory cells, including lymphocytes, macrophages, mast cells, monocytes, eosinophils; and other cells, including endothelial cells, smooth muscle cells and Fibroblasts; and all combinations of these cells.

The present invention also provides formulations of active therapeutic agents that can be delivered in specific formulations that facilitate the introduction and administration of combinations of anti-adhesion, anti-invasive and/or anti-metastatic agents and other agents at the surgical site , and will enhance the delivery, uptake, stability or pharmacokinetics of the combination. The formulation may include, but is not limited to, microparticles, microspheres or nanoparticles composed of proteins, carbohydrates, synthetic organic compounds or inorganic compounds. Examples of formulation molecules include, but are not limited to, lipids capable of forming liposomes or other ordered lipid structures, cationic lipids, hydrophilic polymers, polycations such as protamine, spermidine, and polylysine ), peptide or synthetic ligands and antibodies capable of anchoring substances to specific cell types, gels, sustained release matrices, soluble and insoluble particles, and other formulation components known to those skilled in the art.

The present invention provides for the delivery of anti-tumor adhesion and/or anti-invasion and/or anti-local metastasis drug combinations, either in the form of complex drug actives in a uniform individual delivery vehicle (e.g., individually encapsulated microspheres), or as a discrete mixture in individual delivery vehicles (eg, a set of microspheres encapsulating one or more agents). Examples of formulation molecules include, but are not limited to, hydrophilic polymers, polycations (such as protamine, spermidine, polylysine, and chitosan), peptides or synthetic ligands capable of anchoring substances to specific cell types. bodies and antibodies, gels, sustained-release matrices, soluble and insoluble particles, and formulation ingredients not listed.

The present invention provides the local delivery of anti-tumor cell adhesion drug combination by means of flushing liquid, which contains the above-mentioned drugs at a low therapeutically effective concentration, and can directly deliver the drugs to designated tissues. The irrigating solution containing the above-mentioned medicine can be applied during operation, during operation and before operation, during operation and after operation or before, during and after operation related to surgical operation.

Conventional methods used to deliver drugs require systemic (e.g., oral, intramuscular, intravenous, subcutaneous) administration, and drugs administered to patients must have high concentrations (and high total doses) to be able to Significant therapeutic concentrations are obtained at pathologically affected tissues. Systemic administration also results in high concentrations in tissues other than the target tissue, which is undesirable and may also produce adverse dose-related side effects. These methods of systemic administration subject the drug to secondary metabolism and rapid degradation, thereby limiting the duration of effective therapeutic concentrations. Since the combination of anti-tumor cell adhesion, anti-invasive and anti-metastatic agents (with or without one or more analgesic and/or anti-inflammatory and/or antispasmodic and/or anti-restenotic agents) is administered directly by irrigation At the surgical site, there is no need for vascular infusion to deliver the drug to the target tissue. This significant advantage enables a variety of anti-tumor cell adhesion, anti-local metastasis drugs to be delivered locally, and the total therapeutically effective dose is lower than the drug dose that may be required by other routes of administration.

In another aspect, the flushing solution of the present invention comprises a dilution of at least one tumor cell adhesion, invasion and/or metastasis inhibitor and preferably multiple pain/inflammation inhibitors, antispasmodics and antirestenosis agents in a physiological vehicle liquid. The carrier is a liquid, as used herein is meant to include biocompatible solvents, suspensions, polymerizable and non-polymerizable gels, pastes and ointments. A preferred carrier is an aqueous solution, which may include physiological electrolytes, such as physiological saline or lactated Ringer's solution.

The anti-inflammatory/analgesic agent is selected from (1) serotonin receptor antagonists; (2) serotonin receptor agonists; (3) histamine receptor antagonists; (4) bradykinin receptor antagonists; (5) ) kallikrein inhibitors; (6) tachykinin receptor antagonists, including neurokinin 1 and neurokinin 2 receptor subtype antagonists; (7) calcitonin gene-related peptide (CGRP) receptor Antagonists; (8) Interleukin receptor antagonists; (9) Inhibitors of enzymes that function in the synthetic pathway of arachidonic acid metabolites, including (a) phospholipase inhibitors, including PLA ₂ isotypes type inhibitors and PLC _gamma isoform inhibitors, (b) cyclooxygenase inhibitors, and (c) lipoxygenase inhibitors; (10) prostanoid hormone receptor antagonists, including eicosanoids Acid EP-1 and EP-4 receptor subtype antagonists and thromboxane receptor subtype antagonists; (11) leukotriene receptor antagonists, including leukotriene _B4 receptor subtype antagonists and leukotriene receptor subtype antagonists (12) _opioid receptor agonists, including mu-opioid, delta-opioid, and kappa-opioid receptor subtype agonists; (13) purine Receptor agonists and antagonists, including _P2X receptor antagonists and _P2Y receptor agonists; and (14) adenosine triphosphate (ATP)-sensitive potassium channel openers.

Suitable anti-inflammatory/analgesic agents that may also act as antispasmodics include serotonin receptor antagonists, tachykinin receptor antagonists, ATP-sensitive potassium channel openers and calcium channel antagonists. Other agents that may be used in the solutions of the invention because of their antispasmodic properties include endothelin receptor antagonists, calcium channel antagonists and nitric oxide donors (enzyme activators).

A particularly preferred embodiment of the solution of the present invention suitable for use in cardiovascular and general vascular procedures includes an anti-restenotic agent, most preferably in combination with an antispasmodic agent. Suitable anti-restenotic agents include: (1) antiplatelet agents, including (a) thrombin inhibitors and receptor antagonists, (b) adenosine diphosphate (ADP) receptor antagonists (also known as purine receptor ₁ receptor antagonists), (c) thromboxane inhibitors and receptor antagonists, and (d) platelet membrane glycoprotein receptor antagonists; (2) inhibitors of cell adhesion molecules, including (a) selected Protein inhibitors and (b) integrin inhibitors; (3) anti-chemotactic agents; (4) interleukin receptor antagonists (may also act as analgesic/anti-inflammatory agents); and (5) intracellular signaling inhibition agents, including (a) protein kinase C (PKC) inhibitors and protein tyrosine phosphatases, (b) regulators of intracellular protein tyrosine kinase inhibitors; (c) src homology region ₂ (SH2) structure domain inhibitors, and (d) calcium channel antagonists. These agents help prevent restenosis of arteries undergoing angioplasty, atherectomy, or other cardiovascular or general vascular therapeutic procedures.

The various surgical solutions of the present invention contain agents at concentrations and locally delivered doses that are lower than those required by conventional drug administration methods to achieve a given therapeutic effect. Delivering the same amount of agent by other (ie intravenous, subcutaneous, intramuscular or oral) routes of drug administration would not be possible to achieve equivalent efficacy due to primary and secondary metabolism of the systemically administered drug. The concentration of each agent can be determined based in part on its dissociation constant _Kd . The term dissociation constant as used herein includes the equilibrium dissociation constant corresponding to the agonist-receptor or antagonist-receptor interaction of the agent, respectively, and the equilibrium dissociation constant corresponding to the activator-enzyme or inhibitor-enzyme interaction of the agent, respectively. Equilibrium inhibition constant. With the exception of cyclooxygenase inhibitors, each agent is preferably included at a low concentration of 0.1-10,000 nanomolar Kd _times , with higher concentrations possibly being required depending on the particular inhibitor chosen. Each agent is preferably included at a concentration of 1.0 to 1,000 times _the Kd nanomolar, most preferably about 100 times the _Kd nanomolar. In the absence of metabolic conversion at the site of local delivery, dilution may be considered to adjust the above concentrations as necessary. The exact agents selected for the solutions of the invention are described below, along with their concentrations and modifications for specific applications.

The solution of the present invention may comprise only low concentrations of single or multiple inhibitors of tumor cell adhesion, invasion and/or metastasis, single or multiple inhibitors of pain/inflammation, single or multiple antispasmodics, tumor cell adhesion Anti-restenosis agent combination, anti-restenosis agent combination selected from listed classes. However, multiple agents are preferred due to their synergistic effects as described above and the desire to broadly block pain and inflammation, spasticity and restenosis.

The surgical solution of the present invention establishes a new therapeutic approach by combining multiple pharmacological agents that can act on different receptor and enzyme molecular targets. To date, pharmacological programs have focused on the development of highly specific drugs that are selective for the specific individual receptor subtypes and enzyme isoforms that mediate the response to individual signaling Neurotransmitter and hormone responses. For example, endothelin is one of the most potent vasoconstrictors known. Several pharmaceutical companies are developing selective antagonists specific for several subtypes of the endothelin (ET) receptor for use in the treatment of a number of conditions involving elevated levels of endothelin in the body. Recognizing the potential role of the receptor subtype _ETA in hypertension, these pharmaceutical companies explicitly targeted the development of selective antagonists of _{the ETA} receptor subtype for the proactive treatment of coronary artery spasm. This standard pharmacological regimen, although widely accepted, is not optimal because many other vasoconstrictors (eg, serotonin, prostaglandins, eicosanoids, etc.) may also be responsible for both initiating and maintaining vasospasm. seizures (see Figures 2 and 4). Furthermore, even if a single receptor subtype or enzyme is inactivated while others are activated, the resulting signaling often still triggers a cascade of effects. This explains the notable difficulty of using a single receptor-specific drug to block pathophysiological processes in which multiple transmitters play a role. Therefore, targeting only specific individual receptor subtypes, such as _ETA , is likely to be ineffective.

In contrast to the above-mentioned standard approach to pharmacological treatment, the rationale for the therapeutic approach of the present invention is the necessity to combine drugs that act simultaneously on different molecular targets in order to inhibit the full range of events that are responsible for the development of pathophysiological states. Furthermore, the surgical solution of the present invention consists of multiple drugs that target shared molecular mechanisms operating in different cellular physiological processes involved in the development of pain, inflammation, vasospasm, smooth muscle spasm, and restenosis, rather than targeting only specific affected persons. body subtypes (see Figure 1). In this way the inventive surgical solution minimizes the cascade of other receptors and enzymes in the nociception, inflammation, spasm and restenosis pathways. The surgical solution inhibits the "upstream" and "downstream" cascade effects in these pathophysiological pathways.

Examples of "upstream" inhibition are cyclooxygenase antagonists in the context of pain and inflammation. Cyclooxygenases (COX ₁ and COX ₂ ) catalyze the conversion of arachidonic acid to prostaglandin H, a mediator of inflammation and nociception including prostaglandins, leukotrienes, and thromboxanes in biological intermediates in the synthesis process. The cyclooxygenase inhibitor blocks the formation of these inflammatory and nociceptive mediators "upstream". The program does not need to block the interaction between the above seven subtypes of prostanoid hormone receptors and their natural ligands. A similar "upstream" inhibitor contained in the surgical solution of the present invention is aprotinin, which is a kallikrein inhibitor. Kallikrein is a serine protease that cleaves high molecular weight kininogen in plasma to generate bradykinin, an important mediator of pain and inflammation. Aprotinin effectively inhibits the synthesis of bradykinin by inhibiting kallikrein, thereby effectively inhibiting the above-mentioned inflammatory mediators "upstream".

The surgical solutions of the present invention also utilize "downstream" inhibitors to control pathophysiological pathways. ATP-sensitive potassium channel openers (KCOs) can be concentrated in vascular smooth muscle specimens preconstricted with various neurotransmitters involved in coronary spasm Relaxes smooth muscle in a dependent manner (Quast et al., 1994; Kashiwabara et al., 1994). Thus, KCOs provide a "downstream" antispasmodic effect that confers significant advantages to the surgical solution of the present invention in the context of vasospasm and smooth muscle spasm by providing a "downstream" antispasmodic effect in a manner that is independent of the physiological combination of agonists that initiate the spasm event (See Figures 2 and 4). Likewise, NO donors and voltage-gated calcium channel antagonists inhibit vasospasm and smooth muscle spasm initiated by multiple mediators known to play an early role in the spasm pathway.

II. Anti-inflammatory/Analgesic

Suitable drugs belonging to the classes of anti-inflammatory/analgesic agents described above, and suitable concentrations for use in the solutions of the invention are described below. While not wishing to be bound by theory, the rationale for selecting the classes of agents believed to confer their effectiveness is set forth below.

A. Serotonin receptor antagonists

Serotonin (5-HT) is thought to produce pain by stimulating serotonin 2 (5-HT ₂ ) and/or serotonin 3 (5-HT ₃ ) receptors on peripheral nociceptive neurons. Most researchers agree that 5-HT3 receptors on peripheral nociceptors mediate the immediate pain sensation produced by 5-HT (Richardson et al., 1985). 5-HT ₃ receptor antagonists can inhibit neurogenic inflammation in addition to inhibiting 5-HT-induced pain by inhibiting the activation of nociceptors. See "Modulation of Neurogenic Inflammation: Novel Approaches to Inflammatory Disease" by Barnes PJ et al., Trends in Pharmacological Sciences 11: 185-189 (1990). However, a study in rat ankle joints claimed that 5-HT ₂ receptors cause activation of nociceptors via 5-HT. See "A Study of 5-HT-Receptors Associated with Afferent Nerves Located in Normal and Inflamed Rat Ankle Joints" by Grubb, BD et al. in Agents Actions 25:216-18 (1988). The study of the 5-HT-receptor connected to the afferent nerve)". Thus, activation of 5-HT2 _receptors may also play a role in peripheral pain and neurogenic inflammation.

One use of the solutions of the invention is to block pain and a host of inflammatory processes. Therefore, both 5-HT ₂ and 5-HT ₃ receptor antagonists are suitable to be used alone or in combination in the solution of the present invention, as will be described below. Amitriptyline (Elavil ^™ ) is a 5- _HT2 receptor antagonist suitable for use in the present invention. Amitriptyline has been used clinically for many years as an antidepressant and has been found to have beneficial effects in some patients with chronic pain. Memethopamide (Reglan ^TM ) is clinically used as an antiemetic drug, but it exhibits moderate affinity for the 5-HT ₃ receptor and can inhibit the action of 5-HT on this receptor, which is responsible for pain Inhibition may be due to release of 5-HT from platelets. Therefore, this agent is also suitable for use in the present invention.

Other suitable 5- _HT2 receptor antagonists include imipramine, trazodone, desipramine and ketanserin. Keteserin has been clinically used for its antihypertensive effect. See "Effects of a New Serotonin Antagonist, Ketanserin, in Experimental and Clinical Hypertension" by Hedner, T. et al., Am.J.of Hypertension pp. 317s-23s (July 1988). role in experimental and clinical hypertension)". Other suitable 5- _HT3 receptor antagonists include cisapride and ondansetron. Cardiovascular and general vascular solutions may also contain serotonin _1B (also known as serotonin _1Dβ ) antagonists because serotonin has been shown to cause significant vasospasm by activating serotonin _1B receptors in humans. See "Variable Participation of 5-HT1-Like Receptors and 5-HT2 Receptors nSerotonin-Induced Contract of Human Isolated Coronary Arteries (5-HT1-Like Receptors and 5- _HT2 Receptors nSerotonin-Induced Contract of Variable participation of _-HT2 receptors in serotonin-induced constriction of human isolated coronary arteries)". 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-carbamoyl (“GR127935”) and methiothepin. Table 5 provides these drugs are suitable for Treatments and preferred concentrations of the solutions of the invention.

table 5

Treatment and preferred concentrations of pain/inflammation inhibitors Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) Serotonin ₂ receptor antagonists: amitriptyline imipramine trazodone desipramine ketaserin Serotonin ₃ receptor antagonists: tripixilone metoclopramide cisapride emdansetron serotonin _1B (Human 1D _B ) Antagonist: Yohimbine GR127935methiothepin 0.1-1,0000.1-1,0000.1-2,0000.1-1,0000.1-1,0000.01-10010-10,0000.1-1,0000.1-1,0000.1-1,0000.1-1,0000.1-500 50-50050-50050-50050-50050-5000.05-50200-2,00020-20020-20050-50010-5001-100

B. Serotonin receptor agonists

The 5-HT _1A , 5-HT _1B and 5-HT _1D receptors are all known to inhibit adenylate cyclase activity. Therefore, the addition of low doses of serotonin _1A , serotonin _1B and serotonin _1D receptor agonists to the solutions of the invention should inhibit neurons that mediate pain and inflammation. Agonists of the serotonin _1B and serotonin _1F receptors are thought to have the same effect, since these two receptors also inhibit adenylate cyclase.

Buspirone is a suitable 1A receptor agonist for use in the present invention. Sumatriptan is a suitable 1A, 1B, 1D and 1F receptor agonist. A suitable 1B and 1D receptor agonist is dihydroergotamine. A suitable 1E receptor agonist is ergometrine. Table 6 provides suitable treatments and preferred concentrations for these receptor agonists.

Table 6

Treatment and preferred concentrations of pain/inflammation inhibitors Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) Serotonin _1A agonist: Buspirone Sumatriptan Serotonin _1B agonist: Dihydroergotamine Sumatriptan Serotonin _1D agonist: Dihydroergotamine Sumatriptan Serotonin _1B agonist: Ergonov Base serotonin _1F agonist: sumatriptan 1-1,0001-1,0000.1-1,0001-1,0000.1-1,0001-1,00010-2,0001-1,000 10-20010-20010-10010-20010-10010-200100-1,00010-200

C. Histamine receptor antagonists

Histamine receptors are generally divided into histamine ₁ (H ₁ ) and histamine ₂ (H ₂ ) subtypes. The typical inflammatory response to peripherally administered histamine is mediated by the _H1 receptor. See Douglas, 1985. Accordingly, the solutions of the invention preferably comprise a histamine _H1 receptor antagonist. Promethazine (Phenergan ^TM ) is commonly used as an antiemetic drug, which can effectively block _H1 receptors, and is suitable for use in the present invention. Interestingly, the drug has also been shown to have local anesthetic effects, but at concentrations several orders of magnitude higher than those required to block _H1 receptors, so the two effects are thought to arise through different mechanisms of. The concentration of the histamine receptor antagonist in the solution of the present invention is sufficient to inhibit the _H1 receptors involved in the activation of nociceptors, but does not produce a "local anesthetic" effect, thereby avoiding related systemic side effects.

Histamine receptors are also known to mediate vasomotor tone in coronary arteries. In vitro studies in the human heart have demonstrated that the histamine ₁ receptor subtype mediates contraction of coronary smooth muscle. See "Histamine Provocation of Clinical Coronary Artery Spasm: Implications Concerning Pathogenesis of Variant Angina Pectoris" by Ginsburg, R. et al. in American Heart J. 102:819-822 (1980). Inference of the pathogenesis of angina pectoris)". Certain studies indicate that histamine-induced hypercontractility in the human coronary system is most pronounced in proximal arteries and associated denudation on the arterial endothelium in the setting of atherosclerosis. See Keitoku, M. et al. "Different Histamine Actions in Proximal and Distal Human Coronary Arteries in Vitro" in Cardiovascular Research 24:614-622 (1990) . Accordingly, histamine receptor antagonists may be included in the cardiovascular irrigating solutions of the present invention.

Other suitable _H1 receptor antagonists include terfenadine, diphenhydramine, amitriptyline, mepyramine and pyropyramine. Since amitriptyline is also a potent serotonin ₂ receptor antagonist, it serves a dual function when used in the present invention. Table 7 provides suitable therapeutic and preferred concentrations of these _Hi receptor antagonists.

Table 7

Treatment and preferred concentrations of pain/inflammation inhibitors Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) Histamine ₁ receptor antagonists: Promethazine Diphenhydramine Amitriptyline Terfenadine Mepyramine (Pyramine) Pyrropyramide 0.1-1,0000.1-1,0000.1-1,0000.1-1,0000.1-1,0000.01-100 50-20050-20050-50050-5005-2005-20

D. Bradykinin receptor antagonists

Bradykinin receptors are generally divided into bradykinin ₁ (B ₁ ) and bradykinin ₂ (B ₂ ) subtypes. Studies have found that acute peripheral pain and inflammation produced by bradykinin is mediated by the _B2 subtype, while bradykinin-induced pain in chronic inflammatory situations is mediated by the _B1 subtype. See Perkins, MN et al. "Antinociceptive Activity of the Bradykinin B1 and B2 Receptor Antagonists, des-Arg9, [Leu ⁸ ]-BK and HOE 140, in Two Models of Persistent Hyperalgesia in Pain 53: 191-97 (1993)" Rat (Antagonist of Bradykinin B1 and B2 Receptors, Antinociceptive Activity of des-Arg ⁹ , [Leu ⁸ ]-BK and HOE 140 in Two Rat Models of Persistent Hyperalgesia)”; Dray, A., et al., "Bradykinin and Inflammatory Pain," Trends Neurosci. 16:99-104 (1993), each of which references is incorporated herein by reference.

Currently, bradykinin receptor antagonists have not been used clinically. These drugs are peptides (small proteins) and cannot be taken orally because they cannot be digested. _B2 receptor antagonists block bradykinin-induced acute pain and inflammation. See Dray et al., 1993. _B1 receptor antagonists inhibit the pain that occurs in chronic inflammatory disorders. See Perkins et al., 1993 and Dray et al., 1993. Thus, depending on the application, the solution of the invention preferably includes one or both of the bradykinin _B1 and _B2 receptor antagonists. For example, when arthroscopy is performed in the context of both acute and chronic conditions, the irrigation fluid used may include both _B1 and _B2 receptor antagonists.

Bradykinin receptor antagonists suitable for use in the present invention include the following bradykinin ₁ receptor antagonists: [des-Arg ¹⁰ ] of D-Arg-(Hyp ³ -Thi ⁵ -D-Tic ⁷ -Oic ⁸ )-BK derivatives ("[des-Arg ¹⁰ ] derivatives of HOE140", 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 ("NPC 349"); D- Arg-( ^Hyp3 -D- ^Phe7 )-BK("NPC 567"); and D-Arg-( ^Hyp3 - ^Thi5 -D- ^Tic7 - ^Oic8 )-BK("HOE 140"). These compounds are more fully described in Perkins et al. 1993 and Dray et al. 1993, incorporated by reference above. Table 8 provides suitable treatments and preferred concentrations for these drugs.

Table 8

Treatment and preferred concentrations of pain/inflammation inhibitors Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) Bradykinin ₁ receptor antagonist: [Leu ⁸ ][des-Arg ¹⁰ ] derivative [Leu ⁹ ][des-Arg ¹⁰ ]vasodilator (kalliden) bradykinin ₂ receptor of [Leu ⁸ ]des-Arg 9 -BKHOE140 Antagonist: [D-Phe ⁷ ]-BKNPC 349NPC 567HOE 140 1-1,0001-1,0000.1-500100-10,0001-1,0001-1,0001-1,000 50-50050-50010-200200-5,00050-50050-50050-500

E. Kallikrein Inhibitors

As mentioned above, bradykinin is an important mediator of pain and inflammation. Bradykinin is a cleavage product of kallikrein acting on high molecular weight kininogen in plasma. Therefore, kallikrein inhibitors are considered to be effective in inhibiting bradykinin production and the resulting pain and inflammation. A kallikrein inhibitor suitable for use in the present invention is aprotinin.

Table 9 below provides useful concentrations for solutions of the invention.

Table 9

Treatment and preferred concentrations of pain/inflammation inhibitors Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) Kallikrein Inhibitors: Aprotinin 0.1-1,000 50-500

F. Tachykinin receptor antagonists

Tachykinins (TKs) are a family of structurally related peptides that include substance P, neurokinin A (NKA) and neurokinin B (NKB). Neurons are the main source of TKs in the periphery. An important general effect of TKs is neuronal stimulation, but others include endothelium-dependent vasodilation, plasma protein extravasation, mast cell recruitment and degranulation, and stimulation of inflammatory cells. See Maggi, C.A., Gen. Pharmacol. 22:1-24 (1991). Since the combination of these physiological effects is mediated by TK receptor activation, anchoring TK receptors is a logical approach to promote analgesia and treat neurogenic inflammation.

1. Neurokinin 1 receptor subtype antagonists

Substance P activates a neurokinin receptor subtype known as _NK1 . Substance P is an eleven amino acid polypeptide found in sensory nerve endings. Substance P is known to have multiple effects, including vasodilation, plasma extravasation, and mast cell degranulation, which can produce inflammation and pain in the periphery following C-fiber activation. See "Peptides and the Primary Afferent Nociceptor" by Levine, JD et al. in J. Neurosci. 13:2273 (1993). A suitable substance P antagonist is ([D-Pro ⁹ [spiro-gamma-lactam]Leu ¹⁰ , Trp ¹¹ ]bambowin-(1-11)) ("GR82334"). Other suitable antagonists suitable for use in the present invention and acting on _NK1 receptors are: 1-imino-2-(2-methoxy-phenyl)-ethyl)-7,7-diphenyl-4 - perhydroisoindolinone (3aR, 7aR) ("RP 67580"); and 2S,3S-cis-3-(2-methoxybenzylamino)-2-benzhydrylquinonucine ("RP 67580"); CP 96,345"). Table 10 provides useful concentrations of these agents.

Table 10

Treatment and preferred concentrations of pain/inflammation inhibitors Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) Neurokinin ₁ receptor subtype antagonists: GR 82334CP 96,345RP 67580 1-1,0001-10,0000.1-1,000 10-500100-1,000100-1,000

2. Neurokinin ₂ receptor subtype antagonists

Neurokinin A, a peptide that colocalizes with substance P in sensory neurons, also promotes inflammation and pain. Neurokinin A activates a specific neurokinin receptor known as _NK2 . See Edmonds-Alt, S. et al. "Potent and Selective Non-Peptide Antagonist of the Neurokinin A (NK ₂ ) Receptor (Potent and Selective Non-Peptide Antagonist of the Neurokinin A (NK ₂ ) Receptor" in Life Sci.50: PL101 (1992). and selective non-peptide antagonists)". In the urinary tract, TKs are specific spasmodic agents acting exclusively through _NK2 receptors in the human bladder, and in the human urethra and ureters. See Maggi, CA, Gen. Pharmacol. 22:1-24 (1991). Therefore, an ideal drug to be included in surgical solutions used in urological procedures would contain an _NK2 receptor antagonist to reduce spasticity. Examples of suitable _NK2 receptor antagonists include: ((S)-N-methyl-N-[4-(4-acetamido-4-phenylpiperidinyl)-2-(3,4-dichloro phenyl)butyl]benzamide (“(±)-SR 48968”); Met-Asp-Trp-Phe-Dap-Leu (“MEN 10,627”); and Cyclic (Gln-Trp-Phe-Gly-Leu -Met) ("L659,877"). Table 11 provides suitable concentrations of these agents.

Table 11

Treatment and preferred concentrations of pain/inflammation inhibitors Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) Neurokinin ₂ receptor subtype antagonist: MEN10,627L659,877(±)-SR 48968 1-1,00010-10,00010-10,000 10-1,000100-10,000100-10,000

G. CGRP receptor antagonist

Calcitonin gene-related peptide (CGRP) is also a peptide that co-localizes with substance P in sensory neurons, acting as a vasodilator and enhancing the effect of substance P. See Brain, S.D. et al. "Inflammatory Oedema Induced by Synergism Between Calcitonin Gene-Related Peptide (CGRP) and Mediators of Increased Vascular Permeability (Calcitonin Gene-Related Peptide (CGRP) ) and mediators that can increase vascular permeability induced inflammatory edema)". An example of a suitable CGRP receptor antagonist is α-CGRP-(8-37), which is a truncated model of CGRP. The polypeptide can inhibit the activation of CGRP receptor. Table 12 provides suitable concentrations of this agent.

Table 12

Treatment and preferred concentrations of pain/inflammation inhibitors Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) CGRP receptor antagonist: α-CGRP-(8-37) 1-1,000 10-500

H. Interleukin receptor antagonists

Interleukins are a family of peptides and cytokines that are produced by white blood cells and other cells in response to inflammatory mediators. Interleukins (IL) can be potent peripheral hyperalgesia agents. See "Interleukin-1β as a Potent Hyperalgesic Agent Antagonized by a Tripeptide Analogue" by Ferriera, S.H. et al., Nature 334:698 (1988). An example of a suitable IL-1β receptor antagonist is Lys-D-Pro-Thr, which is a truncated model of IL-1β. This tripeptide inhibits the activation of the IL-1β receptor. Table 13 provides useful concentrations for this agent.

Table 13

Treatment and preferred concentrations of pain/inflammation inhibitors Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) Interleukin Receptor Antagonist: Lys-D-Pro-Thr 1-1,000 10-500

I. Inhibitors of Enzymes Functioning in the Synthetic Pathway of Arachidonic Acid Metabolites

1. Phospholipase inhibitors

The generation of arachidonic acid by phospholipase A ₂ (PLA ₂ ) leads to a cascade of reactions that generate a variety of inflammatory mediators known as eicosanoids. Many stages throughout this pathway can be inhibited, thereby reducing the production of the inflammatory mediators mentioned above. Examples of achieving inhibition at these different stages are provided below.

Inhibition of the enzyme PLA ₂ isoform inhibits the release of arachidonic acid from cell membranes, thereby suppressing the production of prostaglandins and leukotrienes, leading to a reduction in inflammation and pain. See Glaser, KB "Regulation of Phospholipase A2 Enzymes: Selective Inhibitors and Their Pharmacological Potential" in Adv. Pharmacol. 32:31 (1995). An example of a suitable PLA ₂ isoform inhibitor is manoalide. Table 14 provides suitable concentrations of this agent. Inhibition of phospholipase C _gamma (PLC _gamma ) isoforms will also lead to decreased production of prostaglandins and leukotrienes, and thus less pain and inflammation. An example of a PLC _gamma isoform inhibitor is 1-[6-((17β-3-methoxyestradiol-1,3,5(10)-trien-17-yl)amino)hexyl]-1H- Pyrrole-2,5-dione.

Table 14

Treatment and preferred concentrations of pain/inflammation inhibitors Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) PLA ₂ isoform inhibitor: manoalide 100-100,000 500-10,000

2. Cyclooxygenase inhibitors

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used as anti-inflammatory, antipyretic, anticoagulant and analgesic agents. See Lewis, R.A. "Prostaglandins and Leukotrienes" in Textbook of Rheumatology, Third Edition (edited by Kelley W.N. et al.) p. 258 (1989). The molecular targets of these drugs are type I and type II cyclooxygenases (COX-1 and COX-2). Also known as prostaglandin H synthase (PGHS)-1 (constitutive) and -2 (inducible), these enzymes catalyze the conversion of arachidonic acid to prostaglandin H, which is the prostaglandin and Thrombin is an intermediate in the biosynthesis process. Endothelial cells, macrophages and fibroblasts have been determined to contain the COX-2 enzyme. The enzyme is induced by IL-1 and endotoxin, and its expression at sites of inflammation is upregulated. Both constitutive activity of COX-1 and inducible activity of COX-2 lead to the synthesis of prostaglandins, which contribute to pain and inflammation.

NSAIDs currently on the market (diclofenac, naproxen, indomethacin, ibuprofen, etc.) are generally non-selective inhibitors of both COX isoforms, although the selectivity of different compounds for both COX isoforms The ratios vary, but all show selectivity for COX-1 over COX-2. Blocking the formation of prostaglandins using COX-1 and 2 inhibitors represents a more effective treatment than trying to block the interaction between this natural ligand and the seven subtypes of prostanoid hormone receptors mentioned above plan. The reported antagonists of eicosanoid receptors (EP-1, EP-2, EP-3) are quite rare, and only the specific and high-affinity antagonists of thromboxane A2 receptors have been reported . See Wallace, J. and Cirino. G., Trends in Pharm Sci. 15:405-406 (1994).

Oral, intravenous or intramuscular administration of cyclooxygenase inhibitors to patients with ulcers, gastritis or renal failure is contraindicated. In the United States, the only injectable form of this drug available is ketorolac (Toradol ^TM ) available from Syntex Pharmaceuticals, which is usually administered intramuscularly or intravenously to postoperative patients, but is still contraindicated for the above conditions of patients. The use of ketorolac or any other cyclooxygenase inhibitor in the solution of the present invention at doses much lower than those currently required for perioperative use enables the use of the drug in patients with other contraindications. The addition of cyclooxygenase inhibitors to the solutions of the present invention adds a unique mechanism for inhibiting the development of pain and inflammation during arthroscopic or other therapeutic or diagnostic procedures.

Preferred cyclooxygenase inhibitors for use in the present invention are ketorolac and indomethacin. Of these two agents, indomethacin is less preferred because its use requires relatively high doses. Table 15 provides the therapeutic and preferred concentrations of these agents suitable for use in solutions of the invention.

Table 15

Treatment and preferred concentrations of pain/inflammation inhibitors Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) Cyclooxygenase inhibitors: ketorolac, indomethacin 100-10,000 1,000-500,000 500-5,00010,000-200,000

3. Lipoxygenase inhibitors

Inhibition of lipoxygenase inhibits the production of leukotrienes, such as leukotriene _B4 , which are known to be important mediators of inflammation and pain. See "Prostaglandins and Leukotrienes" by Lewis, RA, Textbook of Rheumatology 3rd Ed. (Edited by Kelley WN et al.) p. 258 (1989). An example of a 5-lipoxygenase antagonist is 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodeynyl)-1,4-benzoquinone ("AA861") , Table 16 provides suitable concentrations of this agent.

Table 16

Treatment and preferred concentrations of pain/inflammation inhibitors Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) Lipoxygenase inhibitor: AA861 100-10,000 500-5,000

J. Prostaglandin hormone receptor antagonists

Specific prostanoids produced as metabolites of arachidonic acid mediate their inflammatory effects through activation of prostanoid receptors. Examples of classes of specific prostanoid hormone antagonists are eicosanoid EP-1 and EP-4 receptor subtype antagonists, and thromboxane receptor subtype antagonists. A suitable prostaglandin _E 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 ("SQ 29548"). Table 17 provides suitable concentrations of these agents.

Table 17

Treatment and preferred concentrations of pain/inflammation inhibitors Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) Eicosanoid EP-1 antagonist: SC 19220 100-10,000 500-5,000

K. Leukotriene receptor antagonists

Leukotrienes (LTB ₄ , LTC ₄ and LTD ₄ ) are the products of arachidonic acid metabolized by the 5-lipoxygenase pathway, which are produced enzymatically and have important biological properties. Leukotrienes are involved in a number of pathological conditions, including inflammation. Currently, many pharmaceutical companies are developing specific antagonists for potential therapeutic intervention in the aforementioned pathologies. See Halushka, PV et al., Annu. Rev. Pharmacol. Toxicol. 29:213-239 (1989); Ford-Hutchinson, A., Crit. Rev. Immunol. 10:1-12 (1990). The LTB ₄ receptor is found on certain immune cells, including eosinophils and neutrophils. Binding of LTB ₄ to the aforementioned receptors leads to chemotaxis and release of lysosomal enzymes that contribute to the inflammatory process. Signal transduction processes associated with _LTB4 receptor activation include G-protein-mediated stimulation of phosphatidylinositol (PI) metabolism and increased intracellular calcium (see Figure 2).

An example of a suitable leukotriene _B4 receptor antagonist is SC(+)-(S)-7-(3-(2-(cyclopropylmethyl)-3-methoxy-4-[(methylamino) -Carbonyl]phenoxy(propoxy)-3,4-dihydro-8-propyl-2H-1-chromene-2-propionic acid ("SC 53228"). Table 18 provides the agent Concentrations suitable for the practice of the present invention. Other suitable leukotriene _B receptor antagonists include [3-[-2(7-chloro-2-quinolyl)vinyl]phenyl][[3-( Dimethylamino-3-oxopropyl)thio]methyl]thiopropionic acid ("MK 0571") and the drug LY66,071 and ICI20,3219. MK 0571 may also act as an LTD ₄ receptor subtype antagonist.

Table 18

Treatment and preferred concentrations of pain/inflammation inhibitors Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) Leukotriene _R4 antagonist: SC 53228 100-10,000 500-5,000

L. Opioid receptor agonists

Activation of opioid receptors produces antinociceptive effects, therefore, agonists of these receptors are desirable agents. Opioid receptors include mu-, delta-, and kappa-opioid receptor subtypes. μ-receptors are located on peripheral sensory neuron terminals, and activation of the receptors inhibits sensory neuron activity. See "Opiate analgesia: How Central is a Peripheral Target?" in Basbaum, AI et al., N. Ehgl. J. Med. 325: 1168 (1991). Delta- and kappa-receptors are located on sympathetic afferent terminals and inhibit the release of prostaglandins, thereby inhibiting pain and inflammation. See Taiwo, YO et al., "Kappa- and Delta-Opioids Block Sympathetically Dependent Hyperalgesia" in J. Neurosci. 11:928 (1991). The opioid receptor subtypes are members of the G protein-coupled receptor superfamily. Thus, all opioid receptor agonists interact and signal via their cognate G protein coupled receptors (see Figures 3 and 7). Examples of suitable mu-opioid receptor agonists are fentanyl and Try-D-Ala-Gly-[N-MePhe]-NH( _CH2 )-OH ("DAMGO"). An example of a suitable delta-opioid receptor agonist is [D-Pen ² , D-Pen ⁵ ]enkephalin ("DPDPB"). An example of a suitable kappa-opioid receptor agonist is (trans)-3,4-diamino-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-phenylacetamide ("U50,488"). Table 19 provides suitable concentrations of these agents.

Table 19

Treatment and preferred concentrations of pain/inflammation inhibitors Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) μ-Opioid Agonist: DAMGO Sufentanil Fentanyl PL 017 Delta-Opioid Agonist: DPDPE Kappa-Opioid Agonist: U50,488 0.1-1000.01-500.1-5000.05-500.1-5000.1-500 0.5-201-2010-2000.25-101.0-100l.0-100

M. Purinergic receptor antagonists and agonists

Extracellular ATP acts as a signaling molecule by interacting with _P2 purinoceptors. A large class of purinergic receptors is the P _2X purinergic receptors, which are ligand-gated ion channels with intrinsic ion channels permeable to Na ⁺ , K ⁺ and Ca ²⁺ . _P2X receptors described in sensory neurons are important for primary afferent neurotransmission and nociception. ATP is known to depolarize sensory neurons and plays a role in nociceptor activation, as ATP released from damaged cells stimulates _P2X receptors, leading to depolarization of nociceptive nerve-fiber endings. polarization. The distribution of the _P2X3 receptor is highly restricted (Chen, CC, et al., Natute 377:428-431 (1995)) because it is expressed selectively in sensory C-fiber nerves extending into the spinal cord, of which there are many The C-fibers are thought to carry receptors for pain stimuli. Thus, the highly restricted localized expression of _P2X3 receptor subunits makes these subtypes excellent targets for analgesic effects (see Figures 3 and 7).

_P2X /ATP purinoceptor antagonists suitable for use in the present invention include, for example, suramin and pyridoxal-6-phenylazo-2,4-disulfonic acid ("PPADS"). Table 20 provides suitable concentrations of these agents.

Agonists of _P2Y receptors, a G-protein coupled receptor, are known to affect smooth muscle relaxation by increasing inositol triphosphate ( _IP3 ) levels, which in turn increases intracellular calcium. An example of a _P2Y receptor agonist is 2-me-S-ATP.

Table 20

Treatment and preferred concentrations of pain/inflammation inhibitors Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) Purinergic receptor antagonists: suramin PPADS 100-100,000100-100,000 10,000-100,00010,000-100,000

N. Adenosine triphosphate (ATP)-sensitive potassium channel opener

ATP-sensitive potassium channels are found in many tissues, including vascular and nonvascular smooth muscle and the brain, and studies in conjunction with radiolabeled ligands have confirmed their presence. Opening of these channels results in potassium (K ⁺ ) efflux and hyperpolarization of the cell membrane (see Figure 2). This hyperpolarization leads to a decrease in intracellular free calcium through inhibition of both voltage-dependent calcium (Ca ²⁺ ) channels and receptor-operated Ca ²⁺ channels. These combined actions push cells (eg, smooth muscle cells) into a state of relaxation or a state that is more resistant to activation and, in the case of vascular smooth muscle, result in vasodilation. K ⁺ channel openers (KCOs) are characterized by potent antihypertensive activity in vivo, and vasodilatory activity in vitro (see Figure 4). K ⁺ channel openers (KCOs) have also been shown to prevent stimuli-coupled secretion and are thought to act on preconnected neuronal receptors, thereby inhibiting the consequences of neurostimulation and release of inflammatory mediators. See Quast, U., et al. "Cellular Pharmacology of Potassium Channel Openers in Vascular Smooth Muscle" in Cardiovasc. Res. 28:805-810 (1994).

A synergistic interaction between endothelin (ET _A ) antagonists and ATP-sensitive potassium channel openers (KCOs) is thought to achieve vasodilation or smooth muscle relaxation. The rationale for this dual use is the fact that these drugs have different molecular mechanisms of action in promoting smooth muscle relaxation and preventing vasospasm. In smooth muscle cells, the initial increase in intracellular calcium induced by _ETA receptors subsequently triggers the activation of voltage-dependent channels and the entry of extracellular calcium, which is required for contraction. Antagonists of _{the ETA} receptor specifically block this receptor-mediated effect without blocking the calcium increase triggered by the activation of other G-protein coupled receptors on muscle cells.

Potassium channel opener drugs, such as pinacidil, open these channels, allowing K ⁺ to flow out and hyperpolarize the cell membrane. This hyperpolarization will reduce other receptor-mediated constriction through the following mechanisms: (1) Inhibition of voltage-dependent Ca2 ⁺ channels by reducing the likelihood of opening L-type or T-type calcium channels so that Intracellular free calcium is reduced, (2) by inhibiting inositol triphosphate (IP ₃ ) formation, inhibiting Ca ²⁺ release from intracellular sources induced by agonists (receptor-operated channels), and (3) reducing calcium as Efficiency of contractile protein activators. Thus, the combined action of the above two classes of drugs can push target cells into a state of relaxation or a state that is more resistant to activation.

ATP-sensitive K ^channel openers suitable for implementing the present invention include: (-) pinacidil; Crocarin; Nicorandil; Minoxidil; N-cyano-N'-[1,1 -Dimethyl-[2,2,3,3-3H]propyl]-N″-(3-pyrimidinyl)guanidine (“P1075”); and N-cyano-N′-(2-nitro Ethoxy)-3-pyridinecarboxamidine monomethanesulfonate ("KRN2391"). Table 21 provides the concentrations of these agents.

Table 21

Treatment and preferred concentrations of pain/inflammation inhibitors Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) ATP-sensitive K ⁺ channel openers: Crocalin Nicodil Minoxidil P 1075 KRN 2391(-) Pinacidil 10-10,00010-10,00010-10,0000.1-1,0001-10,0001-10,000 100-10,000100-10,000100-10,00010-1,000100-1,000100-1,000

III. Antispasmodics

A. Multifunctional Potion

Some of the above analgesic/anti-inflammatory agents may also inhibit vasoconstriction or smooth muscle spasm. Therefore, these agents also function as antispasmodics, which can be beneficial in vascular and urological applications. Anti-inflammatory/analgesic agents that may also act as antispasmodics include: serotonin receptor antagonists, especially serotonin 2 receptor antagonists; tachykinin receptor antagonists and ATP-sensitive potassium channel openers.

B. Nitric Oxide Donors

Nitric oxide may also be included in the solutions of the invention due to its antispasmodic activity. Nitric oxide (NO) plays a key role as a molecular mediator of many physiological processes, including vasodilation and regulation of normal vascular tone. In endothelial cells, an enzyme called NO synthase (NOS) catalyzes the conversion of L-arginine to NO, which acts as a diffusible second messenger and mediates response (see Figure 8). NO is continuously formed and released by the vascular endothelium under basal conditions, inhibits contraction and controls basal coronary tone, and is also produced in the endothelium in response to various agonists such as acetylcholine and other endothelium-dependent vasodilators. Therefore, modulation of NO synthase activity and corresponding NO levels is a key molecular target in the control of vascular tone (see Figure 8). See Muramatsu, K., et al., Coron. Artery Dis. 5:815-820 (1994).

A synergistic interaction between NO donors and openers of ATP-sensitive potassium channels (KCOs) is thought to achieve vasodilation or smooth muscle relaxation. The rationale for this dual use is the fact that these drugs have different molecular mechanisms of action in promoting smooth muscle relaxation and preventing vasospasm. Vasoconstrictors that can be demonstrated in cultured coronary artery smooth muscle cells are: vasopressin, angiotensin II and endothelin, which all inhibit K _ATP currents by inhibiting protein kinase A. In addition, it has been reported that K _ATP currents in bladder smooth muscle are inhibited by muscarinic agonists. The role of NO in mediating smooth muscle relaxation occurs through a separate molecular pathway (described above) including protein kinase G (see Figure 8). This indicates that the combination of the above two classes of agents is more effective in relaxing smooth muscle than one class of agents used alone.

Nitric oxide donors suitable for practicing the present invention include nitroglycerin, sodium nitroprusside, the drug FK409, FR 144420, 3-morpholinosyrtonimine or lincidomine hydrochloride, ("SIN-1"); and S-nitroso-N-acetylpenicillamine ("SNAP"). Table 22 provides the concentrations of these agents.

Table 22

Spasticity inhibitor treatment and preferred concentration Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) Nitric oxide donor: Sodium Nitroprusside SIN-1SNAPFK409(NOR-3)FR 144420(NOR-4) 10-10,00010-10,00010-10,00010-10,001-1,00010-10,000 100-1,000100-1,000100-1,000100-1,00010-500100-5,000

C. Endothelin receptor antagonists

Endothelin is a 21 amino acid peptide and one of the most potent vasoconstrictors known. Three different human endothelins, designated ET-1, ET-2, and ET-3, have been described to mediate through at least two receptor subtypes, the ET _A and ET _B receptors. their physiological roles. Cardiac and vascular smooth muscle primarily contain _ETA receptors and this subtype is responsible for contraction in these tissues. In addition, _ETA receptors are often found to mediate contractile responses in isolated smooth muscle specimens. Studies have found that _ETA receptor antagonists are effective antagonists of human coronary artery constriction. Thus, ETA _- receptor antagonists should be therapeutically helpful in the perioperative suppression of coronary vasospasm and may also help in the suppression of smooth muscle contraction in urological applications. See Miller, RC et al. Trends in Pharmacol. Sci. 14:54-60 (1993).

Suitable endothelin receptor antagonists include: cyclic (D-Asp-Pro-D-Val-Leu-D-Trp) ("BQ123"); (N,N-cyclohexamethylene)-aminomethyl Acyl-Leu-D-Trp-(CHO)-D-Trp-OH (“BQ610”); (R)2-([R-2-[(s)-2-([1-hexahydro-1H- Azepanyl]-carbonyl)amino-4-methyl-pentanoyl]amino-3-(3[1-methyl-1H-indolyl])propionylamino-3(2-pyridyl)propanoic acid ("FR139317"); Ring (D-Asp-Pro-D-Ile-Leu-D-Trp) ("JKC301"); Ring (D-Ser-Pro-D-Val-Leu-D-Trp) (" JK302"); 5-(dimethylamino)-N-(3,4-dimethyl-5-isoxazolyl)-1-naphthalenesulfonamide ("BMS182874"); and N-[1-formyl-N -[N-[(Hexahydro-1H-azepin-1-yl)carbonyl]-L-leucyl]-D-tryptophanyl]-D-tryptophan ("BQ610"). Table 23 provides Concentrations of three representative agents among the above-mentioned agents are shown.

Table 23

Spasticity inhibitor treatment and preferred concentration Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) Endothelin receptor antagonist: BQ 123FR 139317BQ 610 0.01-1,0001-100,0000.01 to 10,000 10-1,000100-10,00010-1,000

D. Ca ²⁺ channel antagonists

Calcium channel antagonists are a unique class of drugs that interfere with the transmembrane efflux of specific calcium ions required to activate cellular responses that mediate neuroinflammation. Calcium entry into platelets and leukocytes is a key event mediating the activation of responses in these cells. In addition, the role of bradykinin receptors and neurokinin receptors ( _NK1 and _NK2 ) in mediating neuroinflammatory signaling pathways includes increasing intracellular calcium, leading to activation of calcium channels on the plasma membrane. Calcium channel antagonists, such as nifedipine, reduce the release of arachidonic acid, prostaglandins and leukotrienes induced by various stimuli in many tissues. See Monoada, S., Flower, R. and Vane, J., MacMillan Publ. Inc., Goodman's and Gilman's Pharmacological Basis of Therapeutics (Seventh Edition) (1995), pp. 660-5.

Calcium channel antagonists also interfere with the transmembrane efflux of calcium ions required for vascular smooth muscle contraction. This effect provides a rationale for the perioperative use of calcium channel antagonists during procedures to relieve vasospasm and promote smooth muscle relaxation. Dihydropyridines, including nisoldipine, act as specific inhibitors (antagonists) of the voltage-dependent gating of L-type calcium channel subtypes. Systemic administration of the calcium channel antagonist nifedipine has been used during cardiac surgery to prevent or minimize coronary vasospasm. See Seitelberger, R., et al., Circulation 83:460-468 (1991).

Among the antispasmodics applicable to the present invention, calcium channel antagonists may exhibit a synergistic effect when used in combination with other agents of the present invention. Calcium (Ca ²⁺ ) channel antagonists and nitric oxide (NO) donors may interact in effecting vasodilation or smooth muscle relaxation, ie inhibition of spastic activity. The rationale for this dual use is the fact that the two classes of drugs have different molecular mechanisms of action, are unlikely to be fully effective alone in achieving relaxation, and that the two classes of drugs may have different durations of action. In fact, a large number of studies have shown that the application of calcium channel antagonists alone cannot completely relax the vascular muscles that have been preconstricted by receptor agonists.

The effect of nisoldipine alone and in combination with nitroglycerin on spasm of the internal mammary artery (IMA) showed a large positive synergistic effect of the combination of the two drugs in preventing constriction (Liu et al., 1994) . These studies provide a scientific basis for the combination of calcium channel antagonists and nitric oxide (NO) donors to effectively prevent vasospasm and smooth muscle relaxation. Examples of systemic administration of nitroglycerin and nifedipine to prevent and treat myocardial ischemia or coronary vasospasm during cardiac surgery have been reported (Cohen et al., 1983; Seitelberger et al., 1991).

Calcium channel antagonists have also shown synergistic effects with endothelin receptor subtype A (ET _A ) antagonists. Yanagisawa and co-workers observed that dihydropyridine antagonists blocked the action of ET-1, an endogenous agonist on _ETA receptors in coronary artery smooth muscle, and speculated that ET-1 is an endogenous regulator of voltage-sensitive calcium channels. source agonists. We found that, in smooth muscle cells, the persistence of the increase in intracellular calcium induced by _ETA receptor activation required the participation of extracellular calcium and was at least partially blocked by nicardipine. Therefore, it is expected that when a calcium channel antagonist is added to a surgical solution, the antagonist will synergistically enhance the effect of the _ETA antagonist contained in combination with the surgical solution.

Calcium channel antagonists and ATP-sensitive potassium channel openers have also shown synergy. ATP-sensitive potassium channels (K _ATP ) couple the membrane potential of a cell to the metabolic state of the cell through their sensitivity to adenosine nucleotides. K _ATP channels are inhibited by intracellular ATP but stimulated by intracellular nucleoside diphosphates. The activity of these channels is controlled by electrochemical driving forces on potassium and intracellular signals such as ATP or G-proteins, but is not gated by the membrane potential itself. K _ATP channels hyperpolarize the membrane, thereby enabling it to control the resting potential of the cell. ATP-sensitive potassium currents are found in skeletal muscle, brain, and vascular and nonvascular smooth muscle. Studies in conjunction with radiolabeled ligands have demonstrated the existence of ATP-sensitive potassium channels, which are receptor targets for potassium channel opener drugs, such as pinacidil. The opening of these channels results in potassium efflux and hyperpolarizes the cell membrane. This hyperpolarization (1) reduces intracellular free calcium by reducing the likelihood of opening L-type or T-type calcium channels, inhibiting voltage-dependent Ca2 ⁺ channels, and (2) inhibiting inositol three Phosphate ( _IP3 ) formation, limits Ca2 ⁺ release from intracellular sources under agonist induction (on receptor-operated channels), and (3) reduces the efficiency of calcium as an activator of contractile proteins. The combined action of these two classes of drugs (ATP-sensitive potassium channel openers and calcium channel antagonists) pushes the target cell into a state of relaxation or a state that is more resistant to activation.

Finally, calcium channel antagonists and tachykinin and bradykinin antagonists have been shown to have synergistic effects in mediating neuroinflammation. The role of neurokinin receptors in mediating neuroinflammation has been established. The neurokinin ₁ (NK ₁ ) and neurokinin ₂ (NK ₂ ) receptor (members of the G-protein coupled superfamily) signal transduction pathway involves an increase in intracellular calcium, which leads to activation of calcium channels at the plasma membrane. Likewise, 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 increased intracellular calcium, some of which enters through L-type channels. This is the basis for the synergistic interaction between calcium channel antagonists and antagonists of neurokinin and bradykinin 2 receptors.

Calcium channel antagonists suitable for use in the practice of the present invention include nisoldipine, nifedipine, nimodipine, lacidipine, isradipine and amlodipine. Table 24 provides suitable concentrations of these agents.

Table 24

Spasticity inhibitor treatment and preferred concentration Drug type Therapeutic concentration (nanomolar) Preferred concentration (nanomolar) Calcium channel antagonists: Nisoldipine Nifedipine Nimodipine Lacidipine Isradipine Amlodipine 1-10,0001-10,0001-10,0001-10,0001-10,0001-10,000 100-1,000100-5,000100-5,000100-5,000100-5,000100-5,000

IV. Anti-restenotic agents

Solutions of the present invention for cardiovascular and general vascular procedures may also optionally include anti-restenosis agents, especially for angioplasty, atherectomy or other interventional vascular applications. When the procedure requires limiting restenosis, the aforementioned cardiovascular and general vascular irrigation solutions are suitable for containing the following drugs. In the solutions according to the invention, the anti-restenotic agents mentioned below should preferably be combined with antispasmodic agents, and even more preferably also with analgesic/anti-inflammatory agents.

A. Antiplatelet agents

At sites of arterial injury, platelets adhere to collagen and fibrinogen through specific cell surface receptors and are subsequently activated by several independent mediators. There are various agonists that activate platelets, including collagen, ADP, thrombin A2, epinephrine, and thrombin. Collagen and thrombin act as primary activators at the site of vascular injury, while ADP and thrombin A2 function to recruit other platelets into the growing platelet plug. Activated platelets degranulate and release other substances that can act as chemoattractants and vasoconstrictors, thereby promoting vasospasm and platelet aggregation. Thus, the antiplatelet agent may be an antagonist against any of the agonist-receptor targets described above.

Because of the central role of platelets in the coagulation cascade, oral antiplatelet agents have been routinely administered to patients undergoing vascular procedures. Indeed, the multiplicity of activators and the observation that a single antiplatelet agent is ineffective have led some investigators to assert that combination regimens are necessary to achieve efficacy. More recently, Willerson and co-workers reported the intravenous administration of a combination of 3 agents, lidogrel (antagonist of thromboxane A2), ketosarin (antagonist of serotonin) and clopidogrel (antagonist of ADP). They found that a combination of 3 antagonists inhibited several relevant platelet functions and reduced neointimal proliferation in a canine coronary angioplasty model (JACC Abstracts, Feb. 1995). It is not clear which approach to treat coronary thrombosis is most successful. One possibility would be to include antiplatelet agents and anticoagulants in the cardiovascular and general vascular solutions of the invention.

1. Thrombin inhibitors and receptor antagonists

Thrombin plays a key role in the formation of vascular lesions and is considered to be the main mediator of thrombus formation. Therefore, during and after PTCA (percutaneous transluminal coronary angioplasty) or other vascular procedures, thrombus formation at the vascular lesion site is the key to acute reocclusion and chronic restenosis. This process can be blocked by administration of direct antithrombins, 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 potent growth factor that initiates smooth muscle cell proliferation at sites of vascular injury. In addition, thrombin also plays a role in modulating the action of other growth factors, such as PDGF (platelet-derived growth factor), and thrombin inhibitors have been shown to reduce PDGF mRNA expression after balloon angioplasty-induced vascular injury.

Hirudin is the prototype direct antithrombin drug because it binds to the catalytic site and substrate recognition site (exosite) of thrombin. Animal studies with baboons showed that this proliferative response was reduced by 80% with recombinant hirudin (Ciba-Geigy). Recombinant hirudin (Biogen) is a dodecapeptide model constructed based on hirudin, which can be combined with the active site of thrombin by means of the Phe-Pro-Arg linker molecule. Large clinical trials of hirudin and recombinant hirudin are underway to test their efficacy in reducing vasculopathy after PTCA, and phase II results for these inhibitors are currently positive, and both drugs are considered Suitable for use in the solution of the present invention. Preliminary trial results in 1,200 patients with 6-month repeat angiographic evaluations to detect restenosis showed that hirudin was superior to heparin in the short-term suppression of ischemia. The advantage of this approach is that no significant bleeding complications were reported. Sustained local release of recombinant hirudin therapy has been found to reduce early thrombosis in pigs after arterial stent implantation without thickening the neointima. See "Sustained-Release Local Hirulog Therapy Decreases Early Thrombosis but not Neointimal Thickening After Arterial Stenting" by Muller, D. et al. in Am.Heart J.133(2):211-218 (1996). Arterial stenting reduces early thrombosis without neointimal thickening)". In this study, recombinant hirudin was released from impregnated polymers placed around arteries.

Other active antithrombin agents that were tested and concluded to be suitable for use in the present invention are argatroban (Texas Biotechnology) and efagatran (Lilly).

Table 25

Treatment and preferred concentrations of restenosis inhibitors Drug type Therapeutic/preferred concentration (nanomolar) The most preferred concentration (nanomolar) Thrombin Inhibitors and Receptor Antagonists: Hirudin Recombinant Hirudin 0.00003-3/0.0003-0.30.2-20,000/2-2,000 0.03200

2. ADP receptor antagonists (purine receptor antagonists)

Ticlopidine is an ADP analog that inhibits thromboxane- and ADP-induced platelet aggregation. It is likely that ticlopidine blocks the interaction of ADP with its receptor, thereby inhibiting the signal transduction of G-protein coupled receptors on the platelet membrane surface. Preliminary results show that ticlopidine is more effective than aspirin combined with dipyridamole. However, the clinical application of ticlopidine is still limited because it can cause neutropenia. Clopidogrel, an analog of ticlopidine, is thought to have fewer adverse side effects than ticlopidine and is still under investigation for the prevention of ischemia. It is inferred that these agents may be suitable for the solution of the present invention.

3. Thromboxane inhibitors and receptor antagonists

Currently, conventional approaches to treating thrombosis utilize agents that rely on aspirin, heparin, and plasminogen activators. Aspirin irreversibly acetylates cyclooxygenase and inhibits the synthesis of thromboxane A2 and prostacyclin. Although there is evidence that aspirin is beneficial for PTCA, it is still considered to have only partial or moderate potential efficacy. The reason may be that platelets are activated through a thromboxane A2-independent pathway that is not blocked by aspirin-induced cyclooxygenase acetylation. Platelet aggregation and thrombosis may occur even with aspirin therapy. The study also found that the combination of aspirin and dipyridamole can reduce the incidence of acute complications during PTCA, but it does not reduce the incidence of restenosis.

Two thromboxane receptor antagonists appear to be more potent than aspirin and are considered suitable for use in the solutions and methods of the present invention. Thiampidine inhibits thromboxane- and ADP-induced platelet aggregation. Lidogrel (R68060) is a combined thromboxane B2 synthase inhibitor and thromboxane-prostaglandin endoperoxide receptor blocker. It was compared with salicylate therapy in a specific open-label study of patients receiving PTCA in combination with heparin. See "Ridogrel in the Setting of Percutaneous Transluminal Coronary Angioplasty" by Timmermans, C. et al. Am. J. Cardiol. 68:463-466 (1991). ". The treatment consisted of a slow intravenous injection of 300 mg lidagrel just before the start of the PTCA procedure and continued 12 hours later at a dose of 300 mg twice daily. The study found initial success with the administration of lidagret, with no early acute reocclusion in 30 patients. Bleeding complications did occur in a significant number (34%) of patients, and this appears to be a complication requiring special care. This study confirmed that lidagrel is a long-acting inhibitor of thromboxane B2 synthase.

B. Cell Adhesion Molecule Inhibitors

1. Select protein inhibitors

Selectin inhibitors block the interaction of selectins with their cognate ligands or receptors. Examples of representative selectin targets on which these inhibitors can act include, but are not limited to, E-selectin and P-selectin receptors. Upjohn Co. has approved Cytel Corps to develop a monoclonal antibody that inhibits the activity of P-selectin. The product is CY1748, which is still under preclinical development, and its potential indication is restenosis.

2. Integrin inhibitors

The platelet glycoprotein IIb/IIIa complex is present on the surface of resting as well as activated platelets. It appears to undergo a transformation during platelet activation that allows it to serve as a binding site for fibrinogen and other adhesion proteins. Most promising new antiplatelet agents target this integrin cell surface receptor, suggesting the existence of a final highway for platelet aggregation.

There are several classes of agents that fall into the class of GPIIb/IIIa integrin antagonists. To date, the monoclonal antibody c7E3 (CentoRx; Centocor, Malvern PA) has been intensively studied in a 3,000-patient PTCA study. This antibody is a chimeric human/mouse hybrid. A bolus injection of c7E3 at a dose of 0.25 mg/kg followed by a 12-hour intravenous infusion at a rate of 10 μg/min produced greater than 80% blockade of GPIIb/IIIa receptors during the infusion. This was associated with greater than 80% inhibition of platelet aggregation. Studies have pointed out that when the antibody is co-administered with heparin, it increases the risk of bleeding. Additional data from the EPIC trial showed that the primary endpoint, the combined mortality rate, the incidence of non-fatal myocardial infarction, and the need for coronary revascularization were significantly reduced, suggesting a long-term benefit of the antibody. See Tcheng, Am. Heart J. 130:673-679 (1995). A Phase IV study (EPILOG) is planned or in progress to address safety and efficacy concerns with the c7E3 Fab. The monoclonal antibody can also be classified as an antagonist of the platelet membrane glycoprotein receptor corresponding to the glycoprotein IIb/IIIa receptor.

The platelet glycoprotein IIb/IIIa receptor blocker eptifibatide is a cyclic heptapeptide that is highly specific for its molecular target. Eptifibatide has a short biological half-life (approximately 10 minutes) compared to the above-mentioned antibodies. The safety and efficacy of eptifibatide were initially evaluated in a phase II impact trial. The test employed a 4 or 12 hour intravenous infusion of eptifibatide at a rate of 1.0 [mu]g/kg/min (Topol, E., Am. J. Cardiol, 27B-33B (1995)). Eptifibatide in combination with other agents (heparin, aspirin) showed potent anti-platelet aggregation properties (>80%). A Phase III study of 4000 patients, the Phase II Impact Trial, showed that eptifibatide significantly reduced ischemia in patients undergoing atherectomy (JACCA Abstracts, 1996) . The following table provides concentrations of drugs c7E3 and eptifibatide suitable for use in the present invention.

In addition, two peptidomimetics, MK-383 (Merck) and RO 4483 (Hoffinann-LaRoche), are in phase II clinical studies. Since they are small molecules, they have a short half-life and high potency. But they also appear to interact with other closely related integrins with less specificity. It is speculated that these two peptidomimetics may also be suitable for use in the present invention.

Table 26

Treatment and preferred concentrations of restenosis inhibitors Drug type Therapeutic/preferred concentration (nanomolar) The most preferred concentration (nanomolar) Cell adhesion inhibitor: c7E3 eptifibatide 0.5-50,000/5-5,0000.1-10,000/1-1000xKd 500100xKd

C. Anti-chemotactic agents

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

D. Interleukin receptor antagonists

Interleukin receptor antagonists are agents that block the interaction of an interleukin with its cognate ligand or receptor. Specific receptor antagonists for any of the many interleukin receptors are in the early stages of development. One exception is the naturally occurring secreted form of the IL-1 receptor, known as IL-1 antagonist protein (IL-1AP). This antagonist binds to IL-1 and has been shown to inhibit the biological effects of IL-1 and is presumably suitable for use in the practice of the present invention.

E. Inhibitors of intracellular signaling

1. Protein Kinase Inhibitors

A. Protein Kinase C (PKC) Inhibitors

Protein kinase C (PKC) plays an important role in cell surface signal transduction for a large number of physiological processes. PKC isoenzymes can be activated as downstream targets arising from the initial activation of G-protein coupled receptors (eg, serotonin, endothelin, etc.) or growth factor receptors, such as PDGF. Both types of receptors play an important role in mediating vasospasm and restenosis after coronary balloon angioplasty procedures.

The results of molecular cloning analysis showed that PKC exists as a large family consisting of at least 8 subspecies (isozymes). These isozymes differ substantially in structure and in the specific mechanisms linking receptor activation and changes in specific cellular proliferative responses. Expression of specific isozymes was found in a wide variety of cell types including: platelets, neutrophils, myeloid cells and smooth muscle cells. Therefore, unless a PKC inhibitor is shown to be isozyme specific, it may affect signaling pathways in several cell types. Thus, it can be predicted that PKC inhibitors will effectively block the proliferative response of smooth muscle cells and may also have anti-inflammatory effects in blocking neutrophil activation and subsequent attachment. Several inhibitors have been described, with initial reports indicating an IC50 of ₅₀ μM for calcine C inhibitory activity. G-6203 (also known as Go6976) is a novel and potent PKC inhibitor with high selectivity for certain PKC isoforms with _IC50 values ranging from 2-10 μM. The following table provides the concentrations of the above agents and another drug, GF109203X, also known as Go 6850 or bisindolemaleimide I (available from Warner-Lambert), which are considered suitable for use in this study. invention.

Table 27

Treatment and preferred concentrations of restenosis inhibitors Drug type Therapeutic/preferred concentration (nanomolar) The most preferred concentration (nanomolar) Protein kinase C inhibitor: calcine phosphoprotein CGF 109203XG-6203 (Go 6976) 0.5-50,000/100-5,0000.1-10,000/1-1,0000.1-10,000/1-1,000 500100100

B. Protein tyrosine kinase inhibitors

Despite the great diversity among the many members of the receptor tyrosine-kinase (RTK) family, the signaling mechanisms utilized by these receptors share many common features. Biochemical and molecular genetic studies have shown that ligand binding to the ectodomain of an RTK rapidly activates the catalytic activity of a tyrosine kinase intrinsic to the intracellular domain (see Figure 5). The result of the increased activity is the specific phosphorylation of a large number of intracellular substrates containing common sequence motifs by tyrosine. This results in the activation of numerous "downstream" signaling molecules and the cascade of specific intracellular pathways that regulate phospholipid metabolism, arachidonic acid metabolism, protein phosphorylation (including mechanisms distinct from protein kinases), calcium mobilization, and Transcriptional activation (see Figure 2). Growth factor-dependent tyrosine kinase activity in the cytoplasmic domain of RTKs is the primary mechanism for generating intracellular signals that lead to cell proliferation. Therefore, inhibitors have the potential to block this signaling and, in turn, prevent the proliferative response (see Figure 5).

In the cardiovascular field, since the platelet-derived growth factor (PDGF) receptor is thought to play an important role in both atherosclerosis and restenosis, it is a highly interesting target for inhibition. On the damaged surface of the vascular endothelium, platelets release PDGF as a result of stimulating PDGF receptors on vascular smooth muscle cells. As noted above, this initiates a cascade of intracellular events leading to enhanced proliferation and thickening of the neointima. Inhibitors of PDGF kinase activity are expected to prevent proliferation and increase the success of cardiovascular and general vascular procedures. There are several related tyrosine phosphorylation inhibitor compounds that have potential as specific inhibitors of PDGF-receptor tyrosine kinase activity (in vitro _IC50 range of 0.5-1.0 μM) because of their inhibitory effect on other protein kinases and Other signal transduction systems had little effect. To date, only a few tyrosine phosphorylation inhibitor compounds are commercially available and the following table provides concentrations of these agents suitable for use in the present invention. In addition, it has been reported that staurosporine can effectively inhibit several protein tyrosine kinases in the src subfamily, and the concentration of this agent suitable for the present invention is also provided in the table below.

Table 28

Treatment and preferred concentrations of restenosis inhibitors Drug type Therapeutic/preferred concentration (nanomolar) The most preferred concentration (nanomolar) Protein Kinase Inhibitors: Tonicillin A Tyrosine Phosphorylation Inhibitor AG1296 Tyrosine Phosphorylation Inhibitor AG1295 Staurosporine 10-100,000/100-10,000 10-100,000/100-20,0000-100,000/100-20,0001-100,000/10-10,000 10,00010,00010,0001,000

2. Modulators of intracellular protein tyrosine phosphatases

Non-transmembrane protein tyrosine phosphatases (PTPases) containing the src-homology region ₂ SH2 domain are known and the nomenclature assigns them SE-PTP1 and SH-PTP2. In addition, SH-PTP1 is also known as PTP1C, HCP or SHP. SH-PTP2 is also known as PTP1D or PTP2C. Likewise, SH-PTP1 is expressed at high levels in hematopoietic cells of all lineages and in all stages of differentiation, and the identification of the SH-PTP1 gene as responsible for the phenotype in motheaten(me) mice provides insight into predicting the effect of specific inhibitors. Based on this, the specific inhibitor blocks its own interaction with its cellular substrate. Stimulation of neutrophils with chemotactic peptides is known to result in the activation of tyrosine kinases that mediate neutrophil responses (Cui, et al., J. Immunol. (1994)), and PTPase activity through Reverses the action of tyrosine kinases activated during the initial phase of cell stimulation, modulating agonist-induced activity. Agents that can stimulate PTPase activity can act as anti-inflammatory mediators and have potential therapeutic use.

The aforementioned PTPases have also been shown to regulate the activity of certain RTKs. They appear to balance the actions of activated receptor kinases and thus may present important drug targets. In vitro experiments have shown that injection of PTPase can block the phosphorylation of tyrosyl residues on endogenous proteins stimulated by insulin. Thus, in the case of restenosis, activation of the PDGF-receptor action can be reversed with an activator of PTPase activity and is considered suitable for use in the solution of the invention. In addition, PTPases linked to receptors can also function as extracellular ligands, similar to extracellular ligands of cell adhesion molecules. The functional consequences of ligand binding to the extracellular domain are not well understood, but a reasonable hypothesis is that this binding serves to regulate phosphatase activity within the cell (Fashena and Zinn, Current Biology 5:1367-1369 (1995)). This action blocks adhesion mediated by other cell surface adhesion molecules (NCAMs) and provides anti-inflammatory effects. Drugs suitable for the above applications have not yet been developed.

3. Inhibitors of SH2 domain (src homology region ₂ domain)

The SH2 domain, originally identified in the src subfamily of protein tyrosine kinases (PTKs), is a non-catalytic protein sequence consisting of approximately 100 amino acids that are conserved by various signaling proteins (Cohen, et al., 1995 ). The SH2 domain functions as a phosphotyrosine-binding module, thereby mediating key protein-protein associations in intracellular signal transduction pathways (Pawson, Nature, 573-580, 1995). In particular, the role of the SH2 domain has been well defined as key to receptor tyrosine kinase (RTK)-mediated signaling, such as in the case of the platelet-derived growth factor (PDGF) receptor. On autophosphorylated RTKs, phosphotyrosine-containing sites serve as binding sites for SH2-proteins, which in turn mediate activation of biochemical signaling pathways (see Figure 2) (Carpenter, G., FASEBJ 6:3283-3289 (1992); Sierke, S. and Koland, J. Biochem. 32:10102-10108, (1993)). The SH2 domain is responsible for coupling activated growth factor receptors to specific cellular responses including changes in gene expression and ultimately cell proliferation (see Figure 5). Therefore, inhibitors that selectively block the activation of specific RTKs expressed on the surface of vascular smooth muscle cells are expected to effectively block the proliferation and restenosis processes that occur after PTCA or other vascular procedures. One RTK target currently receiving attention is the PDGF receptor.

At least 20 cytoplasmic proteins have been identified that contain SH2 domains and play a role in intracellular signaling. The distribution of SH2 domains is not restricted to specific protein families, but is also present in several classes of proteins, protein kinases, lipid kinases, protein phosphatases, phospholipases, Ras control proteins and certain transcription factors. Many SH2-containing proteins have known enzymatic activity, while others (Grb2 and Crk) act as 'linkers' and 'adapters' between cell surface receptors and 'downstream' effector molecules function (Marengere, L., et al., Nature 369:502-505 (1994)). Examples of proteins that incorporate an SH2 domain and possess enzymatic activity that can be activated during signal transduction include, but are not limited to, the src subfamily of protein tyrosine kinases (Src( ^pp60c-src ), abl, lck , fyn, fgr, etc.), phospholipase Cγ (PLCγ), phosphatidylinositol 3-kinase (PI-3-kinase), p21-ras GTPase activating protein (GAP) and SH2-containing protein tyrosine phosphatase (SH -PTPase) (Songyang, et al., Cell 72: 767-778 (1993)). Since the above-mentioned various SH2-proteins play an important role in transmitting signals from activated cell surface receptors into specific cascades, inhibitors that can block the binding of specific SH2 proteins are considered as potential therapeutic candidates for many Agents are ideally applied in which the specific cascade described above consists of other molecular interactions that ultimately define the cellular response.

In addition, the regulation of many immune/inflammatory responses is mediated through specific receptors that transmit signals through SH2 domain-containing non-receptor tyrosine kinases. T-cell activation through the antigen-specific T-cell receptor (TCR) initiates a signal transduction cascade leading to lymphokine secretion and T-cell proliferation. One of the earliest biochemical responses following TCR activation is an increase in tyrosine kinase activity. Specifically, neutrophil activation is controlled in part through responses to cell surface immunoglobulin G receptors. Activation of these receptors mediates the activation of certain, though unidentified, tyrosine kinases known to have SH2 domains. There is additional evidence that several src family kinases (lck, blk, fyn) are involved in certain signaling pathways directed by cytokine and integrin receptors, and thus may integrate stimuli from several independent receptor structures. Thus, inhibitors of specific SH2 domains have the potential to block many neutrophil functions and may act as anti-inflammatory mediators.

Currently, efforts to develop drugs targeting the SH2 domain are at the in vitro biochemical and cellular levels. These efforts will be successful and, presumably, corresponding drugs should facilitate the practice of the present invention.

4. Calcium channel antagonists

The above-mentioned calcium channel antagonists related to spasm-inhibiting function can also be used as anti-restenosis agents in the cardiovascular and general blood vessel solutions of the present invention. Activation of growth factor receptors, such as PDGF, is known to lead to increased intracellular calcium (see Figure 2). Studies at the cellular level have shown that calcium channel antagonists effectively inhibit the division of vascular smooth muscle cells.

F. Synergistic Interactions Provided by Therapeutic Combinations of Anti-Restenotic Agents with Other Agents Used in Cardiovascular and General Vascular Solutions

Given the complexity of the known disease process associated with restenosis following PTCA or other cardiovascular or general vascular procedures and the diversity of molecular targets involved, blockade or inhibition of a single molecular target is unlikely to be effective in preventing vasospasm and restenosis (see Figure 2). In fact, many animal studies targeting different individual molecular receptors and/or enzymes have failed to demonstrate efficacy in animal models, or have so far not achieved efficacy against both pathologies in clinical trials. (Freed, M. et al. "An Intensive Poly-pharmaceutical Approach to the Prevention of Restenosis: the Mevacot, Ace Inhibitor, Colchicine (BIG-MAC) Pilot Trial ( An intensive multidrug approach to restenosis prevention: Mevacor, Ace inhibitor, colchicine (BIG-MAC guided trial)"; Serruys, P. et al in J.Am.Coll.of Cardiol.21: "PARK: the Post Angioplasty Restenosis Ketanserin Trial" in 322A (1993). Therefore, a therapeutic combination of several drugs acting on different molecular targets and delivered locally appears to be necessary for clinical efficacy of vasospasm and restenosis therapeutic approaches. As described below, the rationale for this synergistic molecularly anchored therapy arose from new advances in the understanding of the underlying biochemical mechanisms by which vascular smooth muscle cells in the vessel wall deliver and integrate their presence in PTCA or other Stimulation during vascular interventional procedures.

I. "Crosstalk" and convergence in major signaling pathways

The molecular switches responsible for cell signaling have been traditionally divided into two major unconnected signaling pathways, each comprising a unique set of protein families that act as transducers of a specific set of extracellular stimuli and mediate different cellular responses. One such pathway is through the use of intracellular targets of trimeric G proteins and Ca ²⁺ , and transduction of signals from neurotransmitters and hormones via G-protein coupled receptors (GPCRs), resulting in contraction response (see Figure 2). These stimuli and their corresponding receptors mediate smooth muscle contraction and may induce vasospasm in the context of PTCA or other cardiovascular or general vascular therapeutic or diagnostic procedures. Examples of signaling molecules involved in mediating spasm through the GPCR pathway are 5-HT and endothelin, antagonists of which have been included in the GPCR pathway and act through the corresponding G protein-coupled receptors.

The second major pathway transduces signals derived from growth factors, such as PDGF, into the regulation of cell proliferation and differentiation through tyrosine kinases, adapter proteins and Ras proteins (see Figures 2 and 5). This pathway can also be activated during PTCA or other cardiovascular or general vascular procedures, resulting in a high incidence of vascular smooth muscle cell proliferation. An example of a restenosis drug target is the PDGF-receptor.

Signals transmitted by neurotransmitters and hormones stimulate either of two classes of receptors: G-protein coupled receptors consisting of a 7-helical transmembrane domain, or ligand-gated ion channels. "Downstream" signals from these two classes of receptors focus on the regulation of cytoplasmic Ca2 ⁺ concentrations, triggering smooth muscle cell contraction (see Figure 2). Each GPCR transmembrane receptor activates a specific class of trimeric G proteins, including _Gq , _Gi , or many others. G _α and/or G _βγ subunits activate phospholipase C _β , leading to activation of protein kinase C (PKC) and increasing cytosolic calcium levels by releasing intracellular calcium stores.

Growth factor signaling, such as that mediated by PDGF, focuses on the regulation of cell growth. This pathway relies on the phosphorylation of tyrosine residues in both receptor tyrosine kinases and "downstream" enzymes (phospholipase _Cγ , mentioned above in relation to tyrosine kinases). Activation of the PDGF-receptor also leads to PKC stimulation and an increase in intracellular calcium, a step common to GPCRs (see Figure 2). It is now well established that ligand-independent "crosstalk" transactivates the tyrosine kinase receptor pathway in response to GPCR priming. Recently, research work has identified an adapter protein in the tyrosine kinase/Ras pathway, namely Shc, which is a key intermediate protein that transfers information from the above-mentioned GPCR pathway to the tyrosine kinase pathway (see Fig. 2) (Lev et al., Nature 376:737 (1995)). Activation of Shc is calcium dependent. Thus, combinations of selective inhibitors that block transactivation of shared signaling pathways leading to vascular smooth muscle cell proliferation may act synergistically to prevent spasm and restenosis following PTCA or other cardiovascular or general vascular procedures. Specific examples are briefly described below.

2. Synergistic interaction between PKC inhibitors and calcium channel antagonists

In this case, the synergistic interaction of PKC inhibitors and calcium channel antagonists in achieving vasodilation and inhibition of proliferation is due to "crosstalk" between GPCRs and tyrosine kinase signaling pathways (see Figure 2). The rationale for this dual use is the fact that these drugs have different molecular mechanisms of action. As described above, GPCR priming results in the activation of protein kinase C and increases cytosolic calcium levels by releasing intracellular calcium stores. Calcium-activated PKC is a major control point in the transmission of extracellular responses. "Crosstalk" of GPCR activation pathways through PKC can lead to the division of VSMCs, thus calcium channel antagonists have a dual role of directly blocking spasticity and further preventing the activation of proliferation by inhibiting Shc activation. In contrast, PKC inhibitors act on parts of the pathway that lead to contraction.

3. Synergy of PKC inhibitors, 5-HT ₂ antagonists and _ETA antagonists

The 5-HT ₂ receptor family contains three members, 5-HT _2A , 5-HT _2B , and 5-HT _2C , whose common properties are that they can be coupled to the conversion of phosphatidylinositol and increase intracellular Calcium (Hoyer et al., 1998; Hartig et al., 1989). The distribution of these receptors includes vascular smooth muscle and platelets, and because of their localization, these 5-HT receptors have an important role in mediating spasm, thrombosis and restenosis. It has been found that extracellular calcium is required for the duration of the increase in intracellular calcium induced by _ETA receptor activation in smooth muscle cells and is at least partially blocked by nicardipine. Since activation of both the 5- _HT2 receptor and the _ETA receptor is calcium-mediated, it is expected that when the surgical solution contains a PKC inhibitor in combination with the above two receptor antagonists, the PKC inhibitor will be synergistic. Enhance the effect of antagonists on both receptors mentioned above (see Figures 2 and 4).

4. Synergistic effect of protein tyrosine kinase inhibitors and calcium channel antagonists

The mitogenic effects of PDGF (or basic fibroblast growth factor or insulin-like growth factor-1) are mediated through receptors with intrinsic protein tyrosine kinase activity. PDGF phosphorylates many substrates and leads to mitogen-activated protein kinase (MAPK) activation and eventual proliferation (see Figure 5). Endothelin, 5-HT and thrombin receptors that are members of the G-protein coupled superfamily. Both trigger a signal transduction pathway that involves an increase in intracellular calcium, leading to the activation of calcium channels on the plasma membrane. Calcium channel antagonists thus interfere with the common mechanisms used by these GPCRs. Recently, it has been shown that activation of certain GPCRs, including endothelin and bradykinin, leads to a rapid increase in tyrosine phosphorylation of a number of intracellular proteins. Phosphorylation of a subset of these proteins was similar to that of proteins known to be essential for mitogenic excitation. The rapidity of the process allows changes to be detected within seconds, and the affected target likely plays a role in mitosis. These tyrosine phosphorylation events were not blocked by selective PKC inhibitors, or were apparently mediated by increased intracellular calcium. Since two independent pathways, GPCR and tyrosine phosphorylation, drive VSMCs into a proliferative state, it is necessary to block these two independent signaling arms. This is the basis for the synergistic interaction of calcium channel antagonists and tyrosine kinase inhibitors in the surgical solution of the invention. Because the role of protein tyrosine kinase inhibitors in preventing vascular smooth muscle cell proliferation occurs through independent molecular pathways (as described above) that are independent of pathways involving calcium and protein kinase C, it is possible The combination of two classes of drugs, calcium channel antagonists and protein tyrosine kinase inhibitors, is expected to be more effective in inhibiting spasticity and restenosis than either class alone.

5. Synergistic effect of protein tyrosine kinase inhibitors and thrombin receptor antagonists

Thrombin mediates its action through another member of the GPCR superfamily, the thrombin receptor. Binding to this receptor triggers platelet aggregation, smooth muscle cell contraction and mitosis. Signal transduction occurs through several pathways: phospholipase (PLC) activation via G proteins, and tyrosine kinase activation. Activation of tyrosine kinase activity is also required for mitosis in vascular smooth muscle cells. It has been shown that inhibition with specific tyrosine kinase inhibitors has no effect on the PLC pathway when monitored by measuring intracellular calcium, but effectively blocks thrombin-induced mitosis ( Weiss and Nucitelli, J. Biol. Chem. 267:5608-5613 (1992)). Because the effect of the protein tyrosine kinase inhibitor in preventing vascular smooth muscle cell proliferation occurs through an independent molecular pathway (as described above) that is independent of pathways involving calcium and protein kinase C, It is expected that a combination of a protein tyrosine kinase inhibitor and a thrombin receptor antagonist will inhibit platelet aggregation, spasticity and restenosis more effectively than either class alone.

G. Cyclooxygenase-2 (COX-2) Inhibitors

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used as anti-inflammatory, antipyretic, anticoagulant and analgesic agents (Lewis, RA in Textbook of Rheumatology, 3rd ed. (Kelley W. et al. ed.) (1989), p. 258 "Prostaglandins and Leukotrienes (Prostaglandins and Leukotrienes)"). The molecular targets of these drugs are the first enzymes in the prostaglandin synthesis pathway, known as prostaglandin endoperoxide synthase or fatty acid cyclooxygenase. Currently, two forms of cyclooxygenase are recognized, referred to as cyclooxygenase-1 or type 1 (COX-1) and cyclooxygenase-2 or type 2 (COX-2). These isozymes are also known as prostaglandin H synthase (PGHS)-1 and PGHS-2, respectively. Both _enzymes catalyze the conversion of arachidonic acid to unstable intermediates, _PGG2 and _PGH2 , which are intermediates in the biosynthesis of prostaglandins and _thromboxanes . COX-1 is present in platelets and endothelial cells and has been shown to be constitutively active. COX-2 is found in endothelial cells, macrophages and fibroblasts, including synoviocytes after cytokine treatment (induction).

COX-2 isozymes are induced in inflammatory situations by cytokines and inflammatory mediators, such as IL-1, TNF-α and endotoxin, and their expression is upregulated at sites of inflammation. COX-2 activity, above basal COX-1 activity, is dramatically increased with its upregulation, resulting in the synthesis of prostaglandins that contribute to pain and inflammation. Since COX-2 is usually only expressed in inflamed tissues, or only after exposure to inflammatory mediators, selective inhibitors can exhibit anti-inflammatory activity without affecting the components present in platelets and other cell types. This effect is thought to be responsible for the adverse side effects associated with the use of non-selective NSAID drugs (eg, clotting time, bleeding, adverse renal and gastrointestinal side effects).

It has been established that due to the unique pharmacology of the two COX isozymes, it is possible to develop isozyme-specific (selective) cyclooxygenase inhibitors that are useful in anti-inflammatory therapy. Various biochemical, cellular, and animal assays have been developed to assess the relative selectivity of inhibitors for COX-1 and COX-2 isozymes. These assays included the determination of prostaglandin E2 production in microsomes prepared from various cell types and in bioassay systems using intact human cells. For any given drug, compounds have been identified that are selective inhibitors of COX-2, although experimental variations in the degree of selectivity within test systems and between biological sources have been noted. Typically, the criterion defining selectivity is the ratio of COX-1/COX-2 (or COX-2/COX-1 ) inhibition constants obtained for a particular biochemical or cellular assay system. Selectivity ratios account for different absolute _IC50 values that correlate with enzymatic activity obtained between microsomal and cellular assay systems (e.g. platelets and macrophages, cell lines stably expressing recombinant human COX isozymes) Inhibition corresponds.

Many of the conventional NSAIDs currently on the market (naproxen, indomethacin, ibuprofen) are generally non-selective inhibitors of the two COX isoforms and may show The selectivity for COX-1 is greater than that for COX-2. The use of COX-2 inhibitors to block the formation of prostaglandins represents a therapeutic approach that is superior to trying to block endogenous prostanoid ligands, such as PGE2 produced by COX-2 at sites of inflammation, that interact with the eight subclasses of prostaglandins described above. A protocol for interaction with any of the prostaglandin receptors is preferred. This option is currently not feasible due to selectivity for all of the above prostanoid receptors (EP-1, EP-2, EP-3, EP-4, DP, FP, IP and No effective antagonist exists.

A study by Riendeau and co-workers compared more than 45 NSAIDs with selective COX- 2 Selectivity of inhibitors (Can. J. Physiol. Pharmacol. 75:1088-95 (1997)). Among the compounds reported to show selectivity for COX-2 over COX-1 in this study, the ranking order of potency was DuP 697 > SC-58451, Celecoxib > Nimesulide = Mesulide Loxicam = piroxicam = NS-398 = RS-57067 > SC-57666 > SC-58125 > fluthamide > etodolac > L-745,337 > DFU-T-614, _IC50 values range from 7 μM to 17 μM . There is _a good correlation between the IC ₅₀ values for the inhibition of microsomal COX-1 and the inhibition of TXB ₂ production by Ca ²⁺ ionophore stimulated platelets and stable expression The corresponding IC ₅₀ values for the inhibition of prostaglandin E2 produced by human COX-1 in CHO cells. Microsomal assays are more sensitive to inhibition than cell-based assays and are capable of detecting all NSAIDs and selective COX- Inhibition of COX-1 by 2 inhibitors.

From the defined molecular and cellular mechanisms of action and animal studies for selective COX-2 inhibitors, such as celecoxib, it is expected that when the above compounds are administered intraoperatively directly to tissues or joints in the form of irrigation solutions, Will exhibit anti-inflammatory effects. In particular, it is expected that the compounds described above will be effective drugs delivered by irrigation during arthroscopic, urological or general surgical procedures (perioperative). For example, a selective COX-2 inhibitor can be substituted for the relatively non-selective cyclooxygenase inhibitor ketorolac in Examples IV, V & VI. The selective COX-2 inhibitor can be delivered alone or in combination with other small molecule drugs, peptides, proteins, recombinant chimeric proteins, antibodies or gene therapy vectors (viral and non-viral) to joints, In the interstitial spaces of the genitourinary tract, cardiovascular system, or any body cavity. For example, the selective COX-2 inhibitor can act on any cell and specific structure connected to the joint fluid space, including the joint, involved in the normal function of the joint or present due to a pathological condition. These cells and structures include, but are not limited to: synoviocytes, including type A fibroblasts and type B macrophages; cartilage components of the joint, such as chondrocytes; cells associated with bone, including periosteal cells, osteoblasts, Osteoclasts; immunological components, such as inflammatory cells, including lymphocytes, mast cells, monocytes, eosinophils; and other cells similar to fibroblasts; and combinations of these cell types.

Selective COX-2 inhibitors can be delivered in a formulation that facilitates the introduction and administration of the drug into the target tissue or joint and should enhance the delivery, uptake, stability or drug delivery of the inhibitor drug. metabolic kinetics. The formulation may include, but is not limited to, administration using microparticles, microspheres or nanoparticles composed of lipids, proteins, carbohydrates, synthetic organic or inorganic compounds. Examples of formulation molecules include, but are not limited to, lipids capable of forming liposomes or other ordered lipid structures, cationic lipids, hydropolymers, polycations such as protamine, spermidine, and polylysine ), peptide or synthetic ligands and antibodies capable of anchoring substances to specific cell types, gels, sustained release matrices, soluble and insoluble particles, and other formulation components not listed.

The present invention describes the local delivery of a selective COX-2 inhibitor drug using an irrigation solution that contains a low concentration of the selective COX-2 inhibitor drug and enables the drug to be delivered directly to the affected tissue or joint . Irrigate solutions containing this drug are applied perioperatively during surgical procedures. Other conventional methods of facilitating drug delivery all require systemic administration (e.g., intramuscular, intravenous, subcutaneous, oral), and higher concentrations of COX-2 must be administered in order to obtain significant therapeutic concentrations in target tissues or joints Drugs (and higher total doses). Systemic administration also results in high concentrations in tissues other than the target tissue, which is an undesired consequence, and may also produce adverse dose-related side effects (eg, bleeding, ulceration). These systemic delivery methods also subject the drug to secondary metabolism and rapid degradation, limiting the duration of effective therapeutic concentrations. The drug in the flush solution is administered directly to the intended tissue, providing a significant advantage that the therapeutically effective concentration and total therapeutically effective dose of the selective COX-2 inhibitor drug delivered in this manner is lower than that achieved by other routes of administration. The desired therapeutically effective concentration and total dose. Furthermore, the doses of COX-2 inhibitors contained in the solution are much lower than those commonly used in the perioperative period, allowing the drug to be used in patients with other contraindications.

The following table provides compounds suitable for use in the present invention.

Table 29

cyclooxygenase-2 inhibitors compound Therapeutic preferred concentration (μM) The most preferred concentration (μM) DuP 697SC-58451 Celecoxib Meloxicam Nimesulide Diclofenac NS-398L-745,337RS57067SC-58125SC-57666 Fluoxamide 0.3-3,0000.3-3,0000.3-3,0000.5-5,0000.5-5,0000.3-3,0000.3-3,0000.2-10,0000.2-10,0000.2-10,0000.2-10,0000.2-10,000 60060060050050060060010001000100010001000

V. Application method

The solutions of the present invention have application to a variety of surgical/interventional procedures including surgical, diagnostic and therapeutic techniques. The irrigation solution is administered perioperatively during arthroscopic surgical procedures of anatomical joints, urological procedures, cardiovascular and general vascular diagnostic and therapeutic procedures and general surgical procedures. The term "perioperative period" as used herein includes during the procedure, before and during the procedure, during and after the procedure, and before, during and after the procedure. The solution is preferably applied before and/or after operation as well as during operation. These procedures routinely employ physiological irrigation solutions, such as physiological saline or lactated Ringer's solution, applied to the surgical site by techniques well known to those of ordinary skill in the art. The method of the present invention includes substituting the conventionally used flushing liquid with the analgesic/anti-inflammatory/spasmolytic/anti-restenotic flushing liquid of the present invention. The irrigant solution is applied to the wound or surgical site prior to the initiation of the procedure, preferably before causing tissue trauma, and is continued throughout the duration of the procedure to pre-empt the onset of pain and inflammation, spasm and restenosis. The term "irrigation" as used throughout refers to the application of fluid flow to irrigate a wound or anatomical structure. The term "application" includes irrigation and other methods of topical introduction of the solutions of the invention, such as introducing the solution in a gelable form to a surgical site, with the gelling solution remaining at the specific site throughout the procedure. The term "continuously" as used throughout also includes instances where the wound is irrigated repeatedly and frequently at a frequency sufficient to maintain a predetermined therapeutic local concentration of the applied agent, as well as applications where operational techniques may necessitate intermittent interruption of the flow of irrigation fluid.

The concentrations listed corresponding to the respective agents in the solutions of the present invention are the concentrations at which the agents delivered topically to the surgical site achieve a predetermined level of effect at the surgical site in the absence of metabolic conversion. It will be appreciated that the concentration of drug in a particular solution may need to be adjusted to account for local dilution upon delivery. For example, in cardiovascular applications, if the average human coronary blood flow rate is assumed to be 80 cc/min, the average delivery rate of the solution through the local delivery catheter is 5 cc/min (i.e., the ratio of blood flow to solution delivered is 16 : 1), the drug concentration in the solution must be increased to 16 times the predetermined drug concentration in vivo. There is no need to take account of metabolic transformations or to adjust solution concentrations due to dilution by bulk distribution, as local delivery avoids these circumstances, as is the case with oral, intravenous, subcutaneous or intramuscular applications.

Arthroscopy techniques for which the solutions of the present invention may be applied include, but are not limited to, partial meniscectomy and ligament reconstruction in the knee, acromioplasty, cuff debridement, elbow synovectomy, and wrist surgery. and ankle arthroscopy. Irrigation fluid is continuously administered to the joint during surgery at a flow rate sufficient to dilate the joint capsule, remove surgical debris, and enable unobstructed intra-articular imaging.

Example 1 below provides irrigation solutions suitable for controlling pain and edema during this arthroscopic technique. For arthroscopy, a solution comprising a combination of the following agents, and preferably a solution comprising all of the following agents, or any of the following agents is preferred: serotonin ₂ receptor antagonist, serotonin ₃ receptor Antibody antagonists, histamine ₁ receptor antagonists, serotonin receptor agonists acting on 1A, 1B, 1D, 1F and/or 1E receptors, bradykinin ₁ receptor antagonists, bradykinin ₂ receptors Antagonists and cyclooxygenase inhibitors.

Because the agent is applied topically directly to the surgical site during the procedure, the solution allows for very low doses of the aforementioned pain and inflammation inhibitors. For example, less than 0.05 mg of amitriptyline per liter of flushing solution is required to provide the desired effective local tissue concentration that inhibits 5- _HT2 and _H1 receptors (appropriate for _serotonin2 and _histamine1 "dual" receptor antagonists). This dose is extremely low compared to the usual initial dose of 10-25 mg for oral amitriptyline. This same rationale applies to the use of the solutions of the present invention as antispasmodic and antirestenotic agents for reducing spasm associated with urological, cardiovascular, and general vascular procedures, and for inhibiting restenosis associated with cardiovascular and general vascular procedures. For example, less than 0.2 mg of nisoldipine (a suitable calcium channel antagonist) per liter of flush solution is required to provide the desired effective local tissue concentration that inhibits the voltage-dependent gating of L-subtype calcium channels. This dose is extremely low compared to a single oral dose of 20-40 mg of nisoldipine.

In the various surgical solutions of the present invention, the pharmaceutical agents are included in concentrations and doses when delivered locally are lower than those necessary to obtain the desired therapeutic effect by conventional methods of drug administration. It is not possible to achieve equivalent efficacy by delivering the same amount of agent via other routes of drug administration (ie intravenous, subcutaneous, intramuscular or oral) because systemically administered drugs undergo primary and secondary metabolism.

For example, the inventors tested the ability of amitriptyline, a 5-HT ₂ antagonist, to inhibit 5-HT-induced plasma extravasation in rat knees in accordance with the present invention using a rat model of arthroscopy. This study, which is described in more detail in Example XII below, compared therapeutic doses of amitriptyline delivered locally (ie, intra-articularly) and intravenously to the knee. The results confirmed that under the premise of obtaining the same curative effect, the total dose level required for intra-articular administration of amitriptyline is 200 times lower than that required for intravenous administration. Given that only a small fraction of the intra-articularly delivered drug is absorbed by the local synovial tissue, the difference in plasma drug levels between the two routes of administration was much higher than the difference in the total dose level of amitriptyline.

The practice of the present invention should be distinguished from conventional intra-articular injections of opiates and/or local anesthetics when performing arthroscopic or "open" joint (eg, knee, shoulder, etc.) procedures. The solutions of the invention are used for continuous infusion throughout surgical procedures to achieve preemptive suppression of pain and inflammation. In contrast, constant infusion of local anesthetics, such as lidocaine (0.5-2% solution), at the high concentrations necessary for therapeutic efficacy, produces profound systemic toxicity.

In addition to performing the procedures of the present invention, it may be desirable to administer, by injection or otherwise, higher concentrations of pain and inflammation inhibitors at the surgical site as an alternative or in addition to opiates. Same Pain/Inflammation Suppressant as Flush Contains.

The solution of the present invention is also applicable in diagnostic and therapeutic procedures of cardiovascular and common blood vessels, and can potentially reduce vessel wall spasm, platelet aggregation, proliferation of vascular smooth muscle cells and activation of nociceptors produced by vascular manipulation. References herein to arterial treatment include treatment of vein grafts that have been harvested and placed in the arterial system. Example II below discloses a solution suitable for this technique. Cardiovascular and general vascular solutions preferably include any combination of the following agents, and preferably include all of the following agents: 5- _HT receptor antagonists (Saxena, PR et al. in J. Cardiovasc. Pharmacol. 15 (Supp 1.7) : "Cardiovascular Effects of Serotonin Inhibitory Agonists and Antagonists" in S17-S34 (1990); Douglas, 1985); can block sympathetic neurons and C -A 5-HT ₃ receptor antagonist that activates receptors on fiber nociceptive neurons, which has been shown to cause bradycardia and tachycardia (Saxena et al., 1990); bradykinin ₁ receptor antagonists; and cyclooxygenase inhibitors that prevent the production of prostaglandins at sites of tissue damage, thereby reducing pain and inflammation. In addition, cardiovascular and general vascular solutions may also preferably contain serotonin _1B (also known as serotonin _1Dβ ) antagonists, since studies have shown that serotonin can cause significant vasospasm by activating serotonin _1B receptors in humans . See "Variable Participation of 5-HT1-Like Receptors and 5-HT2 Receptors in Serotonin-Induced Contract of Human Isolated Coronary Arteries (5-HT1-like receptors and 5-HT2 Receptors in Serotonin-Induced Contract of -Variable involvement of the -HT2 receptor in serotonin-induced constriction of human isolated coronary arteries)". Excitatory action of this serotonin _IB receptor in the vessel wall results in vasoconstriction, as opposed to the inhibitory action of the serotonin _IB receptor in neurons discussed above. Cardiovascular and general vascular solutions of the present invention may also suitably include one or more of the anti-restenotic agents disclosed herein which reduce the risk of restenosis due to, for example, angioplasty or atherectomy. The incidence of restenosis and reduce its severity.

The solutions of the present invention also have utility in reducing pain and inflammation associated with urological procedures, such as transurethral prostatectomy and similar urological procedures. Reference herein to the application of solutions to the urinary tract or urological structures includes application to the urinary tract itself, the bladder and prostate and associated structures. Studies have shown that serotonin, histamine and bradykinin can cause inflammation in the lower urinary tract tissue. See "Vascular Leakage in the Kidney and Lower Urinary Tract: Effects of Histamine, Serotonin and Bradykinin" by Schwartz, MM et al. in Proc. Soc. Exp. Biol. Med. 140:535-539 (1972). Vascular Leakage: Role of Histamine, Serotonin, and Bradykinin)". Example III below discloses irrigation solutions suitable for use in urological procedures. The solution preferably includes a combination, and preferably all, of the following agents: a histamine ₁ receptor antagonist that inhibits histamine-induced pain and inflammation; Bradykinin ₁ antagonists; Bradykinin ₂ antagonists; and cyclooxygenase inhibitors that reduce prostaglandin-induced pain/inflammation at sites of tissue damage. The solution may also preferably include an antispasmodic agent to prevent spasms in the urinary tract and bladder wall.

Certain solutions of the invention may suitably include a gelling agent to form dilute gels. The gellable solution can be applied, for example, in the urinary tract or arterial vessels to deliver serial, diluted, locally preset concentrations of the agent.

The solutions of the present invention may also be used perioperatively to suppress pain and inflammation in surgical wounds, and to reduce pain and inflammation associated with burns. Burns result in the release of large amounts of biogenic amines that not only produce pain and inflammation but also cause severe plasma extravasation (fluid loss), often a life-threatening component in severe burn situations. See "The Effect of Ketanserin, a Specific Serotonin Antagonist, on Burn Shock Hemodynamic Parameters in a Porcine Burn Model" by Holliman, C.J. et al. in J. Trauma 23:867-871 (1983). Lin on the hemodynamic parameters of burn shock in a porcine burn model)". The solution disclosed in Example I for arthroscopy may also be suitably applied to wounds or burns to control pain and inflammation, and is also suitable for surgical procedures such as arthroscopy. The medicament contained in the solution of Example I may optionally be contained in a paste or ointment base for application to burns or wounds.

VI. Embodiment

Below are several formulations suitable for use in certain surgical procedures according to the invention, followed by an overview of three clinical studies conducted with agents of the invention.

A. Embodiment I

Irrigating solutions for arthroscopy

The following composition is suitable for irrigation of anatomical joints during arthroscopic procedures. Each drug is dissolved in a carrier solution containing physiological electrolytes, such as physiological saline or lactated Ringer's solution, which is also the retention solution described in the subsequent examples.

Drug type drug Concentration (nanomolar): therapeutic concentration preferred concentration optimal concentration Serotonin ₂ antagonist Serotonin ₃ antagonist Histamine ₁ antagonist Serotonin _{1A, 1B, 1D, 1F} agonist Bradykinin ₁ antagonist Bradykinin ₂ antagonist [des-Arg ¹⁰ ] Derivative of Amitriptyline Metoclopramide Amitriptyline Sumatriptan HOE 140 HOE 140 0.1-1,00010-10,0000.1-1,0001-1,0001-1,0001-1,000 50-500200-2,00050-50010-20050-50050-500 1001,00020050200200

B. Example II

Irrigate solutions for cardiovascular and general vascular therapeutic and diagnostic procedures

The following drugs and their concentration ranges in solutions, i.e. physiological carrier solutions, are suitable for irrigation of the surgical site during cardiovascular and general vascular procedures. Drug type drug Concentration (nanomolar): therapeutic concentration preferred concentration optimal concentration Serotonin ₂ antagonists Serotonin ₃ antagonists Serotonin _1B antagonists Bradykinin ₁ antagonists Cyclooxygenase inhibitors Ketorolac, a [des-Arg ¹⁰ ] derivative of trazodone, metoclopramide, and yohimbine HOE 140 0.1-2,00010-10,0000.1-1,0001-1,000100-10,000 50-500200-2,00050-50050-500500-5,000 2001,0002002003,000

C. Example III

Irrigating solutions for urological procedures

The following drugs and their concentration ranges in solution, i.e. physiological carrier solution, are suitable for irrigation of the surgical site during urological procedures.

Drug type drug Concentration (nanomolar): therapeutic concentration preferred concentration optimal concentration Histamine ₁ antagonist Serotonin ₃ antagonist Bradykinin ₁ antagonist Bradykinin ₂ antagonist Cyclooxygenase inhibitor [des-Arg ¹⁰ ] derivative of terfenadine metoclopramide HOE 140 HOE 140 0.1-1,00010-10,0001-1,0001-1,000100-10,000 50-500200-2,00050-50050-500500-5,000   2001,0002002003,000

D. Example IV

Irrigating solutions for arthroscopy, burns, general surgical wounds and oral/dental applications

The following compositions are preferably used in anatomical irrigation during arthroscopy and oral/dental procedures, as well as in the treatment of burns and general surgical wounds. While the solutions disclosed in Example I are suitable for use in the present invention, the solutions described below are still more preferred due to their expected higher potency.

Drug type drug Concentration (nanomolar): therapeutic concentration preferred concentration optimal concentration Serotonin ₂ Antagonist Serotonin ₃ Antagonist Histamine ₁ Antagonist Serotonin _{1A, 1B, 1D, 1F} Agonist Cyclooxygenase Inhibitor Neurokinin ₁ Antagonist Neurokinin ₂ Antagonist Purine _2X Antagonist ATP- Sensitive K ⁺ channel agonist Ca ²⁺ channel antagonist Kallikrein inhibitor Amitriptyline Metoclopramide Amitriptyline Sumatriptan Ketorolac GR82334 (±) SR48968PPADS (-) Pinacidil Nifedipine Aprotinase 0.1-1,00010-10,0000.1-1,0001-1,000100-10,0001-1,0001-1,000100-100,0001-10,0001-10,0000.1-1,000 50-500200-2,00050-50010-200500-5,00010-50010-50010,000-100,000100-1,000100-5,00050-500   2001,0002001003,00020020050,0005001,000200

E. Example V

Optional irrigants for cardiovascular and general vascular therapeutic and diagnostic procedures

The following drugs and their concentration ranges in solutions, ie physiological carrier solutions, are preferably used in the irrigation of surgical sites during cardiovascular and general vascular procedures. Furthermore, this solution is preferred over the solution disclosed in Example II due to its higher potency.

Drug type drug Concentration (nanomolar): therapeutic concentration preferred concentration optimal concentration Serotonin ₂ antagonist Cyclooxygenase inhibitor Endothelin antagonist ATP-sensitive K ⁺⁺ channel agonist Ca ²⁺ channel antagonist Nitric oxide donor Trazodone Ketorolac BQ 123(-)Pinacidil Nisoldipine SIN-1 0.1-2,000100-10,0000.01-1,0001-10,0001-10,00010-10,000 50-500500-5,00010-1,000100-1,000100-1,000100-1,000 2003,000500500500500

F. Example VI

Optional irrigants for urological procedures

The following drugs and their concentration ranges in solution, ie physiological carrier solution, are preferably applied in the irrigation of the surgical site during urological procedures. This solution is believed to have a higher potency than the solution disclosed in Example III above.

Drug type drug Concentration (nanomolar): therapeutic concentration preferred concentration optimal concentration Serotonin ₂ Antagonist Histamine 1 Antagonist Cyclooxygenase Inhibitor Neurokinin ₂ Antagonist Purine _2X Antagonist ATP-sensitive K ⁺ Channel Agonist Ca ²⁺ Channel Antagonist Stimulated Peptidrelase Inhibitor Nitric Oxide Donor LY 53857 Terfenadine Ketorolac SR 48968PPADS(-) Pinacidil Nifedipine Aprotinase SIN-1 0.1-5000.1-1,000100-10,0001-1,000100-100,0001-10,0001-10,0000.1-1,00010-10,000 1-10050-500500-5,00010-50010,000-100,000100-1,000100-5,00050-500100-1,000 502003,00020050,0005001,000200500

G. Example VII

Cardiovascular and General Vascular Anti-Restenosis Irrigate

The following drugs and their concentration ranges in solutions, ie physiological carrier solutions, are preferably applied in irrigation during cardiovascular and general vascular therapeutic and diagnostic procedures. The drug in this preferred solution can also be added at the same concentration to the cardiovascular and general vascular irrigation solutions of Examples II and V above, or to the preferred antispasmodic, antirestenotic, analgesic/anti-inflammatory solution of Example VIII below.

Drug type drug Concentration (nanomolar): therapeutic concentration preferred concentration optimal concentration Thrombin inhibitors Glycoprotein IIb/IIIa receptor blockers PKC inhibitors Protein tyrosine kinase inhibitors Recombinant Hirudin Eptifibatide GF 109203X ^* Tyrosine Phosphorylation Inhibitor AG1296 0.2-20,0000.1-10,000xKdO.1-10,00010-100,000 2-2,0001-1000xKd1-1,000100-20,000 200100xKd20010,000

*Also known as Go 6850 or Bisindolemaleimide I (available from Warnor-Lambert)

H. Example VIII

Optional irrigants for cardiovascular and general vascular therapeutic and diagnostic procedures

Another formulation of the preferred solution for cardiovascular and general vascular therapeutic and diagnostic procedures except nitric oxide (NO donor) SIN-1 is replaced by two agents namely FK 409 (NOR-3) and FR 144420 (NOR -4) except the combination, all the other are identical with the prescription in the above-mentioned example V, and the concentration of medicament is as follows: Drug type drug Concentration (nanomolar): therapeutic concentration preferred concentration optimal concentration NO donor NO donor FK 409(NOR-3)FR 144420(NOR-4) 1-1,000 10-10,000 10-500100-5,000 2501,000

I. Example IX

Optional irrigants for arthroscopy, general surgical wounds, burns and oral/dental applications

The formulation of another preferred irrigant suitable for arthroscopic, general surgical and oral/dental applications is the same as in Example IV above except that the following substitutions, deletions and additions are present at the following concentrations:

1) Amitriptyline was replaced by mepyramine as an _H1 antagonist;

2) Deletion of kallikrein aprotinin;

3) adding bradykinin ₁ antagonist [leu ⁹ ][des-Arg ¹⁰ ] vasodilation;

4) Add bradykinin 2 antagonist HOE 140; and

5) Add the μ-opioid agonist fentanyl. Drug type drug Concentration (nanomolar): therapeutic concentration preferred concentration optimal concentration _H1 antagonists Bradykinin ₁ antagonists Bradykinin ₂ antagonists μ-opioid agonists Mepyramine[leu9][des-Arg10]vasodilator HOE 140 Fentanyl 0.1-1,0000.1-5001-1,0000.1-500 5-20010-20050-50010-200 100100200100

J. Example X

Optional irrigants for urological procedures

The formulation of another preferred irrigation solution suitable for use during urological procedures is the same as in Example VI above except that the following substitutions, deletions and additions are present at the following concentrations.

1) The NO donor SIN-1 was replaced by a combination of two agents:

a) FK 409 (NOR-3); and

b) FR 144420 (NOR-4);

2) Deletion of the kallikrein inhibitor aprotinin;

3) Addition of the bradykinin ₁ antagonist [leu ⁹ ][des-Arg ¹⁰ ] vasodilation; and

4) Add bradykinin ₂ antagonist HOE140.

Drug type drug Concentration (nanomolar): therapeutic concentration preferred concentration optimal concentration NO Donor NO Donor Bradykinin ₁ Antagonist Bradykinin ₂ Antagonist FK409(NOR-3)FR144420(NOR-4)[leu ⁹ ][des-Arg ¹⁰ ]vasodilator HOE140 1-1,00010-10,0000.1-5001-1,000 10-500100-5,00010-20050-500 2501,000100200

K. Example XI

Balloon dilation of normal iliac arteries in New Zealand white rabbits and the effect of histamine/serotonin receptor blockade on this response

The purpose of this study is twofold. First, a new in vivo model was used to study arterial tone. The time course of arterial size changes before and after balloon angioplasty is described below. Second, the joint control of arterial tone by histamine and serotonin in this setting was investigated by selectively infusing histamine and serotonin receptor blockers into arteries before and after injury from angioplasty role.

1. Design Considerations

The aim of this study was to describe the time course of changes in arterial lumen size in one set of arteries and to assess the effect of histamine/serotonin receptor blockade on this change in a second set of similar arteries. In order to facilitate the comparison between two different experimental groups, the two groups of arteries were treated in the same way, but the contents infused during the experiment were different. Control animals (arterial) were infused with saline (the carrier of the test solution). Arteries treated with histamine/serotonin receptor blockade received saline containing the receptor antagonist at the same rate and during the same portion of the procedure as control animals. Specifically, the test solution included: (a) the serotonin ₃ antagonist metoclopramide at a concentration of 16.0 μM; (b) the serotonin ₂ antagonist trazodone at a concentration of 1.6 μM; and (c) the concentration The histamine antagonist promethazine was dissolved in normal saline at 1.0 μM. The drug concentration in this test solution was 16 times higher than that delivered to the surgical site because of the 16:1 flow rate ratio between the iliac artery (80 cc/min) and the solution delivery catheter (5 cc/min). The study was conducted in a scheduled, randomized and double-blind manner. Assignment to specific groups was randomized, and the contents of the infusion solution (normal saline itself or saline containing a histamine/serotonin receptor antagonist) were not known to the investigator until the end of the angiographic analysis.

2. Animal protocol

The protocol was approved by the Seattle Veteran Affairs Medical Center Committee Animal Use and the experimental facility was fully accredited by the American Association for Accreditation of Laboratory Animal Care. The subjects of the study were the iliac arteries of 3-4 kg male New Zealand White rabbits fed regular rabbit chow. Sufficient doses of xylazine (5 mg/kg) and ketamine (35 mg/kg) were administered intravenously, the animals were sedated, and the ventral midline incision of the neck was performed to isolate the carotid artery. The arterial ends were ligated, an arteriotomy was performed, and a 5 French sheath was introduced into the descending aorta. Baseline blood pressure and heart rate were recorded and then recorded remotely on 35mm cine film (15 frames per second) by manual injection of 76% iodoisophthalol (Squibb Diagnostics, Princeton, NJ) into the descending aorta. Angiogram of the lateral aorta and bilateral iliac arteries. Calibrators were placed within the radiographic field of each angiogram to correct for magnification in diameter measurements. A 2.5 French infusion catheter (Advanced Cardiovascular Systems, Santa Clara, CA) was passed through the carotid sheath and placed 1-2 cm above the aortic bifurcation. Infusion of the test solution—either normal saline itself or saline containing a histamine/serotonin receptor antagonist—was initiated at a rate of 5 cc/min and continued for 15 minutes. Five minutes into the infusion, a second angiogram was performed using the technique described above, followed by rapid anterior placement of a 2.5 mm balloon angioplasty catheter (theLightning, Cordis Corp., Miami, FL) under fluoroscopy guidance. artery, and then into the right iliac artery. Using bony landmarks, a balloon catheter was carefully placed between the proximal and distal deep femoral ramus in each ilium and the balloon was inflated for 30 seconds to a pressure of 12 ATM. The balloon catheter is inflated with a diluent of radiographic contrast agent so that the balloon's inflated diameter can be recorded on cine film. The angioplasty catheter was quickly withdrawn and another angiogram was recorded on film a mean of 8 minutes after the start of the infusion. The infusion was continued until the 15 minute time point, and an angiography was performed again (fourth time). The infusion was then terminated (a total of 75 cc of solution had been infused) and the infusion catheter removed. At the 30 minute time point (15 minutes after the infusion was terminated), the last angiogram was recorded as before. Blood pressure and heart rate were recorded immediately before angiography at 15 and 30 min time points, respectively. After the last angiography, an overdose of anesthetic was administered intravenously, the animals were euthanized, and the iliac arteries were harvested and submerged fixed in formalin for histological analysis.

3. Angiographic Analysis

Angiograms were recorded on 35mm film film at a frame rate of 15 frames/sec. For analysis, angiograms were projected at a distance of 5.5 feet using a Vanguard projector. The diameter of the iliac artery at a pre-specified location opposite to the balloon angioplasty operation site was recorded based on hand-held calipers measured with calibrator corrected for magnification. At baseline (before start of test solution infusion), 5 minutes after start of infusion, immediately after balloon angioplasty (mean 8 minutes after start of test solution infusion), 15 minutes (immediately before termination of infusion) and 30 minutes (15 minutes after termination of the infusion) measurements were taken. Diameters were measured at three sites in each iliac artery: proximal to the balloon site, balloon site, and distal to the balloon site.

The diameter measurement is then converted to an area measurement by the following formula:

Area = (Pi) (diameter ² )/4

To calculate vasoconstriction, the maximum area of the artery was expressed as the baseline value and the percent vasoconstriction was calculated as follows: % vasoconstriction = {(baseline area - subsequent time point area)/baseline area} x 100.

4. Statistical method

All values are expressed as mean ± 1 standard error of the mean. The time course of the vasoconstrictor response in control arteries was estimated by correcting for repeated measures using one-way ANOVA. After this, the data between specific time points were compared using the Scheffe assay. Once the time point at which significant vasoconstriction occurs in the control artery is detected, the treatment group can be labeled as an independent variable using multivariate analysis of variance, and the time point at which significant vasoconstriction occurs in the control artery is compared between the control artery and the treated group. Arteries treated with amine/serotonin receptor antagonists. To compensate for the absence of a single prespecified hypothesis, a p value of <0.01 was considered significant. Statistica 4.5 software (Statsoft, Tulsa, OK) running under Windows was used for statistics.

5. Results

The time course of changes in arterial size before and after balloon angioplasty in normal arteries receiving saline infusion was evaluated in 16 arteries derived from 8 animals (Table 30). Three segments of each artery were studied: the proximal segment immediately upstream of the balloon-expanded segment, the balloon-expanded segment, and the distal segment immediately downstream of the balloon-expanded segment. The proximal and distal segments demonstrated a similar pattern of change in arterial size: within each segment, significant changes in arterial diameter were found when comparing all time points (p = 0.0002 for the proximal segment and p < 0.001 for the distal segment, ANOVA). Subsequent trials showed that diameters at the immediate post-angioplasty time point were significantly smaller than at baseline or at the 30-minute time point in each of these segments. On the other hand, at the 5 min, 15 min and 30 min time points, the arterial diameter in each segment approximated the baseline diameter. The balloon-dilated segment showed less change in arterial size than the proximal and distal segments. The segment had a baseline diameter of 1.82 ± 0.05 mm; the balloon used for angioplasty had a nominally inflated diameter of 2.5 mm and the actual measured inflated diameter was 2.20 ± 0.03 mm (p < 0.0001 vs. baseline diameter). Thus, the inflated balloon resulted in stretching of the periphery of the balloon-inflated segment, but only a slight increase in lumen diameter from baseline to the 30 minute time point (1.82±0.05mm-1.94±0.07mm, p=NS by follow-up testing).

Table 30

Lumen diameter determined by angiography at a specific time before and after balloon dilatation of the normal iliac artery segment baseline 5 minutes Immediately after PTA 15 minutes 30 minutes Proximal ¹ Balloon ² Distal ³ 2.18±0.71.82±.051.76±.04 2.03±0.71.77±.031.68±04 ^** 1.81±0.08 ^* 1.79±.051.43±04 ^* 2.00±.081.70±.041.54±.03 2.23±.081.94±.071.69±.06

All measurements are in mm. Mean ± SEM. PTA = percutaneous transluminal angioplasty

¹ p=0.0002 (analysis of variance for intra-group comparison)

² p=0.03 (analysis of variance for intra-group comparison)

³ p<0.0001 (analysis of variance for intra-group comparison). N=16 over all time points

^* p<0.01 vs baseline and 30 min diameter measurements (Scheffe test for subsequent comparisons).

^** p<0.01 vs. measurements immediately after PTA (Scheffe test for subsequent comparisons). All other subsequent comparisons were insignificant at a p<0.01 threshold.

Arterial lumen diameter was used to calculate lumen area, and area measurements were then used to calculate percent vasoconstriction by comparing data at 5 minutes, immediately after angioplasty, 15, and 30 minutes with baseline measurements. Data for the proximal and distal segments expressed as percent vasoconstriction are shown in Figure 9; the amount of vasoconstriction varied significantly over time (p = 0.0008 in the proximal segment; p = 0.0008 in the distal segment 0.0001, ANOVA). Subsequent testing determined that vasoconstriction at the immediate time point after angioplasty was significantly different from vasoconstriction at the 30 minute time point (P<0.001 in both segments). In the distal segment, vasoconstriction was also significantly less immediately after angioplasty than at 5 minutes (p<0.01); subsequent testing found no other differences between the time points to be significant.

Luminal changes in control arteries can be summarized as follows: 1) Vasoconstriction with a loss of approximately 30% of baseline luminal area occurred immediately after balloon expansion, in arterial segments proximal and distal to the segment of balloon expansion. At the pre-dilation and 15-minute time points (approximately 7 minutes after dilation), there was only a small amount of vasoconstriction tendency in the proximal and distal segments, but also at the 30-minute time point (approximately 22 minutes after dilation), the vessels A tendency to dilate supersedes the previous vasoconstriction; 2) In the balloon-expanded segment, there is only a small change in lumen diameter, and even with a balloon whose dilated diameter is significantly greater than the segment's baseline diameter, the lumen of the dilated segment There was no significant increase in diameter. It was concluded from these findings that any effect of histamine/serotonin treatment was presumed to be detectable only at the time point when vasoconstriction occurs, in the proximal and distal segments.

The histamine/serotonin receptor blocking solution was infused into 16 arteries (8 animals); angiographic data were available for all time points in 12 arteries. Heart rate and systolic blood pressure measurements were performed in a group of animals (Table 31). There were no differences in heart rate or systolic blood pressure when comparing the two groups of animals at specific time points. Histamine/serotonin treated animals showed a trend towards lower systolic blood pressure (-14±5 mmHg, p=0.04) and lower heart rate (-26±10, p=0.05) from baseline to 30 minutes. In control animals, the experiment continued to its conclusion with no change in heart rate or systolic blood pressure.

Table 31

Measurements of systolic blood pressure and heart rate in control and histamine/serotonin-treated animals Group Baseline (N) 5 minutes (N) 15 minutes (N) 30 minutes (N) Systolic Blood Pressure Control Histamine/Serotonin Heart Rate Control Amine/Serotonin 83±4(8)93±5(6)221±18(5)232±8(5) 84±4(8)87±9(4)234±18(4)232±8(5) 82±6(8)82±9(6)217±23(5)209±14(5) 80±4(8)80±8(6) ^* 227±22(5) 206±12(5) ^**

Systolic blood pressure is expressed in mm Hg and heart rate is expressed in beats/minute. Mean ± SEM.

^* p=0.04 represents a reduction in systolic blood pressure from baseline to 30 minutes, and

^** p=0.05 represents reduction in heart rate from baseline to 30 minutes in histamine/serotonin treated animals.

Proximal and distal segments of histamine/serotonin-treated arteries were compared to control arteries using measurements of percent vasoconstriction. Figure 10A shows the effect of histamine/serotonin infusion on proximal segment vasoconstriction relative to vasoconstriction present in control arteries. When these two groups of arteries with different treatments were compared at baseline, immediately after angioplasty, and at 15-minute time points, it was found that the histamine/serotonin infusion resulted in significantly less vasoconstriction than the control saline infusion ( p=0.003, two-way ANOVA). A comparison of the distal segments of the two groups of arteries treated differently is shown in Figure 10B. Even though differences in mean diameter measurements were observed in this distal segment, test solution-treated arteries showed less vasoconstriction than saline-treated controls at baseline, immediately after angioplasty, and at the 15-minute time points Arteries, this pattern did not achieve statistical significance (p=0.32, two-way ANOVA). The lack of statistical significance may be due to the measurement of vasoconstriction in the control arteries being less than the expected vasoconstriction.

L. Example XII

Comparison of intra-articular and intravenous amitriptyline in inhibiting serotonin-induced knee plasma extravasation

The purpose of the following study was to compare two routes of administration of the 5- _HT2 receptor antagonist amitriptyline in a rat model of inflammatory knee synovial fluid: 1) continuous intra-articular infusion; versus 2) intravenous injection . The ability of amitriptyline to inhibit 5-HT-induced joint plasma extravasation was determined by comparing the potency and total drug dose of amitriptyline delivered by the two routes.

1. Animals

These studies were approved by the Animal Experimentation Committee of the University of California, San Francisco. Male Sprague-Dawley rats (Bantin and Kingman, Fremont, CA) weighing 300-450 grams were used in the study. Rats were housed under controlled lighting (6A.M.-6P.M. lights) and food and water ad libitum.

2. Plasma extravasation

Rats were anesthetized with pentobarbital sodium (65 mg/kg), and then injected with Evans blue counterstain (50 mg/kg, converted to a volume of 2.5 ml/kg) in the tail vein as a marker for plasma protein extravasation . The knee joint capsule was exposed by excision of the overlying skin, and a 30-gauge needle was inserted into the joint for fluid infusion. The infusion rate (250 μl/min) was controlled by a Sage Instruments Syringe pump (Model 341B, Orion Research Inc., Boston, MA). A 25 gauge needle was also inserted into the joint space and the infusion was withdrawn at a rate of 250 μl/min controlled by a Sage Instruments Syringe pump (Model 351).

Rats were randomly divided into three groups: 1) received 5-HT (1 μM) by intra-articular (IA) route of administration only, 2) received amitriptyline (dose range 0.01-1.0 mg) by intravenous (IV) route of administration /kg), followed by IA 5-HT (1 mM), and 3) received amitriptyline (concentration range 1-100 μM) by the intra-articular (IA) route of administration, followed by IA 5-HT (1 μM) plus IA amitriptyline Forest. In all groups, baseline plasma extravasation levels were obtained by intra-articular infusion of 0.9% saline at the beginning of each experiment, with three infusion samples collected over time (every 5 minutes) over 15 minutes . 5-HT was then administered intra-articularly to the first group for a total of 25 minutes. Infusion samples were collected every 5 minutes. Then, the absorbance at 620nm was measured by a spectrophotometer, and the concentration of the Evans blue counterstain in the sample was analyzed, which was linearly related to the absorbance (Carr and Wilhelm, 1964). Intravenous administration of amitriptyline group was administered during tail vein injection of Evans blue counterstain. Their knees were subsequently infused with saline for 15 minutes (baseline), followed by 25 minutes of 5-HT (1 [mu]M). Infusion samples were collected every 5 minutes. Samples were subsequently analyzed by spectrophotometry. In the intra-articular amitriptyline group, a 10-minute intra-articular infusion of amitriptyline followed by a 15-minute saline infusion was followed by an additional 25-minute infusion of amitriptyline combined with 5-HT. Infusion fluid samples were collected every 5 minutes and analyzed as above.

The study was performed because some rats had knee physical damage to the knee joint or a discrepancy between inflow and outflow (can be detected by blood present in the infusion and high baseline plasma extravasation levels or knee swelling due to improper needle placement). were determined), so these rat knees were excluded.

A. 5-HT-induced plasma extravasation

Baseline plasma extravasation was determined in all knee joints tested (total n=22). Baseline plasma extravasation levels were averagely low, with an average absorbance of 0.022 ± 0.003 absorbance units at 620 nm (mean ± standard error of the mean). This baseline level of extravasation is indicated by the hatching in Figures 11 and 12.

Infusion of 5-HT (1 μM) into rat knee joints produced a time-dependent increase in plasma extravasation above baseline levels. Within 25 minutes of the intra-articular infusion of 5-HT, plasma extravasation peaked at 15 minutes and continued until the end of the infusion at 25 minutes (data not shown). Therefore, the reported levels of 5-HT-induced plasma extravasation are the average of the 15, 20 and 25 min time points during each experiment. 5-HT-induced plasma extravasation averaged 0.192±0.011, about 8 times higher than baseline stimulation. The data are shown in Figures 11 and 12, corresponding to a "0" dose of intravenously administered amitriptyline and a "0" concentration of intra-articularly administered amitriptyline, respectively.

B. Effect of intravenous administration of amitriptyline on 5-HT-induced plasma extravasation

Administration of amitriptyline via tail vein injection resulted in a dose-dependent reduction in 5-HT-induced plasma extravasation, as shown in FIG. 11 . The _IC50 corresponding to the inhibition of 5-HT-induced plasma extravasation by intravenous administration of amitriptyline is approximately 0.025 mg/kg. 5-HT-induced plasma extravasation was completely inhibited by amitriptyline administered intravenously at a dose of 1 mg/kg, with an average plasma extravasation of 0.034±0.010.

C. Effect of intra-articular administration of amitriptyline on 5-HT-induced plasma extravasation

Intra-articular administration of increasing concentrations of amitriptyline alone did not affect the level of plasma extravasation relative to baseline, which averaged 0.018 ± 0.002 (data not shown). Co-infusion of increasing concentrations of amitriptyline with 5-HT resulted in a concentration-dependent reduction in 5-HT-induced plasma extravasation, as shown in FIG. 12 . In the case of intra-articular administration of 3 μM amitriptyline, 5-HT-induced plasma extravasation was not significantly different from that caused by administration of 5-HT alone, but co-infusion of 30 μM amitriptyline with 5-HT , the resulting inhibition was greater than 50%, and 100 μM amitriptyline could completely inhibit 5-HT-induced plasma extravasation. Intra-articular administration of amitriptyline inhibits 5-HT-induced plasma extravasation with an _IC50 of approximately 20 μM.

The main finding of the study was that intra-articular infusion of 5-HT (1 μM) in the knee joint of rats produced approximately 8-fold greater stimulation of plasma extravasation than baseline, regardless of whether 5-HT was administered intravenously or intra-articularly. The receptor antagonist amitriptyline inhibited 5-HT-induced plasma extravasation. However, the total dose of amitriptyline administered was significantly different between the two drug delivery methods. The IC ₅₀ corresponding to the inhibition of 5-HT-induced plasma extravasation by intravenous administration of amitriptyline is 0.025 mg/kg, or 7.5×10 ^-3 mg in 300 g adult rats. The _IC50 corresponding to inhibition of 5-HT-induced plasma extravasation by intra-articular administration of amitriptyline is approximately 20 μM. Since 1 ml of this solution was delivered every 5 minutes for a total of 35 minutes of the experiment, the total dose infused into the knee was 7 ml, ie a total dose of 4.4 x 10 ^-5 mg was infused into the knee. The dose of intra-articular amitriptyline is about 200 times lower than the dose of intravenous amitriptyline. Furthermore, it is likely that only a small amount of intra-articularly infused drug is absorbed systemically, making even greater variability in the total delivered dose of drug.

As mentioned above, since 5-HT has an important role in surgical pain and inflammation, it may be beneficial if administered perioperatively with a 5-HT antagonist, such as amitriptyline. More recently, a study attempted to determine the effect of oral amitriptyline on postoperative pain after plastic surgery (Kerrick et al., 1993). Oral doses as low as 50 mg produced adverse central nervous system side effects such as "decreased well-being". In addition, their study also showed that oral amitriptyline produced higher pain scale scores than placebo (P<0.05) in postoperative patients. Whether this is due to the general discomfort associated with oral amitriptyline is unknown. In contrast, the intra-articular route of administration allows very low concentrations of drug to be delivered locally to the site of inflammation and may yield optimal results with the fewest side effects.

M. Example XIII

Effects of Cardiovascular and Common Vascular Solutions on Rabbit Artery Vasospasm Induced by Atherectomy

1. The solution to be tested

This study employed a wash solution consisting of the agents described in Example V above, with the modifications described below. Nitroprusside replaced SIN-1 as a nitric oxide donor, and nicardipine replaced nisoldipine as a Ca ²⁺ channel antagonist.

The concentration of nitroprusside was selected based on its previously defined pharmacological activity ( _EC50 ). The concentration of other agents in the test solution is determined based on the binding constant of the agent to its cognate receptor. In addition, all concentrations were adjusted based on a blood flow rate of 80 mL/min in the rabbit's distal aorta and a flow rate of 5 mL/min in the solution delivery catheter. The three components were mixed in 1 ml or less of DMSO, and then these components and the remaining three components were mixed in physiological saline to their final concentrations. The control solution used consisted of physiological saline. The infusion rate of the test solution or the control solution was 5 ml/min for 20 minutes. Brief breaks during the infusion were necessary to measure blood pressure so that each animal received approximately 95 ml of solution during the 20 minute treatment period.

2. Animal protocol

The protocol was approved by the Seattle Veteran Affairs Medical Center Committee on Animal Use (Seattle Veterans Affairs Medical Center Animal Application Committee), and was accredited by the American Association for Accreditation of Laboratory Animal Care (American Association for Accreditation of Laboratory Animal Management). The subjects of the study were the iliac arteries of 3-4 kg male New Zealand white rabbits fed a 2% cholesterol rabbit diet for 3-4 weeks. Sufficient doses of xylazine (5 mg/kg) and ketamine (35 mg/kg) were administered intravenously, the animals were sedated, and the ventral midline incision of the neck was performed to isolate the carotid artery. The arterial ends were ligated, atherectomy was performed, and a 5 French sheath was introduced into the descending aorta and placed at the level of the renal arteries. Baseline blood pressure and heart rate were recorded. The distal aorta and bilateral iliac arteries were recorded on 35mm cine film (15 frames/sec) by manual injection of 76% iodoisophthalol (Squibb Diagnostics, Princeton, NJ) into the descending aorta. Angiography.

Calibrators were placed within the radiographic field of each angiogram to correct for magnification when measuring diameter. Infusion of the test solution or saline control solution described above (and delivered to the distal aorta) was initiated through the lateral arm of a 5 French sheath at a rate of 5 mL/min for 20 minutes. At 5 minutes of the infusion, a second angiogram was performed using the technique described previously. A 1.25 mm or 1.50 mm atherectomy drill (Heart Technology/Boston Scientific Inc.) was then advanced into the iliac artery. The atherectomy drill was advanced to more than 3 times the length of the distance cord in each iliac artery at a rotation rate of 150,000-200,000 RPM. Within each iliac, the atherectomy drill was advanced from the distal aorta to the middle of the iliac arteries, between the first and second deep femoral branches. The atherectomy drill was quickly removed and the angiogram was again recorded on film film an average of 8 min after the start of the infusion.

The infusion was continued until the 20 minute time point, and angiography was performed again (fourth time). The infusion was then terminated. A total of about 95 ml of control solution or test solution was infused. At the 30 minute time point (15 minutes after the infusion was terminated), the final angiogram was recorded as before. Blood pressure and heart rate were recorded immediately before angiography at the 15 and 30 minute time points. After the last angiography, the animals were euthanized with an intravenous overdose of anesthetic.

3. Angiographic Analysis

Angiograms were recorded on 35mm film film at a frame rate of 15 frames/sec. Angiograms were reviewed in random order without knowing how the treatments were arranged. For analysis, angiograms were projected at a distance of 5.5 feet using a Vanguard projector. Review the complete angiogram corresponding to each animal to identify the anatomy of the iliac artery and determine the site of most spasm in the iliac artery. Prepare an anatomical map of the iliac area to help ensure that the measurement sites correspond to those on the map. Measurements were performed on the first angiogram recorded 15 minutes after atherectomy and subsequently on other angiograms from that animal in random order. Measurements were made using electronic hand-held calipers (Brown & Sharpe, Inc., N. Kingston, RI). Iliac artery diameter was measured at 3 locations: proximal to the first deep femoral branch of the iliac artery; the site of most spasticity (occurring in all cases between the first and second deep femoral branch); and distal Location (near or distal to the origin of the second deep femoral branch of the iliac artery). At baseline (before start of test solution infusion), 5 minutes after infusion, immediately after atherectomy (mean 8 minutes after start of test solution infusion), and at 20 minutes, immediately after termination of infusion (start Measurements were taken at 15 minutes after atherectomy) and 15 minutes after termination of the infusion (30 minutes after initiation of atherectomy). Calibrators were measured in each angiogram.

The diameter measurement is then converted to an area measurement by the following formula:

Area = (Pi) (diameter ² )/4

To calculate vasoconstriction, the baseline value is used to represent the maximum area of the artery, and the percent vasoconstriction is calculated as follows:

% Vasoconstriction = {(Baseline Area - Subsequent Time Point Area)/Baseline Area} x 100.

4. Statistical method

All values are expressed as mean ± 1 standard error of the mean. Using one-way ANOVA with correction for repeated measures, the time course of the vascular recruiting response in control arteries was estimated. After this, the data between specific time points were compared using the Scheffe assay. Test solution-treated arteries were compared with saline-treated arteries at specific locations and at specific time points in the iliac arteries using multivariate analysis of variance (MANOVA). To compensate for the absence of a single a priori hypothesis, a p-value of <0.01 was considered significant. Statistica 4.5 software (Statsoft, Tulsa, OK) running under Windows was used for statistics.

5. Results

Eight arteries in 4 animals received saline infusion, and 13 arteries in 7 animals received the test solution. In each artery, regardless of the solution used, an atherectomy drill was advanced from the distal aorta to the mid-iliac artery for atherectomy. Therefore, the proximal iliac segment and the segment identified as the site of greatest vasoconstriction were processed with a rotary cutter. The distance cord of the atherectomy catheter passes through the distal segment without the atherectomy drill of the atherectomy catheter itself entering the distal segment.

The iliac artery diameters of the three specific segments in the saline-treated arteries are shown in Table 32. In the proximal segment, arterial diameter did not change significantly over the time course of the experiment (p=0.88, ANOVA). Significant reductions in arterial diameter occurred at the site of greatest vasoconstriction, the mid-iliac artery, with the greatest reduction occurring at the 15-minute time point after atherectomy (p<0.0001, ANOVA comparing all 5 measurement results at a time point). Although there was a tendency for the distal segment to become smaller in diameter immediately after atherectomy and at the 15-minute time point, its diameter did not change significantly over the course of the experiment (p=0.19, ANOVA comparing for all time points).

Table 32

Iliac lumen diameter at specific time points in saline-treated arteries segment Baseline N=8 Infuse for 5 minutes N=8 Immediately after RA N=8 15 minutes after RA N = 8 30 minutes after RA N=8 Proximal ¹ Middle ² Distal ³ 2.40±.182.01±.082.01±.10 2.32±.141.84±.091.86±.08 2.32±0.131.57±.151.79±.08 2.38±.131.24±.131.81±.09 2.34±.07 ^* 1.87±.06 ^** 1.96±.06 ^***

RA = atherectomy

¹ Proximal iliac artery measurement site, close to first deep femoral branch

² Middle part of the iliac artery, located at the site of greatest vasospasm

³ Distal iliac artery measurement site, near or distal to the second deep femoral branch

*p=0.88, results obtained by ANOVA comparing proximal segment diameters at 5 time points.

***p=0.000007, the results obtained by comparing the diameter of the largest part of the degree of vasospasm at 5 time points by ANOVA.

***p=0.19, results obtained by ANOVA comparing distal segment diameters at 5 time points.

The diameter of the iliac artery treated with the test solution is shown in Table 33. Of these arteries, 3 arteries had no recorded angiogram at the 5 min time point after start of the infusion, and 2 arteries (one animal) excluded the 30 min post atherectomy time point Angiographic data above because the animal received an air embolism during angiography at the 15 min time point, resulting in hemodynamic instability. ANOVA statistics were not applied to this data due to the variation in the number of observations at the 5 time points. In each segment of the test solution-treated artery, the magnitude of the change in diameter measurements over the experimental time course was significantly smaller than that measured in the saline-treated artery.

Table 33

Iliac lumen diameter at specific time points in test solution-treated arteries segment Baseline N=13 Infuse for 5 minutes N=10 Immediately after RA N=13 15 minutes after RA N = 13 30 minutes after RA N = 11 Proximal ¹ Middle ² Distal ³ 2.28±.061.97±.062.00±.06 2.07±.071.79±.061.92±.04 2.22±.051.74±.041.90±.04 2.42±.061.95±.072.00±.06 2.39±.081.93±.082.01±.07

RA = atherectomy

¹ Proximal iliac artery measurement site, close to first deep femoral branch

² Middle part of the iliac artery, located at the site of greatest vasospasm

³ Distal iliac artery measurement site, near or distal to the second deep femoral branch

Due to the varying number of observations at different time points, ANOVA was not performed to determine statistical similarity/difference in diameters of specific segments.

The primary endpoint of the study was to compare the amount of vasoconstriction in saline-treated and test solution-treated arteries. Vasoconstriction is based on arterial luminal area deduced from arterial diameter measurements. Relative change in area can be calculated by comparing area values at 5 minutes, immediately after atherectomy, and at subsequent time points with baseline area. The outcome was termed "vasoconstriction" if the luminal area at subsequent time points was smaller than the baseline area, and "vasodilation" if the luminal area at subsequent time points was greater than the baseline area (Tables 27 and 28) . In order to facilitate the statistical analysis of the maximum number of observations that may exist in the two groups with different treatments, the arteries treated with test solution and treated with saline were compared at two time points immediately and 15 minutes after atherectomy. data.

In the proximal segment (Fig. 13), the arterial lumen area treated by the two methods was basically unchanged at the time point immediately after atherectomy, but this segment was significantly higher in the atherectomy There was some degree of vasodilation at the 15-minute time point after atherectomy. The test solution did not alter the results of atherectomy compared to the results of this segment in the saline-treated condition. But in the middle of the artery (Fig. 14), the site of greatest vasoconstriction, the test solution significantly attenuated the atherectomy-induced vasoconstriction in saline-treated arteries (p=0.0004, MANOVA corrected repeated measurements). In the distal segment (FIG. 15), there was slight vasoconstriction in saline-treated arteries, and the test solution did not significantly alter arterial response to atherectomy.

Table 34

Vasoconstriction (negative values) or vasodilation at specific time points in saline-treated arteries

Zhang (positive value) amount segment Infuse for 5 minutes N=8 Immediately after RA N=8 15 minutes after RA N = 8 30 minutes after RA N=8 Proximal ¹ Middle ² Distal ³ -3%±.8%-14%±7%-9%±.10% -1%±10%-35%±10%-14%±.14% 3%±8%-58%±7%-14%±10% 3%±13%-11%±.9% 2%±.12%

¹ Proximal iliac artery measurement site, close to first deep femoral branch

² Middle part of the iliac artery, located at the site of greatest vasospasm

³ Distal iliac artery measurement site, near or distal to the second deep femoral branch

Table 35

Vasoconstriction (negative values) or vasodilation at specific time points in test solution-treated arteries

Zhang (positive value) amount segment Infuse for 5 minutes N=10 Immediately after RA N=13 15 minutes after RA N = 13 30 minutes after RA N = 11 Proximal ¹ Middle ² Distal ³ -17%±.5%-14%±5%-8%±.4% -4%±3%-20%±5%-9%±.4% 14%±6% 0.3%±7% 1%±4% 7%±9%-5%±.5% 3%±.6%

¹ Proximal iliac artery measurement site, close to first deep femoral branch

² Middle part of the iliac artery, located at the site of greatest vasospasm

³ Distal iliac artery measurement site, near or distal to the second deep femoral branch

In saline and test solution treated arteries, the hemodynamic responses are shown in Table 36. Compared to saline-treated animals, test solution-treated animals substantially maintained hypotension and marked tachycardia during infusion of the solution. At 15 minutes after the end of the infusion (or the 30 minute time point after atherectomy), test solution-treated animals showed a partial, but not complete, regression of blood pressure to baseline.

Table 36

Blood pressure and heart rate during the protocol Group Baseline (N) 5 minutes (N) 15 minutes (N) 30 minutes (N) Systolic Blood Pressure Saline Test Solution Heart Rate Saline Test Solution 83±9(4)92±5(7)202±16(3)187±111(7) 93±6(3)35±5(7)204±3(3)246±11(7) 92±11(4)35±5(7)198±22(3)240±5(7) 83±10(4) ^* 46±5(7) ^** 193±29(3) ^* 247±16(7) ^**

*No significant changes in systolic blood pressure or heart rate in this group (p=0.37 vs. systolic blood pressure, p=0.94 vs. heart rate, ANOVA).

**There is a highly significant change in systolic blood pressure or heart rate in this group (p<0.0001 for systolic blood pressure, p=0.002 for heart rate, ANOVA).

6. Research overview

1. Atherectomy of atheroma in hypercholesterolemic New Zealand white rabbits resulted in marked vasospasm in the middle of the iliac artery treated with the atherectomy drill. The vasospasm was most pronounced 15 minutes after atherectomy and persisted in the absence of pharmacologic intervention until 30 minutes after atherectomy before almost completely resolving.

2. Under the treatment conditions of atherectomy of atherosclerotic plaque studied in this scheme, the test solution treatment according to the present invention almost completely eliminated the vasospasm that occurred after the middle part of the iliac artery was treated with the atherectomy drill.

3. In this protocol, the test solution of the present invention with specific component concentrations was used for treatment, resulting in deep hypotension during infusion of the solution. This test solution attenuates vasospasm after atherectomy in the presence of severe hypotension.

While preferred embodiments of the present invention have been illustrated and described, it should be understood that various modifications may be made to the disclosed solutions and methods without departing from the spirit and scope of the invention. For example, varying pain inhibitors and anti-inflammatory and antispasmodic and antirestenotic agents may find it possible to augment or substitute the disclosed agents in the disclosure contained herein. Accordingly, the scope of the patents granted herein should be limited only as defined by the appended claims.

Claims (19)

1. a compositions is used for tumor cell adhesion by wound during the said composition peri-operation period is locally applied to operative site to suppress surgical procedures in preparation, purposes in the medicine of pain and inflammation, wherein said composition comprises at least a antitumor cell adhesive agent and at least a pain/inflammation inhibitor of the treatment effective dose in the interior solution of liquid-carrier, selected these medicaments act on different molecular targets, selected at least a antitumor cell adhesive agent can be sent by the part and pass to operative site, so that the anti-adhesive curative effect is provided under the situation of no metabolic conversion.

2. a compositions is used for adhering to and the purposes of the medicine implanted by the tumor cell in the patient's body lumen that the said composition peri-operation period is locally applied to need to treat during body cavity suppresses surgical procedures in preparation, wherein said composition comprises at least a antitumor cell adhesive agent and at least a pain/inflammation inhibitor of the treatment effective dose in the interior solution of liquid-carrier, selected these medicaments act on different molecular targets, selected at least a antitumor cell adhesive agent can be sent by the part and pass to body cavity, implants curative effect so that provide antitumor cell to adhere to antitumor cell under the situation of no metabolic conversion.

3. a compositions is used for the purposes of the medicine of the tumor cell adhesion by wound during the said composition peri-operation period is locally applied to operative site to suppress surgical procedures in preparation, wherein said composition comprises the first antitumor cell adhesive agent and the second antitumor cell adhesive agent of the treatment effective dose in the interior solution of liquid-carrier, selected these medicaments act on different molecular targets, selected at least a medicament can be sent by the part and pass to operative site, so that the anti-adhesive curative effect is provided under the situation of no metabolic conversion.

4. a compositions is used for adhering to and the purposes of the medicine implanted by the tumor cell in the patient's body lumen that the said composition peri-operation period is locally applied to need to treat during body cavity suppresses surgical procedures in preparation, wherein said composition comprises the first antitumor cell adhesive agent and the second antitumor cell adhesive agent of the treatment effective dose in the interior solution of liquid-carrier, selected these medicaments act on different molecular targets, selected at least a medicament can be sent by the part and pass to body cavity, implants curative effect so that provide antitumor cell to adhere to antitumor cell under the situation of no metabolic conversion.

5. any one purposes among the claim 1-4, wherein the peri-operation period of solution is used and is comprised in operation the using and using before operation or after the operation of solution.

6. any one purposes among the claim 1-5, wherein the peri-operation period of solution is used and is comprised solution using before operation, after the operation neutralization operation.

7. any one purposes among the claim 1-6 comprises the solution continuous administration at operative site or body cavity.

8. any one purposes among the claim 1-7, wherein liquid-carrier is the liquid wash carrier, and solution is sent by the mode of surgical site irrigation or body cavity to pass.

9. any one purposes among the claim 1-7, wherein liquid-carrier comprises lasting release vehicle.

10. any one purposes among the claim 1-7, wherein liquid-carrier comprises gel.

11. any one purposes among the claim 1-10, wherein antitack agent is selected from CD44 receptor antagonist, integrain receptor antagaonists and selection protein receptor antagonist.

12. any one purposes among the claim 1-10, wherein at least a antitack agent comprises the inhibitor of matrix metalloproteinase.

13. the purposes of claim 1 or 2 further comprises multiple pain/inflammation inhibitor.

14. the purposes of claim 3 or 4 further comprises at least a pain/inflammation inhibitor.

15. any one purposes in the claim 1,2,13 or 14, wherein pain/inflammation inhibitor is selected from the serotonin receptor antagonist; Serotonin receptor agonist; Histamine receptor antagonists; Bradykinin receptor antagonists; Kallikrein inhibitor; Tachykinin receptor antagonists comprises neurokinin ₁Receptor subtype antagonist and neurokinin ₂The receptor subtype antagonist; Calcitonin gene related peptide receptor antagonists; Interleukin-1 receptor antagonist; Inhibitor of phospholipase enzymes comprises PLA ₂Isotype inhibitor and PLC _γThe isotype inhibitor; Cyclooxygenase-2 inhibitor; Lipoxygenase inhibitor; The prostanoid receptor antagonist comprises eicosanoid EP-1 receptor subtype antagonist and eicosanoid EP-4 receptor subtype antagonist and thromboxane receptor subtype antagonist; The leukotriene receptor antagonist comprises leukotrienes B ₄Receptor subtype antagonist and leukotriene D ₄The receptor subtype antagonist; Opioid receptor agonist comprises μ-Opioid Receptors subtype agonist, delta-opioid receptor subtype agonist and κ-Opioid Receptors subtype agonist; Purinoceptor agonist and antagonist comprise P _2VReceptor stimulating agent and P _2XReceptor antagonist; With ATP-sensitive potassium channel opener.

16. any one purposes in the claim 1,2,13 or 14, wherein pain/inflammation inhibitor is selected from: the serotonin receptor antagonist; Serotonin receptor agonist; Histamine receptor antagonists; Bradykinin receptor antagonists; Kallikrein inhibitor; Tachykinin receptor antagonists comprises neurokinin 1 receptor subtype antagonist and neurokinin 2 receptor subtype antagonisies; Calcitonin gene related peptide receptor antagonists; Inhibitor of phospholipase enzymes comprises PLA ₂Isotype inhibitor and PLC _γThe isotype inhibitor; Lipoxygenase inhibitor; The prostanoid receptor antagonist comprises eicosanoid EP-1 receptor subtype antagonist and eicosanoid EP-4 receptor subtype antagonist and thromboxane receptor subtype antagonist; The leukotriene receptor antagonist comprises leukotrienes B ₄Receptor subtype antagonist and leukotriene D ₄The receptor subtype antagonist; Opioid receptor agonist comprises μ-Opioid Receptors subtype agonist, delta-opioid receptor subtype agonist and κ-Opioid Receptors subtype agonist; Purinoceptor agonist and antagonist comprise P _2VReceptor stimulating agent and P _2XReceptor antagonist; With ATP-sensitive potassium channel opener.

17. any one purposes among the claim 1-16, wherein the solution of the using concentration or the dosage that comprise each medicament is enough to when local application, provide the inhibitory action level in operative site, and provide identical inhibitory action level required plasma concentration at operative site when making plasma concentration be lower than systemic administration.

18. any one purposes among the claim 1-6, wherein each medicament is sent the concentration of passing to be not more than 100,000 nanomoles by the part in the solution.

19. any one purposes among the claim 1-6, wherein each medicament is sent the concentration of passing to be not more than 10,000 nanomoles by the part in the solution.

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