Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
  • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
  • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

• the present invention relates to surgical irrigation solutions and methods, and particularly for anti-inflammatory, anti-pain, anti-spasm and anti-restenosis surgical irrigation solutions.
• Arthroscopy is a surgical procedure in which a camera, attached to a remote light source and video monitor, is inserted into an anatomic joint (e.g., knee, shoulder, etc.) through a small po ⁇ al incision in the overlying skin and joint capsule. Through similar portal incisions, surgical instruments may be placed in the joint, their use guided by arthroscopic visualization. As arthroscopists' skills have improved, an increasing number of operative procedures, once performed by "open" surgical technique, now can be accomplished arthroscopically. Such procedures include, for example, partial meniscectomies and ligament reconstructions in the knee, shoulder acromioplasties and rotator cuff debridements and elbow synovectomies. As a result of widening surgical indications and the development of small diameter arthroscopes, wrist and ankle arthroscopies also have become routine.
• anatomic joint e.g., knee, shoulder, etc.
• surgical instruments may be placed in the joint, their use guided by arthr
• physiologic irrigation fluid e.g., normal saline or lactated Ringer's
• physiologic irrigation fluid e.g., normal saline or lactated Ringer's
• Irrigation is also used in other procedures, such as cardiovascular and general vascular diagnostic and therapeutic procedures, urologic procedures and the treatment of burns and any operative wounds.
• a physiologic fluid is used to irrigate a wound or body cavity or passage.
• Conventional physiologic irrigation fluids do not provide analgesic, anti-inflammatory, anti-spasm and anti-restenotic effects.
• Alleviating pain and suffering in postoperative patients is an area of special focus in clinical medicine, especially with the growing number of out-patient operations performed each year.
• the most widely used agents, cyclooxygenase inhibitors (e.g., ibuprofen) and opioids (e.g., morphine, fentanyl) have significant side effects including gastrointestinal irritation/bleeding and respiratory depression.
• cyclooxygenase inhibitors e.g., ibuprofen
• opioids e.g., morphine, fentanyl
• therapeutic agents aimed at treating postoperative pain while avoiding detrimental side effects are not easily developed because the molecular targets for these agents are distributed widely throughout the body and mediate diverse physiological actions.
• 5-HT is located in platelets and in central neurons, histamine is found in mast cells, and bradykinin is produced from a larger precursor molecule during tissue trauma, pH changes and temperature changes. Because 5-HT can be released in large amounts from platelets at sites of tissue injury, producing plasma levels 20-fold greater than resting levels (Ashton, J.H., et. al., Serotonin as a Mediator of Cyclic Flow Variations in Stenosed Canine Coronary Arteries, Circulation 73, pp. 572-578 (1986)), it is possible that endogenous 5-HT plays a role in producing postoperative pain, hyperalgesia and inflammation.
• prostaglandins also are known to cause pain and inflammation.
• Cyclooxygenase inhibitors e.g., ibuprofen
• ibuprofen are commonly used in non-surgical and post-operative settings to block the production of prostaglandins, thereby reducing prostaglandin-mediated pain and inflammation.
• Cyclooxygenase inhibitors are associated with some adverse systemic side effects when applied conventionally. For example, indomethacin or ketorolac have well recognized gastrointestinal and renal adverse side effects.
• 5-HT 5-HT
• histamine histamine
• bradykinin and prostaglandins cause pain and inflammation.
• 5-HT and norepinephrine uptake antagonists which includes amitriptyline
• 5-HT and norepinephrine uptake antagonists which includes amitriptyline
• 5-HT and norepinephrine uptake antagonists which includes amitriptyline
• 5-HT and norepinephrine uptake antagonists which includes amitriptyline
• amitriptyline has been used orally with moderate success for chronic pain conditions.
• the mechanisms of chronic versus acute pain states are thought to be considerably different.
• two studies in the acute pain setting using amitriptyline perioperatively have shown no pain-relieving effect of amitriptyline.
• Levine, J.D., et. al. Desipramine Enhances Opiate Postoperative Analgesia, Pain 27, pp. 45-49 (1986); Kerrick, J.M., et.
• Amitriptyline in addition to blocking the uptake of 5-HT and norepinephrine, is a potent 5-HT receptor antagonist. Therefore, the lack of efficacy in reducing postoperative pain in the previously-mentioned studies would appear to conflict with the proposal of a role for endogenous 5-HT in acute pain. There are a number of reasons for the lack of acute pain relief found with amitriptyline in these two studies. (1) The first study (Levine et al., 1986) used amitriptyline preoperatively for one week up until the night prior to surgery whereas the second study (Kerrick et al., 1993) only used amitriptyline postoperatively.
• histaminei (H j ) receptor antagonists have been a few studies demonstrating the ability of extremely high concentrations (1% - 3% solutions — i.e., 10 - 30 mg per milliliter) of histaminei (H j ) receptor antagonists to act as local anesthetics for surgical procedures.
• This anesthetic effect is not believed to be mediated via H j receptors but, rather, due to a non-specific interaction with neuronal membrane sodium channels (similar to the action of lidocaine).
• side effects e.g., sedation
• local administration of histamine receptor antagonists currently is not used in the perioperative setting.
• the present invention provides a solution comprising at least one mitogen-activated protein kinase (MAPK) inhibitor and, preferably, a mixture of multiple agents in low concentrations directed at inhibiting locally the mediators of pain, inflammation, spasm and restenosis in a physiologic electrolyte carrier fluid.
• the invention also provides a method for perioperative delivery of the irrigation solution containing these agents directly to a surgical site, where it works locally at the receptor and enzyme levels to preemptively limit pain, inflammation, spasm and restenosis at the site. Due to the local perioperative delivery method of the present invention, a desired therapeutic effect can be achieved with lower doses of agents than are necessary when employing other methods of delivery (i.e., intravenous, intramuscular, subcutaneous and oral).
• the anti-pain/anti-inflammation agents in the solution include, in addition to the at least one mitogen-activated protein kinase (MAPK) inhibitor(s), agents selected from the following classes of receptor antagonists and agonists and enzyme activators and inhibitors, each class acting through a differing molecular mechanism of action for pain and inflammation inhibition: (1) serotonin receptor antagonists; (2) serotonin receptor agonists; (3) histamine receptor antagonists; (4) bradykinin receptor antagonists; (5) kallikrein inhibitors; (6) tachykinin receptor antagonists, including neurokinin!
• MPK mitogen-activated protein kinase
• CGRP calcitonin gene-related peptide
• interleukin receptor antagonists interleukin receptor antagonists
• inhibitors of enzymes active in the synthetic pathway for arachidonic acid metabolites including (a) phospholipase inhibitors, including PLA 2 isoform inhibitors and PLC ⁇ isoform inhibitors, (b) cyclooxygenase inhibitors, and (c) lipooxygenase inhibitors; (10) prostanoid receptor antagonists including eicosanoid EP-1 and EP-4 receptor subtype antagonists and thromboxane receptor subtype antagonists; (11) leukotriene receptor antagonists including leukotriene B 4 receptor subtype antagonists and leukotriene D 4 receptor subtype antagonists; (12) opioid receptor agonists, including T-opioid, ⁇ -opioid, and P-opioid receptor subtype agonists; (13) purinoceptor agonists and antagonists including P 2
• anti-spasm agents for particular applications.
• anti-spasm agents may be included alone or in combination with anti-pain/anti-inflammation agents in solutions used for vascular procedures to limit vasospasm, and anti-spasm agents may be included for urologic procedures to limit spasm in the urinary tract and bladder wall.
• anti-spasm agents are utilized in the solution.
• an anti-pain/anti-inflammation agent which also serves as an anti-spasm agent may be included.
• Suitable anti-inflammatory/anti-pain agents which also act as anti-spasm agents include serotonin receptor antagonists, tachykinin receptor antagonists, and ATP-sensitive potassium channel openers.
• Other agents which may be utilized in the solution specifically for their anti-spasm properties include calcium channel antagonists, endothelin receptor antagonists and the nitric oxide donors (enzyme activators).
• anti-restenosis agents include: (1) antiplatelet agents including: (a) thrombin inhibitors and receptor antagonists, (b) adenosine diphosphate (ADP) receptor antagonists (also known as purinoceptor ⁇ receptor antagonists), (c) thromboxane inhibitors and receptor antagonists and (d) platelet membrane glycoprotein receptor antagonists; (2) inhibitors of cell adhesion molecules, including (a) selectin inhibitors and (b) integrin inhibitors; (3) anti-chemotactic agents; (4) interleukin receptor antagonists (which also serve as anti-pain/anti-inflammation agents); and (5) intracellular signaling inhibitors including: (a) protein kinase C (PKC) inhibitors and protein tyrosine kinase inhibitors, (b) modulators of intracellular protein
• PDC protein kinase C
• the present invention also provides a method for manufacturing a medicament compounded as a dilute irrigation solution for use in continuously irrigating an operative site or wound during an operative procedure.
• the method entails dissolving in a physiologic electrolyte carrier fluid a plurality of anti-pain/anti-inflammatory agents, and for some applications anti-spasm agents and/or anti-restenosis agents, each agent included at a concentration of preferably no more than 100,000 nanomolar, and more preferably no more than 10,000 nanomolar.
• the method of the present invention provides for the delivery of a dilute combination of multiple receptor antagonists and agonists and enzyme inhibitors and activators directly to a wound or operative site, during therapeutic or diagnostic procedures for the inhibition of pain, inflammation, spasm and restenosis. Since the active ingredients in the solution are being locally applied directly to the operative tissues in a continuous fashion, the drugs may be used efficaciously at extremely low doses relative to those doses required for therapeutic effect when the same drugs are delivered orally, intramuscularly, subcutaneously or intravenously. As used herein, the term "local” encompasses application of a drug in and around a wound or other operative site, and excludes oral, subcutaneous, intravenous and intramuscular administration.
• continuous encompasses uninterrupted application, repeated application at frequent intervals (e.g., repeated intravascular boluses at frequent intervals intraprocedurally), and applications which are uninterrupted except for brief cessations such as to permit the introduction of other drugs or agents or procedural equipment, such that a substantially constant predetermined concentration is maintained locally at the wound or operative site.
• the advantages of local administration of the agents via luminal irrigation or other fluid application are the following: (1) local administration guarantees a known concentration at the target site, regardless of interpatient variability in metabolism, blood flow, etc.; (2) because of the direct mode of delivery, a therapeutic concentration is obtained instantaneously and, thus, improved dosage control is provided; and (3) local administration of the active agents directly to a wound or operative site also substantially reduces degradation of the agents through extracellular processes, e.g., first- and second-pass metabolism, that would otherwise occur if the agents were given orally, intravenously, subcutaneously or intramuscularly. This is particularly true for those active agents that are peptides, which are metabolized rapidly. Thus, local administration permits the use of compounds or agents which otherwise could not be employed therapeutically.
• agents in the following classes are peptidic: bradykinin receptor antagonists; tachykinin receptor antagonists; opioid receptor agonists; CGRP receptor antagonists; and interleukin receptor antagonists.
• Local, continuous delivery to the wound or operative site minimizes drug degradation or metabolism while also providing for the continuous replacement of that portion of the agent that may be degraded, to ensure that a local therapeutic concentration, sufficient to maintain receptor occupancy, is maintained throughout the duration of the operative procedure.
• the term "perioperative” encompasses application intraprocedurally, pre- and intraprocedurally, intra- and postprocedurally, and pre-, intra- and postprocedurally.
• the solutions of the present invention are most preferably applied pre-, intra- and postoperatively.
• the agents of the present solution modulate specific pathways to preemptively inhibit the targeted pathologic process. If inflammatory mediators and processes are preemptively inhibited in accordance with the present invention before they can exert tissue damage, the benefit is more substantial than if given after the damage has been initiated.
• the irrigation solutions of the present invention include combinations of drugs, each solution acting on multiple receptors or enzymes.
• the drug agents are thus simultaneously effective against a combination of pathologic processes, including pain and inflammation, vasospasm, smooth muscle spasm and restenosis.
• the action of these agents is considered to be synergistic, in that the multiple receptor antagonists and inhibitory agonists of the present invention provide a disproportionately increased efficacy in combination relative to the efficacy of the individual agents.
• the synergistic action of several of the agents of the present invention are discussed, by way of example, below in the detailed descriptions of those agents.
• the solution of the present invention may also be applied locally to any human body cavity or passage, operative wound, traumatic wound (e.g., bums) or in any operative/interventional procedure in which irrigation can be performed.
• operative wound e.g., bums
• traumatic wound e.g., bums
• irrigation can be performed.
• procedures include, but are not limited to, urological procedures, cardiovascular and general vascular diagnostic and therapeutic procedures, endoscopic procedures and oral, dental and periodontal procedures.
• wound unless otherwise specified, is intended to include surgical wounds, operative/interventional sites, traumatic wounds and burns.
• the solution should result in a clinically significant decrease in operative site pain and inflammation relative to currently-used irrigation fluids, thereby decreasing the patient's postoperative analgesic (i.e., opiate) requirement and, where appropriate, allowing earlier patient mobilization of the operative site. No extra effort on the part of the surgeon and operating room personnel is required to use the present solution relative to conventional irrigation fluids.
• analgesic i.e., opiate
• FIGURE 1 provides a schematic overview of a generic vascular cell showing molecular targets and flow of signaling information leading to contraction, secretion and/or proliferation.
• the integration of extrinsic signals through receptors, ion channels and other membrane proteins are common to platelets, neutrophils, endothelial cells and smooth muscle cells.
• Representative examples of molecular targets are included for major groups of molecules which are therapeutic targets of drugs included in the solutions of the present invention.
• FIGURE 2 provides a detailed diagram of the signaling pathways illustrating "crosstalk" between G-protein coupled receptor (GPCR) pathways and receptor tyrosine kinase (RTK) pathways in a vascular smooth muscle cell. Only representative proteins in each pathway have been shown to simplify the flow of information. Activation of GPCRs leads to increases in intracellular calcium and increased protein kinase C (PKC) activity and subsequent smooth muscle contraction or spasm. In addition, "crosstalk" to the RTK signaling pathway occurs through activation of PYK2 (a newly discovered protein tyrosine kinase) and PTK-X (an undefined protein tyrosine kinase), triggering proliferation.
• GPCR G-protein coupled receptor
• RTK receptor tyrosine kinase
• GPCR pathway Conversely, while activation of RTKs directly initiates proliferation, "crosstalk" to the GPCR pathway occurs at the level of PKC activity and calcium levels.
• LGR designates ligand-gated receptor
• MAPK designates mitogen-activated protein kinase. These interactions define the basis for synergistic interactions between molecular targets mediating spasm and restenosis.
• the GPCR signaling pathway also mediates signal transduction (FIGURES 3 and 7) leading to pain transmission in other cell types (e.g., neurons).
• FIGURE 3 provides a diagram of the G-Protein Coupled Receptor (GPCR) pathway. Specific molecular sites of action for some drugs in a preferred arthroscopic solution of the present invention are identified.
• GPCR G-Protein Coupled Receptor
• FIGURE 4 provides a diagram of the G-Protein Coupled Receptor (GPCR) pathway including the signaling proteins responsible for ""crosstalk”" with the Growth Factor Receptor signaling pathway. Specific molecular sites of action for some drugs in a preferred cardiovascular and general vascular solution of the present invention are identified. (See also FIGURE 5).
• GPCR G-Protein Coupled Receptor
• FIGURE 5 provides a diagram of the Growth Factor Receptor signaling pathway including the signaling proteins responsible for ""crosstalk”" with the G-Protein Coupled Receptor signaling pathway. Specific molecular sites of action for some drugs in a preferred cardiovascular and general vascular solution of the present invention are identified. (See also FIGURE 4).
• FIGURE 6 provides a diagram of the G-Protein Coupled Receptor pathway including the signaling proteins responsible for ""crosstalk”" with the Growth Factor Receptor signaling pathway. Specific molecular sites of action for some drugs in a preferred urologic solution are identified.
• FIGURE 7 provides a diagram of the G-Protein Coupled Receptor pathway.
• FIGURE 8 provides a diagram of the mechanism of action of nitric oxide (NO) donor drugs and NO causing relaxation of vascular smooth muscle.
• NO nitric oxide
• certain hormones and transmitters can activate a form of NO synthase in the endothelial cell through elevated intracellular calcium resulting in increased synthesis of NO.
• NO donors may generate NO extracellularly or be metabolized to NO within the smooth muscle cell.
• Extracellular NO can diffuse across the endothelial cell or directly enter the smooth muscle cell.
• the primary target of NO is the soluble guanylate cyclase (GC), leading to activation of a cGMP-dependent protein kinase (PKG) and subsequent extrusion of calcium from the smooth muscle cell via a membrane pump.
• GC soluble guanylate cyclase
• PKG cGMP-dependent protein kinase
• NO also hyperpolarizes the cell by opening potassium channels which in turn cause closure of voltage-sensitive calcium channels.
• FIGURES 9, 10A and 10B provide charts of the percent of vasoconstriction versus time in control arteries, in the proximal segment of subject arteries, and in the distal segment of subject arteries, respectively, for the animal study described in EXAMPLE Nil herein demonstrating the effect on vasoconstriction of infusion with histamine and serotonin antagonists, used in the solutions of the present invention, during balloon angioplasty.
• FIGURES 11 and 12 provide charts of plasma extravasation versus dosage of amitriptyline, used in the solutions of the present invention, delivered intravenously and intra-articularly, respectively, to knee joints in which extravasation has been induced by introduction of 5-hydroxytryptamine in the animal study described in EXAMPLE NIII herein.
• the irrigation solution of the present invention is a dilute solution of multiple pain/inflammation inhibitory agents, anti-spasm agents and anti-restenosis agents in a physiologic carrier.
• the carrier is a liquid, which as used herein is intended to encompass biocompatible solvents, suspensions, polymerizable and non-polymerizable gels, pastes and salves.
• the carrier is an aqueous solution that may include physiologic electrolytes, such as normal saline or lactated Ringer's solution.
• the anti-inflammation/anti-pain agents are selected from the group consisting of: (1) serotonin receptor antagonists; (2) serotonin receptor agonists; (3) histamine receptor antagonists; (4) bradykinin receptor antagonists; (5) kallikrein inhibitors; (6) tachykinin receptor antagonists, including neurokinini.
• CGRP calcitonin gene-related peptide
• interleukin receptor antagonists interleukin receptor antagonists
• inhibitors of enzymes active in the synthetic pathway for arachidonic acid metabolites including (a) phospholipase inhibitors, including PLA 2 isoform inhibitors and PLC ⁇ isoform inhibitors (b) cyclooxygenase inhibitors, and (c) lipooxygenase inhibitors; (10) prostanoid receptor antagonists including eicosanoid EP-1 and EP-4 receptor subtype antagonists and thromboxane receptor subtype antagonists; (11) leukotriene receptor antagonists including leukotriene B 4 receptor subtype antagonists and leukotriene D receptor subtype antagonists; (12) opioid receptor agonists, including T-opioid, ⁇ -opioid, and P-opioid receptor subtype agonists; (13) purinoceptor agonists and antagonists including P 2 ⁇ receptor
• Suitable anti-inflammatory/anti-pain agents that also act as anti-spasm agents include serotonin receptor antagonists, tachykinin receptor antagonists, ATP-sensitive potassium channel openers and calcium channel antagonists.
• Other agents that may be utilized in the solution specifically for their anti-spasm properties including endothelin receptor antagonists, calcium channel antagonists and the nitric oxide donors (enzyme activators).
• anti-restenosis agents include: (1) antiplatelet agents including: (a) thrombin inhibitors and receptor antagonists, (b) adenosine diphosphate (ADP) receptor antagonists (also known as purinoceptor ⁇ receptor antagonists), (c) thromboxane inhibitors and receptor antagonists and (d) platelet membrane glycoprotein receptor antagonists; (2) inhibitors of cell adhesion molecules, including (a) selectin inhibitors and (b) integrin inhibitors; (3) anti-chemotactic agents; (4) interleukin receptor antagonists (which also serve as anti-pain/anti-inflammation agents); and (5) intracellular signaling inhibitors including: (a) protein kinase C (PKC) inhibitors and protein tyrosine phosphatases, (b) modulators of intracellular protein tyros
• PDC protein kinase C
• the agents are included in low concentrations and are delivered locally in low doses relative to concentrations and doses required with conventional methods of drug administration to achieve the desired therapeutic effect. It is impossible to obtain an equivalent therapeutic effect by delivering similarly dosed agents via other (i.e., intravenous, subcutaneous, intramuscular or oral) routes of drug administration since drugs given systemically are subject to first- and second-pass metabolism.
• concentration of each agent is determined in part based on its dissociation constant, K ⁇ j
• dissociation constant is intended to encompass both the equilibrium dissociation constant for its respective agonist-receptor or antagonist-receptor interaction and the equilibrium inhibitory constant for its respective activator-enzyme or inhibitor-enzyme interaction.
• Each agent is preferably included at a low concentration of 0.1 to 10,000 times K d nanomolar, except for cyclooxygenase inhibitors, which may be required at larger concentrations depending on the particular inhibitor selected.
• each agent is included at a concentration of 1.0 to 1,000 times K ⁇ nanomolar and most preferably at approximately 100 times K d nanomolar. These concentrations are adjusted as needed to account for dilution in the absence of metabolic transformation at the local delivery site.
• the exact agents selected for use in the solution, and the concentration of the agents varies in accordance with the particular application, as described below.
• a solution in accordance with the present invention can include just a single or multiple pain/inflammation inhibitory agent(s), a single or multiple anti-spasm agent(s), a combination of both anti-spasm and pain/inflammation inhibitory agents, or anti-restenosis agents from the enumerated classes, at low concentration.
• a single or multiple pain/inflammation inhibitory agent(s) a single or multiple anti-spasm agent(s)
• a combination of both anti-spasm and pain/inflammation inhibitory agents or anti-restenosis agents from the enumerated classes, at low concentration.
• multiple agents be utilized.
• the surgical solutions constitute a novel therapeutic approach by combining multiple pharmacologic agents acting at distinct receptor and enzyme molecular targets.
• pharmacologic strategies have focused on the development of highly specific drugs that are selective for individual receptor subtypes and enzyme isoforms that mediate responses to individual signaling neurotransmitters and hormones.
• endothelin peptides are some of the most potent vasoconstrictors known.
• Selective antagonists that are specific for subtypes of endothelin (ET) receptors are being sought by several pharmaceutical companies for use in the treatment of numerous disorders involving elevated endothelin levels in the body.
• the therapeutic approach of the present surgical solutions is based on the rationale that a combination of drugs acting simultaneously on distinct molecular targets is required to inhibit the full spectrum of events that underlie the development of a pathophysiologic state.
• the surgical solutions are composed of drugs that target common molecular mechanisms operating in different cellular physiologic processes involved in the development of pain, inflammation, vasospasm, smooth muscle spasm and restenosis (see FIGURE 1). In this way, the cascading of additional receptors and enzymes in the nociceptive, inflammatory, spasmodic and restenotic pathways is minimized by the surgical solutions. In these pathophysiologic pathways, the surgical solutions inhibit the cascade effect both "upstream” and "downstream".
• upstream inhibition is the cyclooxygenase antagonists in the setting of pain and inflammation.
• the cyclooxygenase enzymes COX and COX ) catalyze the conversion of arachidonic acid to prostaglandin H which is an intermediate in the biosynthesis of inflammatory and nociceptive mediators including prostaglandins, leukotrienes, and thromboxanes.
• the cyclooxygenase inhibitors block "upstream” the formation of these inflammatory and nociceptive mediators. This strategy precludes the need to block the interactions of the seven described subtypes of prostanoid receptors with their natural ligands.
• a similar "upstream" inhibitor included in the surgical solutions is aprotinin, a kallikrein inhibitor.
• the enzyme kallikrein, a serine protease cleaves the high molecular weight kininogens in plasma to produce bradykinins, important mediators of pain and inflammation.
• aprotinin effectively inhibits the synthesis of bradykinins, thereby providing an effective "upstream” inhibition of these inflammatory mediators.
• the surgical solutions also make use of "downstream" inhibitors to control the pathophysiologic pathways.
• vascular smooth muscle preparations that have been precontracted with a variety of neurotransmitters (e.g., serotonin, histamine, endothelin, and thromboxane) implicated in coronary vasospasm
• ATP-sensitive potassium channel openers KCOs
• KCOs produce smooth muscle relaxation which is concentration dependent (Quast et al., 1994; Kashiwabara et al., 1994).
• the KCOs therefore, provide a significant advantage to the surgical solutions in the settings of vasospasm and smooth muscle spasm by providing "downstream" antispasmodic effects that are independent of the physiologic combination of agonists initiating the spasmodic event (see FIGURES 2 and 4).
• NO donors and voltage-gated calcium channel antagonists can limit vasospasm and smooth muscle spasm initiated by multiple mediators known to act earlier in the spasmodic pathway.
• Serotonin (5-HT) is thought to produce pain by stimulating serotonin 2 (5-HT 2 ) and/or serotonin 3 (5-HT 3 ) receptors on nociceptive neurons in the periphery.
• 5-HT 3 receptors on peripheral nociceptors mediate the immediate pain sensation produced by 5-HT (Richardson et al., 1985).
• 5-HT 3 receptor antagonists by inhibiting nociceptor activation, also may inhibit neurogenic inflammation. Barnes P.J., et. al., Modulation ofNeurogenic Inflammation: Novel Approaches to Inflammatory Disease, Trends in Pharmacological Sciences 11, pp. 185-189 (1990).
• 5-HT 2 receptor is responsible for nociceptor activation by 5-HT. Grubb, B.D., et. al., A Study of 5-HT-Receptors Associated with Afferent Nerves Located in Normal and Inflamed Rat Ankle Joints, Agents Actions 25, pp. 216-18 (1988). Therefore, activation of 5-HT 2 receptors also may play a role in peripheral pain and neurogenic inflammation.
• One goal of the solution of the present invention is to block pain and a multitude of inflammatory processes.
• 5-HT 2 and 5-HT 3 receptor antagonists are both suitably used, either individually or together, in the solution of the present invention, as shall be described subsequently.
• Amitriptyline (ElavilTM) is a suitable 5-HT 2 receptor antagonist for use in the present invention.
• Amitriptyline has been used clinically for numerous years as an anti-depressant, and is found to have beneficial effects in certain chronic pain patients.
• Metoclopramide (ReglanTM) is used clinically as an anti-emetic drug, but displays moderate affinity for the 5-HT 3 receptor and can inhibit the actions of 5-HT at this receptor, possibly inhibiting the pain due to 5-HT release from platelets. Thus, it also is suitable for use in the present invention.
• Other suitable 5-HT 2 receptor antagonists include imipramine, trazodone, desipramine and ketanserin. Ketanserin has been used clinically for its anti- hypertensive effects.
• the cardiovascular and general vascular solution also may contain a serotonin j B (also known as serotonin J J n) antagonist because serotonin has been shown to produce significant vascular spasm via activation of the serotonin ⁇ B receptors in humans.
• serotonin j B also known as serotonin J J n
• Suitable serotonin 1B receptor antagonists include yohimbine, N-[-methoxy-3-(4-methyl-l- piperanzinyl)phenyl]-2'-methyl-4'-(5-methyl-l, 2, 4-oxadiazol-3-yl)[l, l-biphenyl]-4- carboxamide ("GR127935") and methiothepin.
• Therapeutic and preferred concentrations for use of these drugs in the solution of the present invention are set forth in Table 1.
• Serotonin 1B Human lDn Antagonists:
• 5-HT 1A , 5-HT 1B and 5-HT ⁇ D receptors are known to inhibit adenylate cyclase activity.
• serotonin 1A , serotonin 1B and serotonin 1D receptor agonists in the solution should inhibit neurons mediating pain and inflammation.
• serotonin 1E and serotonin 1F receptor agonists because these receptors also inhibit adenylate cyclase.
• Buspirone is a suitable IA receptor agonist for use in the present invention.
• Sumatriptan is a suitable 1 A, IB, ID and IF receptor agonist.
• a suitable IB and ID receptor agonist is dihydroergotamine.
• a suitable IE receptor agonist is ergonovine.
• dihydroergotamine 0.1 - 1,000 10 - 100
• dihydroergotamine 0.1 - 1,000 10 - 100
• Histamine receptors generally are divided into histamine! (H ⁇ and histamine 2 (H 2 ) subtypes.
• the classic inflammatory response to the peripheral administration of histamine is mediated via the Hi receptor. Douglas, 1985. Therefore, the solution of the present invention preferably includes a histamine H ⁇ receptor antagonist.
• Promethazine PhenerganTM
• this drug also has been shown to possess local anesthetic effects but the concentrations necessary for this effect are several orders higher than that necessary to block receptors, thus, the effects are believed to occur by different mechanisms.
• the histamine receptor antagonist concentration in the solution is sufficient to inhibit Hi receptors involved in nociceptor activation, but not to achieve a "local anesthetic" effect, thereby eliminating the concern regarding systemic side effects.
• Histamine receptors also are known to mediate vasomotor tone in the coronary arteries. In vitro studies in the human heart have demonstrated that the histamine ⁇ receptor subtype mediates contraction of coronary smooth muscle. Ginsburg, R., et al, Histamine Provocation of Clinical Coronary Artery Spasm: Implications Concerning Pathogenesis of Variant Angina Pectoris, American Heart J., Vol. 102, pp. 819-822, (1980). Some studies suggest that histamine-induced hypercontractility in the human coronary system is most pronounced in the proximal arteries in the setting of atherosclerosis and the associated denudation of the arterial endothelium. Keitoku, M. et al, Different Histamine Actions in Proximal and Distal Human Coronary Arteries in Vitro, Cardiovascular Research 24, pp. 614-622, (1990). Therefore, histamine receptor antagonists may be included in the cardiovascular irrigation solution.
• H j receptor antagonists include terfenadine, diphenhydramine, amitriptyline, mepyramine and tripolidine. Because amitriptyline is also effective as a serotonin 2 receptor antagonist, it has a dual function as used in the present invention. Suitable therapeutic and preferred concentrations for each of these H ⁇ receptor antagonists are set forth in Table 3.
• Bradykinin Receptor Antagonists Bradykinin receptors generally are divided into bradykinin i (B ⁇ and bradykinin 2 (B 2 ) subtypes. Studies have shown that acute peripheral pain and inflammation produced by bradykinin are mediated by the B 2 subtype whereas bradykinin-induced pain in the setting of chronic inflammation is mediated via the Bj subtype.
• Perkins, M.N., et. al Antinociceptive Activity of the Bradykinin BI andB2 Receptor Antagonists, des-Arg 9 , [Leu 8 ]-BK and HOE 140, in Two Models of Persistent Hyperalgesia in the Rat, Pain 53, pp. 191-97 (1993); Dray, A., et. al, Bradykinin and Inflammatory Pain, Trends Neurosci 16, pp. 99-104 (1993), each of which references is hereby expressly incorporated by reference.
• bradykinin receptor antagonists are not used clinically. These drugs are peptides (small proteins), and thus they cannot be taken orally, because they would be digested. Antagonists to B 2 receptors block bradykinin-induced acute pain and inflammation. Dray et. al, 1993. receptor antagonists inhibit pain in chronic inflammatory conditions. Perkins et al, 1993; Dray et. al, 1993. Therefore, depending on the application, the solution of the present invention preferably includes either or both bradykinin B j and B 2 receptor antagonists. For example, arthroscopy is performed for both acute and chronic conditions, and thus an irrigation solution for arthroscopy could include both B ! and B 2 receptor antagonists.
• Suitable bradykinin receptor antagonists for use in the present invention include the following bradykinin j receptor antagonists: the [des-Arg 10 ] derivative of D-Arg-(Hyp 3 -Thi 5 -D-Tic 7 -Oic 8 )-BK ("the [des-Arg 10 ] derivative of HOE 140", available from Hoechst Pharmaceuticals); and [Leu 8 ] des-Arg 9 -BK.
• Suitable bradykinin 2 receptor antagonists include: [D-Phe 7 ]-BK;
• HOE 140 1 - 1,000 50 - 500 E. Kallikrein Inhibitors
• bradykinin is an important mediator of pain and inflammation, as noted previously. Bradykinin is produced as a cleavage product by the action of kallikrein on high molecular weight kininogens in plasma. Therefore kallikrein inhibitors are believed to be therapeutic in inhibiting bradykinin production and resultant pain and inflammation.
• a suitable kallikrein inhibitor for use in the present invention is aprotinin. Suitable concentrations for use in the solutions of the present invention are set forth below in Table 5.
• Tachykinins are a family of structurally related peptides that include substance P, neurokinin A (NKA) and neurol nin B (NKB). Neurons are the major source of TKs in the periphery. An important general effect of TKs is neuronal stimulation, but other effects include endothelium-dependent vasodilation, plasma protein extravasation, mast cell recruitment and degranulation and stimulation of inflammatory cells. Maggi, C.A., Gen. Pharmacol., Vol. 22, pp. 1-24 (1991). Due to the above combination of physiological actions mediated by activation of TK receptors, targeting of TK receptors is a reasonable approach for the promotion of analgesia and the treatment of neurogenic inflammation.
• Substance P activates the neurokinin receptor subtype referred to as NK j .
• Substance P is an undecapeptide that is present in sensory nerve terminals.
• Substance P is known to have multiple actions which produce inflammation and pain in the periphery after C-fiber activation, including vasodilation, plasma extravasation and degranulation of mast cells.
• Levine, J.D., et. al Peptides and the Primary Afferent Nociceptor, J. Neurosci. 13, p. 2273 (1993).
• a suitable Substance P antagonist is ([D-Pro 9 [spiro-gamma-lactam]Leu 10 ,Trp 11 ]physalaemin-(l-l l)) ("GR 82334").
• Other suitable antagonists for use in the present invention which act on the N j receptor are: l-imino-2-(2-methoxy-phenyl)-ethyl)-7,7-diphenyl-4- perhydroisoindolone(3aR,7aR) (“RP 67580”); and 2S,3S-cis-3-(2- methoxybenzylamino)-2-benzhydrylquinuclidine (“CP 96,345").
• Suitable concentrations for these agents are set forth in Table 6.
• Neurokinin A is a peptide which is colocalized in sensory neurons with substance P and which also promotes inflammation and pain. Neurokinin A activates the specific neurokinin receptor referred to as NK 2 . Edmonds-Alt, S., et. al, A Potent and Selective Non-Peptide Antagonist of the Neurokinin A (NKz) Receptor, Life Sci. 50:PL101 (1992). In the urinary tract, TKs are powerful spasmogens acting through only the NK 2 receptor in the human bladder, as well as the human urethra and ureter. Maggi, C.A., Gen. Pharmacol., Vol. 22, pp. 1-24 (1991).
• NK 2 antagonists include: ((S)-N-methyl-N-[4-(4-acetylamino-4- phenylpiperidino)-2- (3,4-dichlorophenyl)butyl]benzamide ("( ⁇ )-SR 48968"); Met-
• Calcitonin gene-related peptide is a peptide which is also colocalized in sensory neurons with substance P, and which acts as a vasodilator and potentiates the actions of substance P.
• CGRP Calcitonin Gene-Related Peptide
• An example of a suitable CGRP receptor antagonist is I-CGRP-(8-37), a truncated version of CGRP. This polypeptide inhibits the activation of CGRP receptors. Suitable concentrations for this agent are provided in Table 8.
• Interleukins are a family of peptides, classified as cytokines, produced by leukocytes and other cells in response to inflammatory mediators. Interleukins (IL) may be potent hyperalgesic agents peripherally. Ferriera, S.H., et. al, Interleukin- 1 & as a Potent Hyperalgesic Agent Antagonized by a Tripeptide Analogue, Nature 334, p. 698 (1988).
• An example of a suitable IL-l ⁇ receptor antagonist is Lys-D-Pro-Thr, which is a truncated version of IL-l ⁇ . This tripeptide inhibits the activation of IL-l ⁇ receptors. Suitable concentrations for this agent are provided in Table 9.
• PKA 2 phospholipase A 2
• PLA 2 isoform inhibits the release of arachidonic acid from cell membranes, and therefore inhibits the production of prostaglandins and leukotrienes resulting in decreased inflammation and pain.
• Glaser, K.B. Regulation of Phospholipase A2 Enzymes: Selective Inhibitors and Their Pharmacological Potential, Adv. Pharmacol. 32, p. 31 (1995).
• An example of a suitable PLA 2 isoform inhibitor is manoalide. Suitable concentrations for this agent are included in Table 10.
• Inhibition of the phospholipase C ⁇ (PLC r ) isoform also will result in decreased production of prostanoids and leukotrienes, and, therefore, will result in decreased pain and inflammation.
• PLC ⁇ isoform inhibitor is l-[6-((17 ⁇ -3- methoxyestra- 1,3,5(10)-trien- 17-yl)amino)hexyl]- lH-pynole-2, 5-dione.
• PLA Isoform Inhibitor manoalide 100-100,000 500-10,000
• Nonsteroidal anti-inflammatory drugs are widely used as anti-inflammatory, anti-pyretic, anti-thrombotic and analgesic agents.
• Lewis, R.A. is widely used as anti-inflammatory, anti-pyretic, anti-thrombotic and analgesic agents.
• Prostaglandins and Leukotrienes In: Textbook of Rheumatology, 3d ed. (Kelley W.N., et. al, eds.), p. 258 (1989).
• the molecular targets for these drugs are type I and type II cyclooxygenases (COX-1 and COX-2). These enzymes are also known as Prostaglandin H Synthase (PGHS)-l (constitutive) and -2 (inducible), and catalyze the conversion of arachidonic acid to Prostaglandin H which is an intermediate in the biosynthesis of prostaglandins and thromboxanes.
• PGHS Prostaglandin H Synthase
• -2 inducible
• the COX-2 enzyme has been identified in endothelial cells, macrophages, and fibroblasts. This enzyme is induced by IL-1 and endotoxin, and its expression is upregulated at sites of inflammation. Constitutive activity of COX-1 and induced activity of COX-2 both lead to synthesis of prostaglandins which contribute to pain and inflammation.
• NSAIDs currently on the market (diclofenac, naproxen, indomethacin, ibuprofen, etc.) are generally nonselective inhibitors of both isoforms of COX, but may show greater selectively for COX-1 over COX-2, although this ratio varies for the different compounds.
• COX-1 and 2 inhibitors to block formation of prostaglandins represents a better therapeutic strategy than attempting to block interactions of the natural ligands with the seven described subtypes of prostanoid receptors.
• Reported antagonists of the eicosanoid receptors (EP-1, EP-2, EP-3) are quite rare and only specific, high affinity antagonists of the thromboxane A2 receptor have been reported. Wallace, J. and Cirino, G. Trends in Pharm. Sci., Vol. 15 pp. 405-406 (1994).
• the oral, intravenous or intramuscular use of cyclooxygenase inhibitors is contraindicated in patients with ulcer disease, gastritis or renal impairment.
• ketorolac In the United States, the only available injectable form of this class of drugs is ketorolac (ToradolTM), available from Syntex Pharmaceuticals, which is conventionally used intramuscularly or intravenously in postoperative patients but, again, is contraindicated for the above-mentioned categories of patients.
• ketorolac, or any other cyclooxygenase inhibitor(s) in the solution in substantially lower dosages than currently used perioperatively may allow the use of this drug in otherwise contraindicated patients.
• the addition of a cyclooxygenase inhibitor to the solutions of the present invention adds a distinct mechanism for inhibiting the production of pain and inflammation during arthroscopy or other therapeutic or diagnostic procedure.
• Prefened cyclooxygenase inhibitors for use in the present invention are keterolac and indomethacin. Of these two agents, indomethacin is less preferred because of the relatively high dosages required. Therapeutic and preferred concentrations for use in the solution are provided in Table 11. Table 11
• ketorolac 100 - 10,000 500 - 5,000 indomethacin 1,000 - 500,000 10,000 - 200,000
• lipooxygenase inhibits the production of leukotrienes, such as leukotriene B 4 , which is known to be an important mediator of inflammation and pain.
• leukotriene B 4 which is known to be an important mediator of inflammation and pain.
• Lewis, R.A. Prostaglandins and Leukotrienes, In: Textbook of Rheumatology, 3d ed. (Kelley W.N., et. al, eds.), p. 258 (1989).
• An example of a 5- lipooxygenase antagonist is 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-l,4- benzoquinone ("AA 861”), suitable concentrations for which are listed in Table 12.
• prostanoids produced as metabolites of arachidonic acid mediate their inflammatory effects through activation of prostanoid receptors.
• classes of specific prostanoid antagonists are the eicosanoid EP-1 and EP-4 receptor subtype antagonists and the thromboxane receptor subtype antagonists.
• a suitable prostaglandin E 2 receptor antagonist is 8-chlorodibenz[b,f][l,4]oxazepine-10(l lH)-
• SUBST ⁇ JTE S RULE carboxylic acid, 2-acetylhydrazide (“SC 19220")
• a suitable thromboxane receptor subtype antagonist is [15-[l ⁇ , 2 ⁇ (5Z), 3 ⁇ , 4 ⁇ ]-7-[3-[2-(phenylamino)-carbonyl] hydrazino] methyl]-7-oxobicyclo-[2,2,l]-hept-2-yl]-5-heptanoic acid (“SQ 29548").
• Suitable concentrations for these agents are set forth in Table 13.
• LTB 4 , LTC 4 , and LTD 4 are products of the leukotrienes
• LTB 4 receptor is found in certain immune cells including eosinophils and neutrophils. LTB 4 binding to these receptors results in chemotaxis and lysosomal enzyme release thereby contributing to the process of inflammation.
• the signal transduction process associated with activation of the LTB 4 receptor involves G-protein-mediated stimulation of phosphotidylinositol (PI) metabolism and elevation of intracellular calcium (see FIGURE 2).
• a suitable leukotriene B 4 receptor antagonist is SC (+)-(S)-7- (3-(2-(cyclopropylmethyl)-3-methoxy-4-[(methylamino)-carbonyl]phenoxy(propoxy)- 3,4-dihydro-8-propyl-2H-l-benzopyran-2-propanoic acid ("SC 53228").
• SC 53228 3-(2-(cyclopropylmethyl)-3-methoxy-4-[(methylamino)-carbonyl]phenoxy(propoxy)- 3,4-dihydro-8-propyl-2H-l-benzopyran-2-propanoic acid
• leukotriene B 4 receptor antagonists include [3-[-2(7-chloro-2-quinolinyl)ethenyl]phenyl] [[3-(dimethylamino-3-oxopropyl)thio] methyl]thiopropanoic acid ("MK 0571”) and the drugs LY 66,071 and ICI 20,3219. MK 0571 also acts as a LTD 4 receptor subtype antagonist.
• Opioid receptors include the ⁇ -, ⁇ - and ⁇ -opioid receptor subtypes.
• the ⁇ -receptors are located on sensory neuron terminals in the periphery and activation of these receptors inhibits sensory neuron activity.
• A- and P-receptors are located on sympathetic efferent terminals and inhibit the release of prostaglandins, thereby inhibiting pain and inflammation.
• the opioid receptor subtypes are members of the G-protein coupled receptor superfamily. Therefore, all opioid receptor agonists interact and initiate signaling through their cognate G-protein coupled receptor (see FIGURES 3 and 7).
• suitable T-opioid receptor agonists are fentanyl and Try-D-Ala-Gly-[N-MePhe]- NH(CH 2 )-OH (“DAMGO").
• Suitable ⁇ -opioid receptor agonist is [D-Pen 2 ,D-Pen 5 ]enkephalin ("DPDPE").
• DPDPE D-Pen 2 ,D-Pen 5 ]enkephalin
• P-opioid receptor agonist is (trans)-3,4-dichloro-N-methyl-N-[2-(l-pynolidnyl)cyclohexyl]-benzene acetamide (“U50,488"). Suitable concentrations for each of these agents are set forth in Table 15. Table 15
• Extracellular ATP acts as a signaling molecule through interactions with P 2 purinoceptors.
• P 2x purinoceptors which are ligand-gated ion channels possessing intrinsic ion channels permeable to Na + , K + , and Ca 2+ .
• P x receptors described in sensory neurons are important for primary afferent neurotransmission and nociception.
• ATP is known to depolarize sensory neurons and plays a role in nociceptor activation since ATP released from damaged cells stimulates P 2 ⁇ receptors leading to depolarization of nociceptive nerve-fiber terminals.
• the P2X 3 receptor has a highly restricted distribution (Chen, CC, et. al, Nature, Vol.
• Suitable antagonists of P ⁇ /ATP purinoceptors for use in the present invention include, by way of example, suramin and pyridoxylphosphate-6-azophenyl- 2,4-disulfonic acid ("PPADS"). Suitable concentrations for these agents are provided in Table 16.
• Agonists of the P 2 ⁇ receptor are known to effect smooth muscle relaxation through elevation of inositol triphosphate (IP 3 ) levels with a subsequent increase in intracellular calcium.
• IP 3 inositol triphosphate
• An example of a P 2Y receptor agonist is 2-me-S-ATP.
• ATP-Sensitive Potassium Channel Openers ATP-sensitive potassium channels have been discovered in numerous tissues, including vascular and non-vascular smooth muscle and brain, and binding studies using radiolabeled ligands have confirmed their existence. Opening of these channels causes potassium (K + ) efflux and hyperpolarizes the cell membrane (see FIGURE 2).
• K + channel openers KCOs
• KCOs K + channel openers
• FIGURE 4 K + channel openers
• KCOs also have been shown to prevent stimulus coupled secretion and are considered to act on prejunctional neuronal receptors and thus will inhibit effects due to nerve stimulation and release of inflammatory mediators.
• ATP-sensitive potassium channels are expected in achieving vasorelaxation or smooth muscle relaxation.
• a rationale for dual use is based upon the fact that these drugs have different molecular mechanisms of action in promoting relaxation of smooth muscle and prevention of vasospasm.
• An initial intracellular calcium elevation in smooth muscle cells induced by the ET A receptor subsequently triggers activation of voltage-dependent channels and the entry of extracellular calcium which is required for contraction.
• Antagonists of the ET A receptor will specifically block this receptor mediated effect but not block increases in calcium triggered by activation of other G-protein coupled receptors on the muscle cell.
• Potassium-channel opener drugs such as pinacidil, will open these channels causing K + efflux and hyperpolarization of the cell membrane.
• This hyperpolarization will act to reduce contraction mediated by other receptors by the following mechanisms: (1) it will induce a reduction in intracellular free calcium through inhibition of voltage-dependent Ca 2+ channels by reducing the probability of opening L-type or T-type calcium channels, (2) it will restrain agonist induced (receptor operated channels) Ca 2+ release from intracellular sources through inhibition of inositol triphosphate (IP 3 ) formation, and (3) it will lower the efficiency of calcium as an activator of contractile proteins. Consequently, combined actions of these two classes of drugs will clamp the target cells into a relaxed state or one which is more resistant to activation.
• Suitable ATP-sensitive K + channel openers for the practice of the present invention include: (-)pinacidil; cromakalim; nicorandil; minoxidil; N-cyano-N'-[l,l- dimethyl-[2,2,3,3- 3 H]propyl]-N"-(3-pyridinyl)guanidine (“P 1075”); and N-cyano-N- (2-nitroxyethyl)-3-pyridinecarboximidamide monomethansulphonate ("KRN 2391"). Concentrations for these agents are set forth in Table 17. Table 17
• Anti-inflammatory/anti-pain agents that also serve as anti- spasm agents include: serotonin receptor antagonists, particularly, serotonin 2 antagonists; tachykinin receptor antagonists and ATP-sensitive potassium channel openers.
• Nitric Oxide donors may be included in the solutions of the present invention particularly for their anti-spasm activity.
• Nitric oxide (NO) plays a critical role as a molecular mediator of many physiological processes, including vasodilation and regulation of normal vascular tone.
• NOS NO synthase
• NO is continuously formed and released by the vascular endothelium under basal conditions which inhibits contractions and controls basal coronary tone and is produced in the endothelium in response to various agonists (such as acetylcholine) and other endothelium dependent vasodilators.
• various agonists such as acetylcholine
• other endothelium dependent vasodilators are key molecular targets controlling vascular tone (see FIGURE 8).
• FIGURE 8 Muramatsu, K., et. al, Coron. Artery Dis., Vol. 5, pp. 815-820 (1994).
• Synergistic interactions between NO donors and openers of ATP-sensitive potassium channels (KCOs) are expected to achieve vasorelaxation or smooth muscle relaxation.
• vasoconstrictors vasopressin, angotensin II and endothelin, all inhibit K ATP currents through inhibition of protein kinase A.
• K AT p cunent in bladder smooth muscle is inhibited by muscarinic agonists.
• the actions of NO in mediating smooth muscle relaxation occur via independent molecular pathways (described above) involving protein kinase G (see FIGURE 8). This suggests that the combination of the two classes of agents will be more efficacious in relaxing smooth muscle than employing a single class of agent alone.
• Suitable nitric oxide donors for the practice of the present invention include nitroglycerin, sodium nitroprusside, the drug FK 409, FR 144420, 3-morpholinosydnonimine , or linsidomine chlorohydrate, ("SIN-1"); and S-nitroso- N-acetylpenicillamine (“SNAP"). Concentrations for these agents are set forth in Table 18.
• Endothelin is a 21 amino acid peptide that is one of the most potent vasoconstrictors known.
• Three different human endothelin peptides, designated ET-1, ET-2 and ET-3 have been described which mediate their physiological effects through at least two receptor subtypes referred to as ET A and ET B receptors.
• ET A receptors have often been found to mediate contractile responses in isolated smooth muscle preparations.
• Antagonists of ET A receptors have been found to be potent antagonists of human coronary artery contractions.
• antagonists to the ET A receptor should be therapeutically beneficial in the perioperative inhibition of coronary vasospasm and may additionally be useful in inhibition of smooth muscle contraction in urological applications. Miller, R.C., et. al, Trends in Pharmacol. Sci., Vol. 14, pp. 54-60 (1993).
• Suitable endothelin receptor antagonists include: cyclo(D-Asp-Pro-D-Val- Leu-D-Trp) ("BQ 123"); (N,N-hexamethylene)-carbamoyl-Leu-D-Trp-(CHO)-D-Trp- OH ("BQ 610"); (R)2-([R-2-[(s)-2-([l-hexahydro-lH-azepinyl]-carbonyl]amino-4- methyl-pentanoyl) amino-3-(3[l-methyl-lH-indodyl])propionylamino-3(2-pyridyl) propionic acid (“FR 139317”); cyclo(D-Asp-Pro-D-Ile-Leu-D-Trp) ("JKC 301”); cyclo(D-Ser-Pro-D-Val-Leu-D-Trp) ("JK 302"); 5-
• Calcium channel antagonists are a distinct group of drugs that interfere with the transmembrane flux of calcium ions required for activation of cellular responses mediating neuroinflammation. Calcium entry into platelets and white blood cells is a key event mediating activation of responses in these cells. Furthermore, the role of bradykinin receptors and neurol inin receptors (NKi and NK 2 ) in mediating the neuroinflammation signal transduction pathway includes increases in intracellular calcium, thus leading to activation of calcium channels on the plasma membrane. In many tissues, calcium channel antagonists, such as nifedipine, can reduce the release of arachidonic acid, prostaglandins, and leukotrienes that are evoked by various stimuli. Moncada, S., Flower, R. and Vane, J. in Goodman 's and Gilman 's Pharmacological Basis of Therapeutics, (7th ed.), MacMillan Publ Inc., pp. 660-5 (1995).
• Calcium channel antagonists also interfere with the transmembrane flux of calcium ions required by vascular smooth muscle for contractions. This effect provides the rationale for the use of calcium channel antagonists perioperatively during procedures in which the goal is to alleviate vasospasm and promote relaxation of smooth muscle.
• the dihydropyridines, including nisoldipine act as specific inhibitors (antagonists) of the voltage-dependent gating of the L-type subtype of calcium channels.
• Systemic administration of the calcium channel antagonist nifedipine during cardiac surgery previously has been utilized to prevent or minimize coronary artery vasospasm.
• Calcium channel antagonists which are among the anti-spasm agents useful in the present invention, exhibit synergistic effect when combined with other agents of the present invention.
• Calcium (Ca 2+ ) channel antagonists and nitric oxide (NO) donors interact in achieving vasorelaxation or smooth muscle relaxation, i.e., in inhibiting spasm activity.
• NO nitric oxide
• a rationale for dual use is based upon the fact that these two classes of drugs have different molecular mechanisms of action, may not be completely effective in achieving relaxation used alone, and may have different time periods of effectiveness. In fact, there are numerous studies showing that calcium channel antagonists alone cannot achieve complete relaxation of vascular muscle that has been precontracted with a receptor agonist.
• nisoldipine used alone and in combination with nitroglycerin
• IMA internal mammary artery
• ET-1 endothelin receptor subtype A
• Dihydropyridine antagonists blocked effects of ET-1, an endogenous agonist at the ET A receptor in coronary arterial smooth muscle, and hence speculated that ET-1 is an endogenous agonist of voltage-sensitive calcium channels. It has been found that the sustained phase of intracellular calcium elevation in smooth muscle cells induced by ET A receptor activation requires extracellular calcium and is at least partially blocked by nicardipine. Thus, the inclusion of a calcium channel antagonist would be expected to synergistically enhance the actions of an ET A antagonist when combined in a surgical solution.
• K A ⁇ p Potassium channels that are ATP-sensitive (K A ⁇ p) couple the membrane potential of a cell to the cell's metabolic state via sensitivity to adenosine nucleotides. K ATP channels are inhibited by intracellular ATP but are stimulated by intracellular nucleotide diphosphates. The activity of these channels is controlled by the electrochemical driving force to potassium and intracellular signals (e.g., ATP or a G-protein), but are not gated by the membrane potential per se. K A ⁇ p channels hyperpolarize the membrane and thus allow them to control the resting potential of the cell.
• K A ⁇ p channels hyperpolarize the membrane and thus allow them to control the resting potential of the cell.
• ATP-sensitive potassium cunents have been discovered in skeletal muscle, brain, and vascular and nonvascular smooth muscle. Binding studies with radiolabeled ligands have confirmed the existence of ATP-sensitive potassium channels which are the receptor targets for the potassium-channel opener drugs such as pinacidil. Opening of these channels causes potassium efflux and hyperpolarizes the cell membrane. This hyperpolarization (1) induces a reduction in intracellular free calcium through inhibition of voltage-dependent Ca 2+ channels by reducing the probability of opening L-type or T-type calcium channels, (2) restrains agonist induced (at receptor operated channels) Ca 2+ release from intracellular sources through inhibition of inositol triphosphate (EP 3 ) formation, and (3) lowers the efficiency of calcium as an activator of contractile proteins.
• ATP-sensitive potassium channels which are the receptor targets for the potassium-channel opener drugs such as pinacidil. Opening of these channels causes potassium efflux and hyperpolarizes the cell membrane. This hyperpolarization (1) induces a reduction in intracellular free calcium through inhibition of voltage-dependent Ca
• ATP-sensitive potassium channel openers and calcium channel antagonists will clamp the target cells into a relaxed state or one which is more resistant to activation.
• calcium channel antagonists and tachykinin and bradykinin antagonists exhibit synergistic effects in mediating neuroinflammation.
• the role of neurokinin receptors in mediating neuroinflammation has been established.
• the neurokinini (NKi) and neurokinin 2 (NK 2 ) receptor (members of the G-protein coupled superfamily) signal transduction pathway includes increases in intracellular calcium, thus leading to activation of calcium channels on the plasma membrane.
• activation of bradykinin 2 (BK 2 ) receptors is coupled to increases in intracellular calcium.
• calcium channel antagonists interfere with a common mechanism involving elevation of intracellular calcium, part of which enters through L-type channels. This is the basis for synergistic interaction between calcium channel antagonists and antagonists to neurokinin and bradykinin 2 receptors.
• Suitable calcium channel antagonists for the practice of the present invention include nisoldipine, nifedipine, nimodipine, lacidipine, isradipine and amlodipine. Suitable concentrations for these agents are set forth in Table 20.
• Solutions of the present invention utilized for cardiovascular and general vascular procedures may optionally also include an anti-restenosis agent, particularly for angioplasty, rotational atherectomy and other interventional vascular uses.
• an anti-restenosis agent particularly for angioplasty, rotational atherectomy and other interventional vascular uses.
• the following drugs are suitable for inclusion in the previously described cardiovascular and general vascular irrigation solutions when limitation of restenosis is indicated.
• the following anti-restenosis agents would preferably be combined with anti-spasm, and still more preferably also with anti-pain/anti-inflammation agents in the solutions of the present invention.
• Antiplatelet Agents At sites of arterial injury, platelets adhere to collagen and fibrinogen via specific cell surface receptors, and are then activated by several independent mediators. A variety of agonists are able to activate platelets, including collagen, ADP, thromboxane A2, epinephrine and thrombin. Collagen and thrombin serve as primary activators at sites of vascular injury, while ADP and thromboxane A2 act to recruit additional platelets into a growing platelet plug. The activated platelets degranulate and release other agents which serve as chemoattractants and vasoconstrictors, thus promoting vasospasm and platelet accumulation. Thus, antiplatelet agents can be antagonists drawn from any of the above agonist-receptor targets.
• Thrombin plays a central role in vascular lesion formation and is considered the principal mediator of thrombogenesis.
• thrombus formation at vascular lesion sites during and after PTC A (percutaneous transluminal coronary angioplasty) or other vascular procedure is central to acute reocclusion and chronic restenosis.
• This process can be interrupted by application of direct anti-thrombins, including hirudin and its synthetic peptide analogs, as well as thrombin receptor antagonist peptides (Harker, et al, 1995, Am. J. Cardiol 75, 12B).
• Thrombin is also a potent growth factor which initiates smooth muscle cell proliferation at sites of vascular injury.
• thrombin also plays a role in modulating the effects of other growth factors such as PDGF (platelet-derived growth factor), and it has been shown that thrombin inhibitors reduce expression of PDGF mRNA subsequent to vascular injury induced by balloon angioplasty.
• PDGF platelet-derived growth factor
• Hirudin is the prototypic direct antithrombin drug since it binds to the catalytic site and the substrate recognition site (exosite) of thrombin. Animal studies using baboons have shown that this proliferative response can be reduced 80% using recombinant hirudin (Ciba-Geigy). Hirulog (Biogen) is a dodecapeptide modeled after hirudin, and binds to the active site of thrombin via a Phe-Pro-Arg linker molecule.
• active anti-thrombin agents being tested which are theorized to be suitable for the present invention are argatroban (Texas Biotechnology) and efegatran (Lilly).
• Thrombin Inhibitors and Receptor Antagonists hirudin 0.00003-3/0.0003-0.3 0.03 hirulog 0.2-20,000/2-2,000 200 b.
• ADP Receptor Antagonists Purinoceptor Antagonists Ticlopidine, an analog of ADP, inhibits both thromboxane and ADP-induced platelet aggregation. It is likely that ticlopidine blocks interaction of ADP with its receptor, thereby inhibiting signal transduction by this G-protein coupled receptor on the surface of platelet membranes. A preliminary study showed it to be more effective than aspirin in combination with dipyridamole. However, the clinical use of ticlopidine has been limited because it causes neutropenia.
• Clopidogrel a ticlopidine analog
• Clopidogrel a ticlopidine analog
• Clopidogrel is thought to have fewer adverse side effects than ticlopidine and is cunently being studied for prevention of ischemic events. It is theorized that these agents may be suitable for use in the solutions of the present invention.
• Thromboxane Inhibitors and Receptor Antagonists Agents cunently utilized for conventional methods of treatment of thrombosis rely upon aspirin, heparin and plasminogen activators. Aspirin irreversibly acetylates cyclooxygenase and inhibits the synthesis of thromboxane A2 and prostacyclin.
• Ridogrel (R68060) is a combined thromboxane B2 synthetase inhibitor and thromboxane-prostaglandin endoperoxide receptor blocker. It has been compared with salicylate therapy in an open-pilot study of patients undergoing PTCA administered in combination with heparin. Timmermans, C, et al, Ridogrel in the Setting of Percutaneous Transluminal Coronary Angioplasty, Am. J. Cardiol 68, pp. 463-466, (1991).
• ridogrel was found to be primarily successful since no early acute reocclusion occurred in 30 patients. Bleeding complications did occur in a significant number (34%) of patients, and this appears to be a complicating factor that would require special care. The study confirmed that ridogrel is a potent long-lasting inhibitor of thromboxane B2 synthetase.
• Selectin inhibitors block the interaction of a selectin with its cognate ligand or receptor.
• Representative examples of selectin targets at which these inhibitors would act include, but are not limited to, E-selectin and P-selectin receptors.
• Upjohn Co. has licensed rights to a monoclonal antibody developed by Cytel Corporation that inhibits the activity of P-selectin.
• the product, CY 1748, is in preclinical development, with a potential indication being restenosis.
• the platelet glycoprotein Ilb/IIIa complex is present on the surface of resting as well as activated platelets. It appears to undergo a transformation during platelet activation which enables it to serve as a binding site for fibrinogen and other adhesive proteins. Most promising new antiplatelet agents are directed at this integrin cell surface receptor which represents a final common pathway for platelet aggregation.
• GPIIb/TIIa integrin antagonists Several types of agents fit into the class of GPIIb/TIIa integrin antagonists.
• a monoclonal antibody, c7E3, (CentoRx; Centocor, Malvern PA) has been intensively studied to date in a 3,000 patient PTCA study. It is a chimeric human/murine hybrid.
• a 0.25 mg/kg bolus of c7E3 followed by 10 ⁇ g/min intravenous infusion for 12 hrs produced greater than 80% blockade of GPIl IIa receptors for the duration of the infusion. This was conelated with a greater than 80% inhibition of platelet aggregation.
• the antibody was coadministered with heparin and an increased risk of bleeding was noted.
• the platelet glycoprotein Ilb/IIIa receptor blocker, integrelin is a cyclic heptapeptide that is highly specific for this molecular target. In contrast to the antibody, it has a short biologic half-life (about 10 minutes).
• the safety and efficacy of integrelin was first evaluated in the Phase II Impact trial. Either 4 or 12 hour intravenous infusions of 1.0 ⁇ g/kg/min of integrelin were utilized (Topol, E., 1995 Am. J. Cardiol, 27B-33B). It was provided in combination with other agents (heparin, aspirin) and was shown to exhibit potent anti-platelet aggregation properties (>80%).
• Anti-chemotactic agents prevent the chemotaxis of inflammatory cells.
• Representative examples of anti-chemotactic targets at which these agents would act include, but are not limited to, F-Met-Leu-Phe receptors, IL-8 receptors, MCP-1 receptors, and MtP-l-I/RANTES receptors. Drugs within this class of agents are early in the development stage, but it is theorized that they may be suitable for use in the present invention.
• Interleukin receptor antagonists are agents which block the interaction of an interleukin with its cognate ligand or receptor. Specific receptor antagonists for any of the numerous interleukin receptors are early in the development process. The exception to this is the naturally occurring existence of a secreted form of the IL-1 receptor, referred to as IL-1 antagonist protein (IL-IAP). This antagonist binds TL-1 and has been shown to suppress the biological actions of IL-1, and is theorized to be suitable for the practice of the present invention. 5. Intracellular Signaling Inhibitors a. Protein Kinase Inhibitors i.
• PKC Protein Kinase C
• PKC Protein kinase C
• G-protein coupled receptors e.g., serotonin, endothelin, etc.
• growth-factor receptors such as PDGF. Both of these receptor classes play important roles in mediating vascular spasm and restenosis subsequent to coronary balloon angioplasty procedures.
• PKC exists as a large family consisting of at least 8 subspecies (isozymes). These isozymes differ substantially in structure and mechanism for linking receptor activation to changes in the proliferative response of specific cells. Expression of specific isozymes is found in a wide variety of cell types, including: platelets, neutrophils, myeloid cells, and smooth muscle cells. Inhibitors of PKC are therefore likely to effect signaling pathways in several cell types unless the inhibitor shows isozyme specificity. Thus, inhibitors of PKC can be predicted to be effective in blocking the proliferative response of smooth muscle cells and may also have an anti-inflammatory effect in blocking neutrophil activation and subsequent attachment.
• G-6203 (also known as Go 6976) is a new, potent PKC inhibitor with high selectivity for certain PKC isotypes with IC 50 values in the 2-10 nM range.
• Concentrations of these and another drug, GF 109203X, also known as Go 6850 or bisindoylmaleimide I (available from Warner-Lambert), that are believed to be suitable for use in the present invention are set forth below.
• calphostin C 0.5-50,000/100-5,000 500
• G-6203 (Go 6976) 0.1-10,000/1-1,000 100 ii.
• Protein tyrosine kinase inhibitors Although there is a tremendous diversity among the numerous members of the receptors tyrosine-kinase (RTK) family, the signaling mechanisms used by these receptors share many common features. Biochemical and molecular genetic studies have shown that binding of the ligand to the extracellular domain of the RTK rapidly activates the intrinsic tyrosine kinase catalytic activity of the intracellular domain (see FIGURE 5). The increased activity results in tyrosine-specific phosphorylation of a number of intracellular substrates which contain a common sequence motif.
• PDGF-receptor tyrosine kinase activity IC50S in vitro in the 0.5-1.0 ⁇ M range
• Protein Kinase Inhibitors lavendustin A 10-100,000/100-10,000 10,000 tyrphostin 10-100,000/100-20,000 10,000 AG1296 tyrphostin 10-100,000/100-20,000 10,000 AG1295 staurosporine 1-100,000/10-10,000 1,000 iii.
• MAP Kinase Inhibitors lavendustin A 10-100,000/100-10,000 10,000 tyrphostin 10-100,000/100-20,000 10,000 AG1296 tyrphostin 10-100,000/100-20,000 10,000 AG1295 staurosporine 1-100,000/10-10,000 1,000 iii.
• the mitogen-activated protein (MAP) kinases are a group of protein serine/threonine kinases that are activated in response to a variety of extracellular stimuli and function in transducing signals from the cell surface to the nucleus.
• the MAP kinase cascade is one of the major signaling pathways that transmit signals from growth factors, hormones and inflammatory cytokines to intermediate early genes. In combination with other signaling pathways, these activated mitogen-activated protein-kinases (MAPKs) differentially alter the phosphorylation state and activity of transcription factors, and ultimately regulate cell proliferation, differentiation and cellular response to environmental stress.
• MAPKs mediate the major signal transduction pathways from the potent inflammatory cytokine, IL-1, leading to induction of cyclooxygenase-2 (COX-2) in stimulated macrophages, acting through cis-acting factors involved in the transcriptional regulation of the COX-2 gene.
• COX-2 cyclooxygenase-2
• MAP kinases including the extracellular signal-regulated kinases (ERKs), ERK1 and ERK2 (p44MAPK and p42MAPK, respectively); stress-activated protein kinases (SAPKs/JNKs); and p38 MAP kinase (also known as stress-activated kinase (SAPK)-2, reactivating kinase and cytokine-suppressive binding protein).
• ERKs extracellular signal-regulated kinases
• p44MAPK and p42MAPK stress-activated protein kinases
• SAPKs/JNKs stress-activated protein kinases
• p38 MAP kinase also known as stress-activated kinase (SAPK)-2, reactivating kinase and cytokine-suppressive binding protein.
• G-proteins heterotrimeric GTP-binding proteins
• mitogen-activated protein/ERK kinase induced by G-protein-coupled receptors is mediated by both G ⁇ and G ⁇ K subunits involving a common signaling pathway with receptor-tyrosine-kinases.
• G ⁇ K-mediated mitogen-activated protein kinase activation is mediated by activation of phosphoinositide 3-kinase, followed by a tyrosine phosphorylation event, and proceeds in a sequence of events that involve functional association with the adaptor proteins She, Grb2, and Sos.
• SAPKs Stress-activated protein kinases
• JNKs JNKs
• p38 MAPK are able to be activated by G ⁇ K proteins in a pathway involving Rho family proteins including RhoA and Racl.
• a class of pyridinyl imidazoles inhibit p38 MAP kinase ((Lee, J. et al. (1994)
• SB203580 inhibitory action was demonstrated by its failure to inhibit 12 other protein kinases in vitro (including ERKs) and by its lack of effect on the activation of RK kinase and other MAP kinase cascades in vivo.
• SB 203580 has become useful for identifying the physiological roles and targets of p38 MAP kinase.
• the role of p38 mitogen-activated protein kinase (MAPK) in biochemical inflammatory responses of human fibroblasts and vascular endothelial cells to IL-1 was investigated by use of SB203580, which specifically inhibits the enzyme.
• MAPK mitogen-activated protein kinase
• IL-1 that are selectively controlled by p38 MAPK are the regulation of prostaglandin H synthase-2 (also known as COX-2), metalloproteinases, and IL-6 at different levels.
• SB203580 inhibited (50% inhibitory concentration approximately 0.5 ⁇ M) H-l-induced phosphorylation of hsp 27 (an indicator of p38 MAPK activity) in fibroblasts without affecting the other known IL-1 -activated protein kinase pathways (p42/p44 MAPK, p54 MAPK c-Jun N-terminal kinase).
• SB203580 significantly inhibited IL-1 -stimulated IL-6, (30 to 50% at 1 ⁇ M) but not IL-8 production from human fibroblasts (gingival and dermal) and umbilical vein endothelial cells. IL-1 induction of steady state level of IL-6 mRNA was not significantly inhibited, which is consistent with p38 MAPK regulating IL-6 production at the translational level. Importantly, SB203580 strongly inhibited IL-1 -stimulated prostaglandin production by fibroblasts and human umbilical vein endothelial cells. This was associated with the inhibition of the induction of COX-2 protein and mRNA.
• the MAPK inhibitor is expected to exhibit anti-inflammatory activity against all of these cellular types.
• MAPK inhibitors may also be effective as cartilage protective agents when applied locally to tissues of the joint in a variety of inflammatory or pathophysiological conditions.
• SB203580 was found to inhibit the stimulation of collagenase-1 and stromelysin-1 production by IL-1 without affecting synthesis of tissue inhibitor metalloproteinases (TIMP)-l. Furthermore, SB203580 prevented the increase in collagenase-1 and stromelysin-1 mRNA stimulated by IL-1. In a model of cartilage breakdown, short-term IL-1 -stimulated proteoglycan resorption and inhibition of proteoglycan synthesis were unaffected by SB 203580, while longer term collagen breakdown was prevented.
• p38 MAP kinase is involved in tumor necrosis factor (TNF)-induced cytokine expression and drugs which function as inhibitors of p38 MAP kinase activity block the production of proinflammatory cytol ines, as described below (Beyaert, R. et al, EMBO J. 1996 15:1914-23). TNF treatment of cells activated the p38 MAPK pathway, as revealed by increased phosphorylation of p38 MAPK itself, activation of the substrate protein MAPKAP kinase-2, and phosphorylation of the heat shock protein 27 (hsp27).
• TNF tumor necrosis factor
• SB 203580 possessed therapeutic activity in collagen-induced arthritis in DBA/LACJ mice with a dose of 50 mg/kg resulting in significant inhibition of paw inflammation and serum amyloid protein levels. Antiarthritic activity was also observed in adjuvant-induced arthritis in the Lewis rat when SB203580 was administered p.o. at 30 and 60 mg/kg. Additional evidence was obtained for beneficial effects on bone resorption with an IC50 of 0.6 ⁇ M. In keeping with the inhibitory effects on lipopolysaccharide-induced TNF- ⁇ in mice.
• p38 MAPK plays an important role in the regulation of responses to IL-1, TNF- ⁇ and LPS and it is involved in the regulation of mRNA levels of some inflammatory-responsive genes, such as COX-2.
• Inhibitors of p38 block the production of proinflammatory cytokines and appear effective as anti-inflammatory drugs in animal models of arthritis and bone resorption.
• a large number of inflammatory mediators have been implicated in producing synovitis of the joint, including arachidonic acid metabolites (particularly PGE2), vasoactive amines, and cytokines such as TNF- ⁇ , IL-1, IL-6 and neuropeptides.
• cytokines are found in the synovial fluid of acutely injured knee joints and remain elevated in patients for at least 4 weeks. These cytokines are produced locally in the joint from several activated cell types, including synovial fibroblasts, synovial macrophages, as well as chondrocytes.
• Pain and hyperalgesia commonly associated with inflammatory conditions in the joint are in part due to activation of nociceptive sensory neurons in the joint by PGE2 released as a result of the inflammatory process.
• the ability to block the actions of key proinflammatory cytokines, such as IL-1 and TNF- ⁇ , will have downstream effects on many cell types in the joint (synovial fibroblasts and chondrocytes) thus inhibiting subsequent pathological effects such as infiltration of inflammatory cells into the joint, synovial hyperplasia, synovial cell activation, cartilage breakdown and inhibition of cartilage matrix synthesis.
• a MAPK inhibitor should block the propagation of the inflammatory response by the aforementioned cytokines, and thereby interrupt the disease process.
• MAPK inhibitors are expected to be effective drugs delivered by an irrigation solution during an arthroscopic, urologic, or general surgical procedure (periprocedurally).
• the MAPK inhibitor may be delivered alone, or in combination with other small molecule drugs, peptides, proteins, recombinant chimeric proteins, antibodies, or gene therapy vectors (viral and nonviral) to the spaces of the joint, urogenital tract, or any cavity of the body.
• the MAPK inhibitor can exert its actions on any cells associated with the fluid spaces of the joint and structures comprising the joint and are involved in the normal function of the joint or are present due to a pathological condition.
• These cells and structures include, but are not limited to: synovial cells including both Type A fibroblast and type B macrophage cells; the cartilaginous components of the joint such as chondrocytes; cells associated with bone, including periosteal cells, osteoblasts, osteoclasts; the immunological components such as inflammatory cells including lymphocytes, mast cells, monocytes, eosinophils; and other cells like fibroblasts; and combinations of the above.
• the MAPK inhibitor may be delivered in a formulation useful for introduction and administration of the drug into the targeted tissue or joint that would enhance the delivery, uptake, stability or pharmacokinetics of the inhibitor drug.
• the formulation could include, but is not limited to, administration using microparticles, microspheres or nanoparticles composed of lipids, proteins, carbohydrates, synthetic organic compounds, or inorganic compounds.
• formulation molecules include, but, are not limited to, lipids capable of forming liposomes or other ordered lipid structures, cationic lipids, hydrophilic polymers, polycations (e.g.
• the present invention describes the local delivery of a MAPK drug using an irrigation solution containing the drug which is present at low concentration and which enables the drug to be delivered directly to the affected tissue or joint.
• the drug-containing irrigation solution is employed perioperatively during a surgical procedure.
• Other conventional methods used for drug delivery have required systemic administration (intramuscular, intravenous, subcutaneous) which necessitates high concentrations of drugs (and higher total dose) to be administered in order to achieve significant therapeutic concentrations in the targeted tissue or joint.
• Systemic administration also results in high concentrations in tissues other than the targeted tissue which is undesirable and, depending on the dose, may result in adverse side effects.
• These systemic methods subject the drug to second pass metabolism and rapid degradation, thereby limiting the duration of the effective therapeutic concentration. Since the drug is administered directly to the desired tissue, it does not depend upon vascular perfusion to carry the drug to the targeted tissue. This significant advantage allows for the delivery of the MAPK drug using a therapeutically effective lower concentration and lower therapeutically effective total dose.
• MAPK inhibitors delivered intravascularly is also expected to have applications in the cardiovascular field, including but not limited to, use in the treatment of restenosis.
• Restenosis may be defined as the post-injury neointimal hyperplasia seen in arteries following various angioplasty procedures.
• the MAP kinase cascade is one of the major signaling pathways that transmit signals from growth factors, such as EGF, PDGF, bFGF and others, resulting in cellular proliferation.
• PDGF and bFGF have been identified as important regulators in the process of neointimal formation.
• insulinlike growth factor and transforming growth factor- ⁇ have also been identified as growth factors which act through their respective tyrosine kinase receptors and are implicated in the pathophysiology of restenosis.
• Use of a MAPK inhibitor would be expected to block the proliferative response induced by any one or combination of the above growth factor activated receptors and thereby inhibit initimal hyperplasia.
• MAPK inhibitors are suitable for use in the arthroscopic, urologic, and general surgical applications of the cunent invention, delivered either as a single agent or in combination with other anti-pain and/or anti-inflammatory agents, to inhibit pain and inflammation.
• MAPK inhibitors are also suitable in the cardiovascular surgical solution of the current invention, delivered either as a single agent or in combination with other anti-pain, anti-inflammatory, anti-spasm, and/or anti-restenotic agents to inhibit restenosis.
• a MAPK inhibitor could be included in Example VII.
• MAPK inhibitor compounds suitable for the invention include, for example, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4- pyridyl)- lH-imidazole (SB203580), 4-(3-Iodophenyl)-2-(4-methylsulfinylphenyl)-5-(4- pyridyl)-lH-imidazole (SB203580-iodo), 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5- (4- pyridyl)-lH-imidazole (SB202190), 4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4- pyridyl)-lH-imidazole (PD 169316), and 2'-amino-3'-methoxyflavone (PD98059). Representative useful dosages for these compounds are listed
• Non-transmembrane protein tyrosine phosphatases containing src-homology SH2 domains are known and nomenclature refers to them as SH-PTPl and SH-PTP2.
• SH-PTPl is also known as PTP1C, HCP or SHP.
• SH-PTP2 is also known as PTP1D or PTP2C Similarly, SH-PTPl is expressed at high levels in hematopoietic cells of all lineages and all stages of differentiation, and the SH-PTPl gene has been identified as responsible for the motheaten (me) mouse phenotype and this provides a basis for predicting the effects of inhibitors that would block its interaction with its cellular substrates.
• PTPase activity modulates agonist induced activity by reversing the effects of tyrosine kinases activated in the initial phases of cell stimulation.
• Agents that could stimulate PTPase activity could have potential therapeutic applications as anti-inflammatory mediators.
• PTPases have also been shown to modulate the activity of certain RTKs. They appear to counter-balance the effect of activated receptor kinases and thus may represent important drug targets.
• PTPase blocks insulin stimulated phosphorylation of tyrosyl residues on endogenous proteins.
• activators of PTPase activity could serve to reverse activation of PDGF-receptor action in restenosis, and are believed to be useful in the solutions of the present invention.
• receptor-linked PTPases also function as extracellular ligands, similar to those of cell adhesion molecules.
• SH2 domains originally identified in the src subfamily of protein tyrosine kinases (PTKs), are noncatalytic protein sequences and consist of about 100 amino acids conserved among a variety of signal transducing proteins (Cohen, et al, 1995). SH2 domains function as phosphotyrosine-binding modules and thereby mediate critical protein-protein associations in signal transduction pathways within cells (Pawson, Nature, 573-580, 1995). In particular, the role of SH2 domains has been clearly defined as critical for receptor tyrosine kinase (RTK) mediated signaling such as in the case of the platelet-derived growth factor (PDGF) receptor.
• RTK receptor tyrosine kinase
• Phosphotyrosine-containing sites on autophosphorylated RTKs serve as binding sites for SH2-proteins and thereby mediate the activation of biochemical signaling pathways (see FIGURE 2) (Carpenter, G., FASEB J. 6:3283-3289, 1992; Sierke, S. and Koland, J. Biochem. 32:10102-10108, 1993).
• the SH2 domains are responsible for coupling the activated growth-factor receptors to cellular responses which include alterations in gene expression, and ultimately cellular proliferation (see FIGURE 5).
• inhibitors that will selectively block the effects of activation of specific RTKs expressed on the surface of vascular smooth muscle cells are predicted to be effective in blocking proliferation and the restenosis process after PTCA or other vascular procedure.
• One RTK target of current interest is the PDGF receptor.
• cytosolic proteins that contain SH2 domains and function in intracellular signaling.
• the distribution of SH2 domains is not restricted to a particular protein family, but found in several classes of proteins, protein kinases, lipid kinases, protein phosphatases, phospholipases, Ras-controlling proteins and some transcription factors.
• Many of the SH2-containing proteins have known enzymatic activities while others (Grb2 and Crk) function as "linkers” and "adapters” between cell surface receptors and "downstream” effector molecules (Marengere, L., et al, Nature 369:502-505, 1994).
• proteins containing SH2 domains with enzymatic activities that are activated in signal transduction include, but are not limited to, the src subfamily of protein tyrosine kinases (src (pp60 c"src ), abl, lck, fyn, fgr and others), phospholipaseC ⁇ (PLC ⁇ ), phosphatidylinositol 3-kinase (PI-3 -kinase), p21-ras GTPase activating protein (GAP) and SH2 containing protein tyrosine phosphatases (SH-PTPases) (Songyang, et al, Cell 72, 767-778, 1993).
• inhibitors which block specific SH2 protein binding are desirable as agents for a variety of potential therapeutic applications.
• T-cell activation via the antigen specific T-cell receptor (TCR) initiates a signal transduction cascade leading to lymphokine secretion and T-cell proliferation.
• TCR antigen specific T-cell receptor
• One of the earliest biochemical responses following TCR activation is an increase in tyrosine kinase activity.
• neutrophil activation is in part controlled through responses of the cell surface immunoglobulin G receptors. Activation of these receptors mediates activation of unidentified tyrosine kinases which are known to possess SH2 domains.
• Calcium channel antagonists previously described with relation to spasm inhibitory function, also can be used as anti-restenotic agents in the cardiovascular and general vascular solutions of the present invention.
• Studies at the cellular level have shown that actions of calcium channel antagonists are effective at inhibiting mitogenesis of vascular smooth muscle cells.
• the molecular switches responsible for cell signaling have been traditionally divided into two major discrete signaling pathways, each comprising a distinct set of protein families that act as transducers for a particular set of extracellular stimuli and mediating distinct cell responses.
• One such pathway transduces signals from neurotransmitters and hormones through G-protein coupled receptors (GPCRs) to produce contractile responses using intracellular targets of trimeric G proteins and Ca 2+ (see FIGURE 2).
• GPCRs G-protein coupled receptors
• These stimuli and their respective receptors mediate smooth muscle contraction and may induce vasospasm in the context of PTCA or other cardiovascular or general vascular therapeutic or diagnostic procedure.
• Examples of signaling molecules involved in mediating spasm through the GPCR pathway are 5-HT and endothelin for which antagonists have been included acting via their respective G-protein coupled receptors.
• a second major pathway transduces signals from growth factors, such as PDGF, through tyrosine kinases, adaptor proteins and the Ras protein into regulation of cell proliferation and differentiation (see FIGURES 2 and 5).
• This pathway may also be activated during PTCA or other cardiovascular or general vascular procedure leading to a high incidence of vascular smooth muscle cell proliferation.
• An example of a restenosis drug target is the PDGF-receptor.
• Signals transmitted from neurotransmitters and hormones stimulate either of two classes of receptors: G-protein-coupled receptors, composed of seven-helix transmembrane regions, or ligand-gated ion channels.
• Downstream signals from both kinds of receptors converge on controlling the concentration of cytoplasmic Ca 2+ which triggers contraction in smooth muscle cells (see FIGURE 2).
• Each GPCR transmembrane receptor activates a specific class of trimeric G proteins, including G q , G j or many others.
• Gj and/or G ⁇ ⁇ subunits activate phospholipase C $ , resulting in activation of protein kinase C (PKC) and an increase in the levels of cytoplasmic calcium by release of calcium from intracellular stores.
• PKC protein kinase C
• Growth factor signaling such as mediated by PDGF, converges on regulation of cell growth. This pathway depends upon phosphorylation of tyrosine residues in receptor tyrosine kinases and "downstream" enzymes (phospholipase ⁇ , discussed above with regard to tyrosine kinases). Activation of the PDGF-receptor also leads to stimulation of PKC and elevation of intracellular calcium, common steps shared by the GPCRs (see FIGURE 2). It is now recognized that ligand-independent "crosstalk" can transactivate tyrosine kinase receptor pathways in response to stimulation of GPCRs.
• the 5-HT 2 receptor family contains three members designated 5-HT 2A , 5-HT 2B , and 5-HT 2 c, all of which share the common property of being coupled to phosphotidylinositol turnover and increases in intracellular calcium (Hoyer et al, 1988, Hartig et al, 1989).
• the distribution of these receptors includes vascular smooth muscle and platelets and, due to their localization, these 5-HT receptors are important in mediating spasm, thrombosis and restenosis. It has been found that the sustained phase of intracellular calcium elevation in smooth muscle cells induced by ET A receptor activation requires extracellular calcium and is at least partially blocked by nicardipine.
• the mitogenic effect of PDGF is mediated through receptors that possess intrinsic protein tyrosine kinase activity.
• the substrates for PDGF phosphorylation are many and lead to activation of mitogen-activated protein kinases (MAPK) and ultimately proliferation (see FIGURE 5).
• MEPK mitogen-activated protein kinases
• the endothelin, 5-HT and thrombin receptors which are members of the G-protein coupled superfamily, trigger a signal transduction pathway which includes increases in intracellular calcium, leading to activation of calcium channels on the plasma membrane.
• calcium channel antagonists interfere with a common mechanism employed by these GPCRs.
• Thrombin mediates its action via the thrombin receptor, another member of the GPCR superfamily. Binding to the receptor stimulates platelet aggregation, smooth muscle cell contraction and mitogenesis. Signal transduction occurs through multiple pathways: activation of phospholipse (PLC) through G proteins and activation of tyrosine kinases. The activation of tyrosine kinase activity is also essential for mitogenesis of the vascular smooth muscle cells.
• PLC phospholipse
• tyrosine kinase activity is also essential for mitogenesis of the vascular smooth muscle cells.
• the solution of the present invention has applications for a variety of operative/interventional procedures, including surgical, diagnostic and therapeutic techniques.
• the irrigation solution is perioperatively applied during arthroscopic surgery of anatomic joints, urological procedures, cardiovascular and general vascular diagnostic and therapeutic procedures and for general surgery.
• the term "perioperative” encompasses application intraprocedurally, pre- and intraprocedurally, intra- and postprocedurally, and pre-, intra- and postprocedurally.
• the solution is applied preprocedurally and/or postprocedurally as well as intraprocedurally.
• irrigation Such procedures conventionally utilize physiologic irrigation fluids, such as normal saline or lactated Ringer's, applied to the surgical site by techniques well known to those of ordinary skill in the art.
• the method of the present invention involves substituting the anti-pain/anti-inflammatory/anti-spasm/anti-restenosis irrigation solutions of the present invention for conventionally applied irrigation fluids.
• the irrigation solution is applied to the wound or surgical site prior to the initiation of the procedure, preferably before tissue trauma, and continuously throughout the duration of the procedure, to preemptively block pain and inflammation, spasm and restenosis.
• irrigation is intended to mean the flushing of a wound or anatomic structure with a stream of liquid.
• the term "application” is intended to encompass irrigation and other methods of locally introducing the solution of the present invention, such as introducing a gellable version of the solution to the operative site, with the gelled solution then remaining at the site throughout the procedure.
• the term “continuously” is intended to also include situations in which there is repeated and frequent irrigation of wounds at a frequency sufficient to maintain a predetermined therapeutic local concentration of the applied agents, and applications in which there may be intermittent cessation of irrigation fluid flow necessitated by operating technique.
• the concentrations listed for each of the agents within the solutions of the present invention are the concentrations of the agents delivered locally, in the absence of metabolic transformation, to the operative site in order to achieve a predetermined level of effect at the operative site.
• the drug concentrations in a given solution may need to be adjusted to account for local dilution upon delivery.
• a local delivery catheter i.e., a blood flow-to-solution delivery ratio of 16 to 1
• Solution concentrations are not adjusted to account for metabolic transformations or dilution by total body distribution because these circumstances are avoided by local delivery, as opposed to oral, intravenous, subcutaneous or intramuscular application.
• Arthroscopic techniques for which the present solution may be employed include, by way of non-limiting example, partial meniscectomies and ligament reconstructions in the knee, shoulder acromioplasties, rotator cuff debridements, elbow synovectomies, and wrist and ankle arthroscopies.
• the irrigation solution is continuously supplied intraoperatively to the joint at a flow rate sufficient to distend the joint capsule, to remove operative debris, and to enable unobstructed intra- articular visualization.
• a suitable irrigation solution for control of pain and edema during such arthroscopic techniques is provided in Example I herein below.
• the solution include a combination, and preferably all, or any of the following: a serotoni ⁇ receptor antagonist, a serotonin 3 receptor antagonist, a histaminej receptor antagonist, a serotonin receptor agonist acting on the IA, IB, ID, IF and/or IE receptors, a bradykinin j receptor antagonist, a bradykinin 2 receptor antagonist, and a cyclooxygenase inhibitor.
• This solution utilizes extremely low doses of these pain and inflammation inhibitors, due to the local application of the agents directly to the operative site during the procedure.
• amitriptyline a suitable serotonin 2 and histamine ⁇ "dual" receptor antagonist
• irrigation fluid to provide the desired effective local tissue concentrations that would inhibit 5-HT 2 and H ⁇ receptors.
• This dosage is extremely low relative to the 10-25 mg of oral amitriptyline that is the usual starting dose for this drug.
• This same rationale applies to the anti-spasm and anti-restenosis agents which are utilized in the solution of the present invention to reduce spasm associated with urologic, cardiovascular and general vascular procedures and to inhibit restenosis associated with cardiovascular and general vascular procedures.
• nisoldipine a suitable calcium channel antagonist
• irrigation fluid For example, less than 0.2 mg of nisoldipine (a suitable calcium channel antagonist) is required per liter of irrigation fluid to provide the desired effective local tissue concentrations that would inhibit the voltage-dependent gating of the L-subtype of calcium channels.
• This dose is extremely low compared to the single oral dose of nisoldipine which is 20 to 40 mg.
• the agents are included in low concentrations and are delivered locally in low doses relative to concentrations and doses required with conventional methods of drug administration to achieve the desired therapeutic effect. It is impossible to obtain an equivalent therapeutic effect by delivering similarly dosed agents via other (i.e., intravenous, subcutaneous, intramuscular or oral) routes of drug administration since drugs given systemically are subject to first- and second-pass metabolism.
• other routes of drug administration since drugs given systemically are subject to first- and second-pass metabolism.
• the inventors examined the ability of amitriptyline, a 5-HT2 antagonist, to inhibit 5-HT-induced plasma extravasation in the rat knee in accordance with the present invention.
• Example XII compared the therapeutic dosing of amitriptyline delivered locally (i.e., intra-articularly) at the knee and intravenously.
• the difference in plasma drug levels between the two routes of administration is much greater than the difference in total amitriptyline dosing levels.
• Practice of the present invention should be distinguished from conventional intra-articular injections of opiates and/or local anesthetics at the completion of arthroscopic or "open" joint (e.g., knee, shoulder, etc.) procedures.
• the solution of the present invention is used for continuous infusion throughout the surgical procedure to provide preemptive inhibition of pain and inflammation.
• the high concentrations necessary to achieve therapeutic efficacy with a constant infusion of local anesthetics, such as lidocaine (0.5-2% solutions) would result in profound systemic toxicity.
• the solution of the present invention also has application in cardiovascular and general vascular diagnostic and therapeutic procedures to potentially decrease vessel wall spasm, platelet aggregation, vascular smooth muscle cell proliferation and nociceptor activation produced by vessel manipulation.
• Reference herein to arterial treatment is intended to encompass the treatment of venous grafts harvested and placed in the arterial system.
• a suitable solution for such techniques is disclosed in Example II herein below.
• the cardiovascular and general vascular solution preferably includes any combination, and preferably all, of the following: a 5-HT 2 receptor antagonist (Saxena, P. R., et. al, Cardiovascular Effects of Serotonin Inhibitory Agonists and Antagonists, J Cardiovasc Pharmacol 15 (Suppl 7), pp.
• the cardiovascular and general vascular solution also preferably will contain a serotonin 1B (also known as serotonin jrjo) antagonist because serotonin has been shown to produce significant vascular spasm via activation of the serotonin 1B receptors in humans.
• serotonin 1B also known as serotonin jrjo
• the cardiovascular and general vascular solution of the present invention also may suitably include one or more of the anti-restenosis agents disclosed herein that reduce the incidence and severity of post-procedural restenosis resulting from, for example, angioplasty or rotational atherectomy.
• the solution of the present invention also has utility for reducing pain and inflammation associated with urologic procedures, such as trans-urethral prostate resection and similar urologic procedures.
• References herein to application of solution to the urinary tract or to the urological structures is intended to include application to the urinary tract per se, bladder and prostate and associated structures. Studies have demonstrated that serotonin, histamine and bradykinin produce inflammation in lower urinary tract tissues. Schwartz, M.M., et.
• the solution preferably includes a combination, and preferably all, of the following: a histamine ⁇ receptor antagonist to inhibit histamine-induced pain and inflammation; a 5-HT 3 receptor antagonist to block activation of these receptors on peripheral C-fiber nociceptive neurons; a bradykinin antagonist; a bradykinin 2 antagonist; and a cyclooxygenase inhibitor to decrease pain/inflammation produced by prostaglandins at the tissue injury sites.
• an anti-spasm agent is also included to prevent spasm in the urethral canal and bladder wall.
• Some of the solutions of the present invention may suitably also include a gelling agent to produce a dilute gel.
• This gellable solution may be applied, for example, within the urinary tract or an arterial vessel to deliver a continuous, dilute local predetermined concentration of agents.
• the solution of the present invention may also be employed perioperatively for the inhibition of pain and inflammation in surgical wounds, as well as to reduce pain and inflammation associated with burns.
• Burns result in the release of a significant quantity of biogenic amines, which not only produce pain and inflammation, but also result in profound plasma extravasation (fluid loss), often a life-threatening component of severe burns.
• Holliman, C.J., et. al The Effect of Ketanserin, a Specific Serotonin Antagonist, on Burn Shock Hemodynamic Parameters in a Porcine Burn Model, J Trauma 23, pp. 867-871 (1983).
• Example I for arthroscopy may also be suitably applied to a wound or burn for pain and inflammation control, and for surgical procedures such as arthroscopy.
• the agents of the solution of Example I may alternately be included in a paste or salve base, for application to the burn or wound.
• composition is suitable for use in anatomic joint irrigation during arthroscopic procedures.
• Each drug is solubilized in a carrier fluid containing physiologic electrolytes, such as normal saline or lactated Ringer's solution, as are the remaining solutions described in subsequent examples.
• physiologic electrolytes such as normal saline or lactated Ringer's solution
• Prefened Preferred serotonin 2 antagonist amitriptyline 0.1-1,000 50-500 100 serotonin 3 antagonist metoclopramide 10-10,000 200-2,000 1,000 histamine ! antagonist amitriptyline 0.1-1,000 50-500 200 serotonin 1A; 1B 1D; ⁇ sumatriptan 1-1,000 10-200 50 agonist bradykinin antagonist [des-Arg 10 ] 1-1,000 50-500 200 derivative of HOE 140 bradykinin 2 antagonist HOE 140 1-1,000 50-500 200 B.
• the following drugs and concentration ranges in solution in a physiologic carrier fluid are suitable for use in irrigating operative sites during cardiovascular and general vascular procedures.
• Class of Agent Drug Nanomolar: Most Therapeutic Preferred Preferred serotonin 2 antagonist trazodone 0.1-2,000 50-500 200
• bradykinin ! antagonist [des-Arg 10 ] 1-1,000 50-500 200 derivative of
• ketorolac 100-10,000 500-5,000 3,000
• the following drugs and concentration ranges in solution in a physiologic carrier fluid are suitable for use in irrigating operative sites during urologic procedures.
• histamine ! antagonist terfenadine 0.1-1,000 50-500 200
• bradykinin! antagonist [des-Arg 10 ] 1-1,000 50-500 200 derivative of HOE 140
• cyclooxygenase inhibitor 100-10,000 500-5,000 3,000
• composition is preferred for use in anatomic irrigation during arthroscopic and oral/dental procedures and the management of burns and general surgical wounds. While the solution set forth in Example I is suitable for use with the present invention, the following solution is even more preferred because of expected higher efficacy.
• Class of Agent Drug (Nanomolar): Most Therapeutic Prefened Preferred serotonin 2 antagonist amitriptyline 0.1 - 1,000 50 - 500 200 serotonin 3 antagonist metoclopramide 10 - 10,000 200 - 2,000 1,000 histamine ! antagonist amitriptyline 0.1 - 1,000 50 - 500 200 serotonin 1A ⁇ B> m sumatriptan 1 - 1,000 10 - 200 100 1F agonist cyclooxygenase ketorolac 100 - 10,000 500 - 5,000 3,000 inhibitor neurokinin ! antagonist GR 82334 1 - 1,000 10 - 500 200 neurokinin 2 antagonist ( ⁇ ) SR 48968 1 - 1,000 10 - 500 200 purine 2X antagonist PPADS 100 - 100,000 10,000- 50,000 100,000
• the following drugs and concentration ranges in solution in a physiologic carrier fluid are prefened for use in irrigating operative sites during urologic procedures.
• the solution is believed to have even higher efficacy than the solution set forth in prior Example III.
• Cardiovascular and General Vascular Anti-Restenosis Irrigation Solution The following drugs and concentration ranges in solution in a physiologic carrier fluid are preferred for use in irrigation during cardiovascular and general vascular therapeutic and diagnostic procedures.
• the drugs in this preferred solution may also be added at the same concentration to the cardiovascular and general vascular irrigation solutions of Examples II and V described above or Example VIII described below for prefened anti-spasmodic, anti-restenosis, anti-pain/anti-inflammation solutions.
• Therapeutic Preferred Prefened thrombin inhibitor hirulog 0.2-20,000 2-2,000 200 glycoprotein Ilb/TIIa integrelin 0.1-10,000 x Kd 1-1000 x Kd 100 x Kd receptor blocker
• PKC inhibitor GF 109203X* 0.1-10,000 1-1,000 200 protein tyrosine tyrphostin 10-100,000 100-20,000 10,000 kinase inhibitor AG1296
• nitric oxide (NO donor) SIN-1 is replaced by a combination of two agents, FK 409 (NOR-3) and FR 144420 (NOR-4), at the concentrations set forth below:
• NO donor FR 144420 (NOR-4) 10-10,000 100-5,000 1,000
• HOE 140 a bradyl inin 2 antagonist
• bradykinin [leu 9 ] [des-Arg 10 ] 0.1-500 10-200 100 antagonist kalliden
• SIN-1 is replaced as the NO donor by a combination of two agents: a) FK 409 (NOR-3); and b) FR 144420 (NOR-4);
• HOE 140 a bradykinin 2 antagonist
• the test solution included: (a) the serotonin 3 antagonist metoclopramide at a concentration of 16.0 ⁇ M; (b) the serotonin 2 antagonist trazodone at a concentration of 1.6 ⁇ M; and (c) the histamine antagonist promethazine at concentrations of 1.0 ⁇ M, all in normal saline.
• Drug concentrations within the test solution were 16- fold greater than the drug concentrations delivered at the operative site due to a 16 to 1 flow rate ratio between the iliac artery (80 cc per minute) and the solution delivery catheter (5 cc per minute). This study was performed in a prospective, randomized and blinded manner. Assignment to the specific groups was random and investigators were blinded to infusion solution contents (saline alone or saline containing the histamine/serotonin receptor antagonists) until the completion of the angiographic analysis.
• Baseline blood pressure and heart rate were recorded and then an angiogram of the distal aorta and bilateral iliac arteries was recorded on 35 mm cine film (frame rate 15 per second) using hand injection of iopamidol 76% (Squibb Diagnostics, Princeton, NJ) into the descending aorta.
• a calibration object was placed in the radiographic field of view to allow for correction for magnification when diameter measurements were made.
• a 2.5 French infusion catheter (Advanced Cardiovascular Systems, Santa Clara, CA) was placed through the carotid sheath and positioned 1-2 cm above the aortic bifurcation.
• Infusion of the test solution either saline alone or saline containing the histamine/serotonin receptor antagonists ⁇ was started at a rate of 5 cc per minute and continued for 15 minutes.
• a second angiogram was performed using the previously described technique then a 2.5 mm balloon angioplasty catheter (the Lightning, Cordis Corp., Miami, FL) was rapidly advanced under fluoroscopic guidance into the left and then the right iliac arteries.
• the balloon catheter was carefully positioned between the proximal and distal deep femoral branches using bony landmarks and the balloon was inflated for 30 seconds to 12 ATM of pressure.
• the balloon catheter was inflated using a dilute solution of the radiographic contrast agent so that the inflated balloon diameter could be recorded on cine film.
• the angioplasty catheter was rapidly removed and another angiogram was recorded on cine film at a mean of 8 minutes after the infusion was begun. The infusion was continued until the 15 minute time point and another angiogram (the fourth) was performed. Then the infusion was stopped (a total of 75 cc of solution had been infused) and the infusion catheter was removed. At the 30 minute time point (15 minutes after the infusion was stopped), a final angiogram was recorded as before. Blood pressure and heart rate were recorded at the 15 and 30 minute time points immediately before the angiograms. After the final angiogram, the animal was euthanized with an overdose of the anesthetic agents administered intravenously and the iliac arteries were retrieved and immersion fixed in formation for histologic analysis. 3. Angiographic Analysis
• the angiograms were recorded on 35 mm cine film at a frame rate of 15 per second. For analysis, the angiograms were projected from a Vanguard projector at a distance of 5.5 feet. Iliac artery diameters at prespecified locations relative to the balloon angioplasty site were recorded based on hand held caliper measurement after conection for magnification by measurement of the calibration object. Measurements were made at baseline (before test solution infusion was begun), 5 minutes into the infusion, immediately post balloon angioplasty (a mean of 8 minutes after the test solution was begun), at 15 minutes Q st before the infusion was stopped) and at 30 minutes (15 minutes after the infusion was stopped). Diameter measurements were made at three sites in each iliac artery: proximal to the site of balloon dilatation, at the site of balloon dilatation and just distal to the site of balloon dilatation.
• % Vasoconstriction ⁇ (Baseline area - Later time point area)/Baseline area ⁇ xlOO. 4.
• the time course of arterial dimension changes before and after balloon angioplasty in normal arteries receiving saline infusion was evaluated in 16 arteries from 8 animals (Table 23). Three segments of each artery were studied: the proximal segment immediately upstream from the balloon dilated segment, the balloon dilated segment and the distal segment immediately downstream from the balloon dilated segment.
• the arterial diameters in each segment at the 5 minute, 15 minute and 30 minute time points were similar to the baseline diameters.
• the balloon dilated segment showed lesser changes in arterial dimension than the proximal and distal segments.
• the baseline diameter of this segment was 1.82 ⁇ 0.05 mm; the nominal inflated diameter of the balloon used for angioplasty was 2.5 mm and the actual measured inflated diameter of the balloon was 2.20 ⁇ 0.03 mm (pO.OOOl vs. baseline diameter of the balloon treated segment).
• Post hoc testing identifies the vasoconstriction at the immediate post angioplasty time point as significantly different from that present at the 30 minute time point (P ⁇ 0.001 in both segments).
• the immediate post angioplasty vasoconstriction was also significantly less than that at 5 minutes (p ⁇ 0.01); no other differences in intra-time point comparisons were significant by post hoc testing.
• the luminal changes in control arteries can be summarized as follows: 1) Vasoconstriction with loss of approximately 30% of baseline luminal area occurs in the segments of artery proximal and distal to the balloon dilated segment immediately after balloon dilatation. There are trends to smaller amounts of vasoconstriction in the proximal and distal segments before dilatation and at the 15 minute time point (approximately 7 minutes after dilatation) also but, by the 30 minute time point (approximately 22 minutes after dilatation), a trend towards vasodilatation has replaced the previous vasoconstriction; 2) In the balloon dilated segment, only minor changes in lumen dimensions are present, and, despite the use of a balloon with a significantly larger inflated diameter than was present in this segment at baseline, there was no significant increase in lumen diameter of the dilated segment. These findings lead to a conclusion that any effects of the putative histamine/serotonin treatment would only be detectable in the proximal and distal segments
• the histamine/serotonin receptor blockade solution was infused into 16 arteries (8 animals); angiographic data was available at all time points in 12 arteries.
• **p 0.05 for decrease in heart rate from baseline to 30 minutes within the histamine/serotonin treated animals.
• FIGURE 10A shows the effects of the histamine/serotonin infusion on proximal segment vasoconstriction relative to the vasoconstriction present in the control arteries.
• Rats (Bantin and Kingman, Fremont, CA) weighing 300 - 450 g were used in these studies. Rats were housed under controlled lighting conditions (lights on 6 A.M. to 6 P.M.), with food and water available ad libitum.
• Rats were anesthetized with sodium pentobarbital (65 mg/kg) and then given a tail vein injection of Evans Blue dye (50 mg/kg in a volume of 2.5 ml/kg), which is used as a marker for plasma protein extravasation.
• the knee joint capsule was exposed by excising the overlying skin, and a 30-gauge needle was inserted into the joint and used for the infusion of fluid.
• the infusion rate 250 ⁇ l/min
• a 25-gauge needle was also inserted into the joint space and perfusate fluid was extracted at 250 ⁇ l/min, controlled by a Sage Instruments Syringe pump (Model 351).
• the rats were randomly assigned to three groups: 1) those receiving only intra-articular (I A) 5-HT (1 ⁇ M), 2) those receiving amitriptyline intravenously (IV) (doses ranging from 0.01 to 1.0 mg/kg) followed by IA 5-HT (1 mM), and 3) those receiving amitriptyline intra-articularly (IA) (concentrations ranging from 1 to 100 nM) followed by I A 5-HT (1 ⁇ M) plus IA amitriptyline.
• I A intra-articular
• IV amitriptyline intravenously
• IA amitriptyline intra-articularly
• baseline plasma extravasation levels were obtained at the beginning of each experiment by perfusing 0.9% saline intra-articularly and collecting three perfusate samples over a 15 min period (one every 5 min).
• the first group was then administered 5-HT IA for a total of 25 min. Perfusate samples were collected every 5 min for a total of 25 min. Samples were then analyzed for Evans Blue dye concentration by spectrophotometric measurement of absorbance at 620 nm, which is linearly related to its concentration (Carr and Wilhelm, 1964). The IV amitriptyline group was administered the drug during the tail vein injection of the Evans Blue dye. The knee joints were then perfused for 15 min with saline (baseline), followed by 25 min perfusion with 5-HT (1 ⁇ M). Perfusate samples were collected every 5 min for a total of 25 min. Samples were then analyzed using spectrophotometry.
• amitriptyline was perfused intra-articularly for 10 min after the 15 min saline perfusion, then amitriptyline was perfused in combination with 5-HT for an additional 25 min. Perfusate samples were collected every 5 min and analyzed as above.
• 5-HT (1 ⁇ M) perfused into the rat knee joint produces a time-dependent increase in plasma extravasation above baseline levels.
• maximum levels of plasma extravasation were achieved by 15 min and continued until the perfusion was terminated at 25 min (data not shown). Therefore, 5-HT-induced plasma extravasation levels reported are the average of the 15, 20 and 25 min time points during each experiment.
• 5-HT-induced plasma extravasation averaged 0.192 + 0.011, approximately an 8-fold stimulation above baseline. This data is graphed in FIGURES 11 and 12, corresponding to the "0" dose of IV amitriptyline and the "0" concentration of IA amitriptyline, respectively.
• 5-HT-induced plasma extravasation in the presence of 3 nM IA amitriptyline was not significantly different from that produced by 5-HT alone, however, 30 nM amitriptyline co-perfused with 5-HT produced a greater than 50% inhibition, while 100 nM amitriptyline produced complete inhibition of 5-HT-induced plasma extravasation.
• the IC 50 for IA amitriptyline inhibition of 5-HT-induced plasma extravasation is approximately 20 nM.
• 5-HT (1 uM) perfused intra- articularly in the rat knee joint produces a stimulation of plasma extravasation that is approximately 8-fold above baseline levels and that either intravenous or intra- articular administration of the 5-HT 2 receptor antagonist, amitriptyline, can inhibit 5-HT-induced plasma extravation.
• the total dosage of administered amitriptyline differs dramatically between the two methods of drug delivery.
• the IC50 for IV amitriptyline inhibition of 5-HT-induced plasma extravasation is 0.025 mg/kg, or 7.5 x 10 "3 mg in a 300 g adult rat.
• the IC 50 for IA amitriptyline inhibition of 5-HT- induced plasma extravasation is approximately 20 nM. Since 1 ml of this solution was delivered every five minutes for a total of 35 min during the experiment, the total dosage perfused into the knee was 7 ml, for a total dosage of 4.4 x 10 "5 mg perfused into the knee. This IA amitriptyline dose is approximately 200-fold less than the IV amitriptyline dose. Furthermore, it is likely that only a small fraction of the IA perfused drug is systemically absorbed, resulting in an even greater difference in the total delivered dose of drug.
• 5-HT may play an important role in surgical pain and inflammation
• 5-HT antagonists such as amitriptyline may be beneficial if used during the perioperative period.
• a recent study attempted to determine the effects of oral amitriptyline on post-operative orthopedic pain (Kerrick et al, 1993).
• An oral dose as low as 50 mg produced undesirable central nervous system side-effects, such as a "decreased feeling of well-being".
• Their study in addition, also showed that oral amitriptyline produced higher pain scale scores than placebo (P ⁇ 0.05) in the postoperative patients. Whether this was due to the overall unpleasantness produced by oral amitriptyline is not known.
• an intra-articular route of administration allows an extremely low concentration of drug to be delivered locally to the site of inflammation, possibly resulting in maximal benefit with minimal side-effects.
• Example V Example V. above, with the following exceptions. Nitroprusside replaced SIN-1 as the nitric oxide donor and nicardipine replaced nisoldipine as the Ca 2+ channel antagonist.
• the concentration of nitroprusside was selected based on its previously- defined pharmacological activity (EC50).
• the concentrations of the other agents in this test solution were determined based on the binding constants of the agents with their cognate receptors. Furthermore, all concentrations were adjusted based on a blood flow rate of 80 cc per minute in the distal aorta of the rabbit and a flow rate of 5 cc per minute in the solution delivery catheter. Three components were mixed in one cc or less DMSO, and then these components and the remaining three components were mixed to their final concentrations in normal saline. A control solution consisting of normal saline was utilized. The test solution or the control solution was infused at a rate of 5 cc per minute for 20 minutes. A brief pause in the infusion was necessary at the times blood pressure measurements were made, so each animal received about 95 cc of the solution in the 20 minute treatment period.
• An angiogram of the distal aorta and bilateral iliac arteries was recorded on 35 mm cine film (frame rate 15 per second) using hand injection of iopamidol 76% (Squibb Diagnostics, Princeton, NJ) into the descending aorta.
• a calibration object was placed in the radiographic field of view to allow for conection for magnification when diameter measurements were made.
• Infusion of either the above described test solution or a saline control solution was started through the side arm of the 5 French sheath (and delivered to the distal aorta) at a rate of 5 cc per minute and continued for 20 minutes.
• a second angiogram was performed using the previously described technique. Then a 1.25 mm or a 1.50 mm rotational atherectomy burr (Heart Technology/Boston Scientific Inc.) was advanced to the iliac arteries.
• the rotational atherectomy burr was advanced three times over a guide wire in each of the iliac arteries at a rotation rate of 150,000 to 200,000 RPM. In each iliac, the rotational atherectomy burr was advanced from the distal aorta to the mid portion of the iliac artery between the first and second deep femoral branches. The rotational atherectomy burr was rapidly removed and another angiogram was recorded on cine film at a mean of 8 minutes after the infusion was begun.
• the infusion was continued until the 20 minute time point, and another angiogram (the fourth) was performed. Then the infusion was stopped. A total of about 95 cc of the control or test solution had been infused. At the 30 minute time point (15 minutes after the infusion was stopped), a final angiogram was recorded as before. Blood pressure and heart rate were recorded at the 15 and 30 minute time points immediately before the angiograms. After the final angiogram, the animal was euthanized with an overdose of the anesthetic agents administered intravenously.
• the angiograms were recorded on 35 mm cine film at a frame rate of 15 per second. Angiograms were reviewed in random order without knowledge of treatment assignment. For analysis, the angiograms were projected from a Vanguard projector at a distance of 5.5 feet. The entire angiogram for each animal was reviewed to identify the anatomy of the iliac arteries and to identify the sites of greatest spasm in the iliac arteries. A map of the iliac anatomy was prepared to assist in consistently identifying sites for measurement. Measurements were made on the 15 minute post rotational atherectomy angiogram first, then in random order on the remaining angiograms from that animal. Measurements were made using an electronic handheld caliper (Brown & Sharpe, Inc., N. Springfield, RI).
• Iliac artery diameters were measured at three locations: proximal to the first deep femoral branch of the iliac artery; at the site of most severe spasm (this occurred between the first and second deep femoral artery branches in all cases); and at a distal site (near or distal to the origin of the second deep femoral artery branch of the iliac artery). Measurements were made at baseline (before test solution infusion was begun), 5 minutes into the infusion, immediately post rotational atherectomy (a mean of 8 minutes after the test solution was begun), at 20 minutes just after the infusion was stopped (this was 15 minutes after the rotational atherectomy was begun) and at 15 minutes after the infusion was stopped (30 minutes after the rotational atherectomy was begun). The calibration object was measured in each angiogram. The diameter measurements were then converted to area measurements by the formula:
• % Vasoconstriction ⁇ (Baseline area - Later time point area) Baseline area ⁇ xlOO.
• Iliac artery diameters in saline treated arteries at the three specified segments are summarized in Table 25.
• mid-iliac artery at the site of maximal vasoconstriction there was a significant reduction in diameter with the largest reduction occurring at the 15 minute post-rotational atherectomy time point (p ⁇ 0.0001, ANOVA comparing measurements at all 5 time points).
• Iliac artery lumen diameters at specified time points in saline treated arteries are Iliac artery lumen diameters at specified time points in saline treated arteries.
• RA rotational atherectomy l
• the diameters of iliac arteries treated with the test solution are shown in Table 26. Angiograms were not recorded in three of these arteries at the 5 minute post-initiation of the infusion time point and angiographic data were excluded from two arteries (one animal) at the 30 minute post-rotational atherectomy time point because the animal received an air embolus at the 15 minute angiogram that resulted in hemodynamic instability. Because there is a variable number of observations at the five time points, no ANOVA statistic was applied to this data. Still it is apparent that the magnitude of change in the diameter measurements within segments in the test solution treated arteries over the time course of the experiment is less than was seen in the saline treated arteries. Table 26
• Iliac artery lumen diameters at specified time points in Test Solution treated arteries are measured.
• RA rotational atherectomy ! Proximal iliac artery measurement site, proximal to the first deep femoral branch
• the primary endpoint for this study was the comparison of the amounts of vasoconstriction in saline treated and test solution treated arteries.
• Vasoconstriction was based on arterial lumen areas derived from artery diameter measurements. Area values at the 5 minute, immediate post-rotational atherectomy and later time points were compared to the baseline area values to calculate the relative change in area. The results were termed "vasoconstriction” if the lumen area was smaller at the later time point than at baseline, and “vasodilatation” if the lumen area was larger at the later time point compared to the baseline area (Tables 27 and 28). To facilitate statistical analysis with the largest number of observations possible in both treatment groups, the test solution and saline treated artery data were compared at the immediate post- and at the 15 minute postrotational atherectomy time points.
• vasoconstriction negative values or vasodilatation Coositive values
• Amount of vasoconstriction (negative values or vasodilatation (positive values at specified time points in Test Solution treated arteries.
• test solution treated animals sustained substantial hypotension and significant tachycardia during the solution infusion.
• test solution treated animals showed some partial, but not complete, return of blood pressure towards baseline.
• Rotational atherectomy in hypercholesterolemic New Zealand white rabbits results in prominent vasospasm in the mid-portion of iliac arteries subjected to the rotating bun.
• the vasospasm is most apparent 15 minutes after rotational atherectomy treatment and has almost completely resolved without pharmacologic intervention by 30 minutes after rotational atherectomy.
• test solution treatment in accordance with the present invention almost completely abolishes the vasospasm seen after the mid-iliac artery is subjected to the rotating bun.

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