Solutions and methods for inhibiting pain, cartilage inflammation and destruction

FIELD: medicine.

SUBSTANCE: method involves introducing solutions into articulation to inhibit cartilage destruction. The solutions contain: (a) therapeutically effective amount of anabolic chondroprotective agent selected from a group composed of interleukine antagonists stimulating anabolic processes in cartilage, members of superfamily transforming growth β-factor including TGF-β agonists and agonists of morphogenous bone proteins stimulating anabolic processes in cartilage, insulin-like fibroblast growth factors stimulating anabolic processes in cartilage; (b) therapeutically effective amount of a cartilage catabolism inhibitor selected from a group composed of antagonists of interleukine-1-receptors, antagonists of TGF-α-receptors, specific cyclo-oxygenase-2 inhibitors, nitrogen oxide synthase inhibitors, nuclear kB factor inhibitors, matrix metalloproteinase inhibitors, cell adhesion molecules including integrin agonists and integrin antagonists, anti-chemotaxis agents, intracellular signal transmission inhibitors including protein kinase C inhibitors and tyrosine protein kinase inhibitors, intracellular (protein-tyrosine)-phosphatases and SH2-domain inhibitors inhibiting cartilage catabolism. The solution is locally supplied.

EFFECT: stimulated integration and modulation of anti-inflammatory synoviocyte and chondrocyte responses.

54 cl, 9 dwg, 30 tbl

 

I. SCOPE of the INVENTION

The present invention relates to treatment fluids and to methods of treatment and, in particular, the solutions against inflammations, against pains and against the destruction of the cartilage and to methods of their treatment.

II. BACKGROUND of the INVENTION

Arthroscopy is a surgical procedure in which the camera is connected to a remote light source and a video monitor, is introduced into the anatomical joint (e.g. knee, shoulder etc) through a small portal incision in the skin and joint capsule. Through this portal incisions in the joint, you can enter surgical instruments, their use is regulated by arthroscopic visualization. As the technology arthroscopy increasing number of surgical procedures previously carried out "open" surgery, it is now possible to conduct arthroscopy. Such procedures include, for example, partial meniscectomy and restoration of the ligaments in the knee, acromioplasty shoulder and cuff rehabilitation of rotation of the shoulder and synovectomy elbow. The enlargement of the surgical indications and development of arthroscopes with a small diameter has also become a common arthroscopy of the wrist and ankle.

Every arthroscopy physiological irrigating fluid (i.e. normal saline or lachinova the hydrated ringer's solution) is continuously passed through the joint, expanding the joint capsule and removing surgical residues, thus providing a clearer visualization of the inside of the joint. In U.S. patent 4504493 Marshall revealed smolyary solution of glycerin in water as non-conductive and optically pure irrigation solution for arthroscopy. Normal physiological irrigating fluid do not provide analgesic, anti-inflammatory effects and effect against the destruction of cartilage.

Relief of pain and suffering in patients in the postoperative period is an area of special attention in clinical medicine, especially with the growing number of outpatient surgical procedures performed annually. The most widely used system tools, cyclo-oxygenase inhibitors (eg, ibuprofen) and opioids (e.g. morphine, fentanyl) have significant side effects, including irritation of the stomach and intestines/bleeding and respiratory depression. The high frequency of symptoms of nausea and vomiting associated with opioids, is a special problem in the postoperative period. A therapeutic agent aimed at relief of pain in the postoperative period, with a simultaneous lack of harmful side effects, it is difficult to develop due to the fact that the molecular targets for these agents are widely distributed in the body and mediate various physiological functions. Despite significant clinical need in suppressing pain and inflammation and destruction of cartilage, not developed methods of delivery of inhibitors of pain, inflammation and destruction of cartilage in effective dosages, when to minimize this adverse systemic effects. As an example, normal (i.e. intravenous, oral, subcutaneous or intramuscular) routes of administration of opiates in therapeutic doses are often associated with significant side effects, including severe respiratory depression, changes in mood, confusion, severe nausea and vomiting.

In previous studies it was shown that the ability of endogenous agents, such as serotonin (5-hydroxytryptamine, sometimes referred to in the description as "5-HT"), bradykinin and histamine that cause pain and inflammation. Sicuteri F. et al., Serotonin-Bradykinin Potentiation in the Pain Receptors in Man, Life Sci. 4, pp.309-316 (1965); S.R. Rosenthal, Histamine as the Chemical Mediator for Cutaneous Pain, J. Invest. Dermat. 69, pp.98-105 (1977); Richardson V.R. et. al., Identification of Serotonin M-Receptor Subtypes and their Specific Blockade by a New Class of Drugs, Nature 316, pp.126-131 (1985); Whalley E.T. et. al., The Effect of Kinin Agonists and Antagonists, Naunyn-Schmiedeb Arch. Pharmacol. 36, pp.652-57 (1987); E. Lang et. al., Chemo-Sensitivity of Fine Afferents from Rat Skin In Vitro, J. Neurophysiol. 63, pp.887-901 (1990).

For example, it was shown that 5-HT caused to the man on the skin, covered with blisters (bare skin), causes pain, which can be suppressed by antagonists of 5-HT3receptors:Richardson et. al., (1985). Similarly applied in peripheral tissues bradykinin causes pain, which can be blocked by antagonists bradykininase receptors: Sicuteri et. al., 1965; Whalley et. al., 1987; A. Dray et. al., Bradykinin and Inflammatory Pain, Trends Neurosci. 16, pp.99-104 (1993). Used in peripheral tissues histamine causes vasodilatation, itching and pain, which can be suppressed by antagonists of histamine receptors: Rosenthal, 1977; Douglas W.W., "Histamine and 5-Hydroxytryptamine (Serotonin) and their Antagonists", in L.S. Goodman et. al., ed. The Pharmacological Basis of Therapeutics, MacMillan Publishing Company, New York, pp.605-638 (1985); Rumore M.M. et. al., Analgesic Effects of Antihistaminics, Life Sci 36, pp.403-416 (1985). It was shown that the combination of these three agonists (5-HT, bradykinin and histamine), used together, have a synergistic causing pain effect, causing a long and intense pain signal. Sicuteri et. al., 1965; Richardson et. al., 1985; Kessler W. et. al., "Excitation of Cutaneous Afferent Nerve Endings In Vitro by a Combination of Inflammatory Mediators and Conditioning Effect of Substance P", Exp. Brain Res. 91: 467-476 (1992).

In the body of 5-HT is found in platelets and Central neurons, histamine was detected in the fat cells, and bradykinin is produced from a larger precursor molecules during tissue injury, changes in pH and temperature changes. Because 5-HT may be released in large quantities of platelets in areas of tissue injury, leading to concentrations in plasma in excess of 20 times that is new in the state of rest (Ashton, J.H. et. al., "Serotonin as a Mediator of Cyclic Flow Variations in Stenosed Canine Coronary Arteries", Circulation 73: 572-578 (1986), it is possible that endogenous 5-HT plays a role in the occurrence of postoperative pain, hyperalgesia and inflammation. In fact, it was shown that activation of platelets leads to the excitation of peripheral nocireceptors in vitro. Ringkamp M. et. al., 'Activated Human Platelets in Plasma Excite Nociceptors in Rat Skin, In Vitro, Neurosci. Lett. 170: 103-106 (1994). Similarly, histamine and bradykinin also released in tissue injury. Kimura E. et. al., "Changes in Bradykinin Level in Coronary Sinus Blood After the Experimental Occlusion of a Coronary Artery", Am. Heart J. 85: 635-647 (1973); Douglas, 1985; Dray et. al. (1993).

In addition, it is known that prostaglandins also cause pain and inflammation. Usually cyclo-oxygenase inhibitors, such as ibuprofen, are used when non-surgical and postoperative interventions in order blocking the production of prostaglandins, thereby reducing mediated by prostaglandins pain and inflammation: Flower R.J. et al., Analgesic-Antipyretics and Anti-Inflammatory Agents; Drugs Employed in the Treatment of Gout, in Goodman L.S. et. al., The Pharmacological Basis of Therapeutics, MacMillan Publishing Company, New York, pp. 674-715 (1985). Cyclo-oxygenase inhibitors are associated with some adverse systemic effects when applied in the usual way. For example, indomethacin or Ketorolac has recognised side effects gastro-intestinal tract and kidneys.

As absurd the elk, 5-HT, histamine, bradykinin and prostaglandins cause pain and inflammation. Known and/or discussed in the last two decades, a variety of receptors through which these agents mediate their effects in peripheral tissues. Most research was done on rats or other animal models. However, there are differences in pharmacology and sequences of receptors in humans and animals.

In addition, antagonists of these mediators is not currently used for pain relief in the postoperative period. A group of drugs called antagonists absorption of 5-HT and norepinephrine, which includes amitriptyline, used orally with an average success in chronic pain. However, I believe that the mechanisms of chronic and acute pain differ considerably. In fact, in two studies of the suppression of acute perioperative pain with the use of amitriptyline was shown by the absence of debilitating pain of action in amitriptyline: J.D. Levine et. al., "Desipramine Enhances Postoperative Opiate Analgesia", Pain 27: 45-49 (1986); Kerrick J.M. et. al., "Low-Dose Amitriptyline as an Adjunct to Opioids for Postoperative Orthopedic Pain: a Placebo-Controlled Trial Period, Pain 52: 325-30 (1993). In both studies the drug was administered orally. In the second study it was noted that oral introduced amitriptyline actually causes the lower the percent the total sensitivity in patients in the postoperative period, that may be due to the affinity of the drug for many amine receptors in the brain.

Amitriptyline in addition to blocking the absorption of 5-HT and norepinephrine, is a strong antagonist of 5-HT receptors. Therefore, the lack of efficacy in reducing pain in the postoperative period in the two previously mentioned studies contradict the assumption of the role of endogenous 5-HT in the development of acute pain. There are a number of reasons in respect of the lack of attenuation of acute pain under the action of amitriptyline in these two studies. (1) In the first study (Levine et. al., 1986) amitriptyline was used prior to surgery in one week till the night prior to surgery at the time, as in the second study (Kerrick et. al., 1993) amitriptyline was used only in the postoperative period. Therefore, do not know the level of amitriptyline available in the tissues at the surgical site during the phase of actual injury to tissues, and the time when 5-HT starts to be released. (2) it is Known that amitriptyline is actively metabolized in the liver. When administered orally in a second study, the concentration of amitriptyline in the tissues at the surgical site may not be high enough over a long enough period of time for inhibiting the activity of released 5-HT in the postoperative period. (3) is since there are numerous mediators of inflammation, and studies have shown synergism mediators of inflammation, blocking only one substance (5-HT) can inhibit the inflammatory response to tissue injury is not sufficient.

There have been several studies that have shown the ability of antagonists of histamine receptors in very high concentrations (1%-3% solutions, i.e. 10-30 mg per milliliter) to act as local anesthetics during surgical procedures. I believe that this anesthetic action is mediated not through Hi-receptors, and to a greater extent due to nonspecific interaction with sodium channels of membranes of neurons (similar to the action of lidocaine). Having side effects (e.g. sedation)related to data high "anesthetic" concentrations of antagonists of histamine receptors, local application of antagonists of histamine receptors is not currently practiced in the perioperative period.

III. BRIEF description of the INVENTION

The present invention provides a solution comprising a mixture of many agents in low concentrations, intended for local inhibition of mediators of pain, inflammation and destruction of cartilage in physiological containing electrolytes the carrier fluid. The invention also provides a method perioperative remove the key irrigating solution containing these agents directly into the surgical site, where it acts locally at the level of receptors and enzymes for proactive suppression of pain, inflammation and destruction of cartilage in the surgical site. Due to the way local perioperative delivery according to the present invention, the desired therapeutic effect can be achieved at lower doses of agents than necessary when using other delivery methods (i.e. intravenous, intramuscular, subcutaneous and oral). Agents against pain, and/or against inflammation, and/or against the destruction of cartilage in the solution include agents selected from the following groups of antagonists and agonists of the receptors and activators and inhibitors of enzymes, where each group works through a different molecular mechanism of suppression of pain and/or inflammation and/or destruction of the cartilage. Typical agents for suppressing pain and/or inflammation include, for example: (1) antagonists of serotonin receptors; (2) agonists of serotonin receptors; (3) antagonists of histamine receptors; (4) antagonists bradykininase receptors; (5) inhibitors of kallikrein; (6) antagonists tachykinin receptors, including antagonists of receptor subtypes neirokinina1and neirokinina2; (7) receptor antagonists of the peptide linked to the genome of calcitonin (CGRP); (8) antagonists of interle is of kinowych receptors; (9) inhibitors of enzymes active in the synthesis of metabolites of arachidonic acid, including (a) inhibitors of phospholipases, including inhibitors of the isoform PLA2and inhibitors of PLC isoforms; (b) inhibitors of cyclooxygenase and (C) inhibitors of lipoxygenase; (10) antagonists prostanoid receptors, including antagonists of receptor subtype eicosanoid EP-1 and EP-4 and subtype antagonists of thromboxane receptor; (11) leukotriene receptor antagonists, including antagonists subtype receptor leukotriene D4 antagonists subtype receptor leukotriene D4; (12) opioid receptor agonists, including agonists of receptor subtypes μ-opioid δ-opioid and κ-opioid; (13) antagonists purinoceptors, including receptor antagonists P2Xand receptor antagonists P2Y; (14) calcium channel antagonists. Each of these agents acts as either agent against inflammation, or against sensitivity, i.e. against pain or analgesic agent. The choice of agents of these groups of compounds are related to a particular application. Typical agents for suppressing the destruction of cartilage include, for example: (1) receptor antagonists family protein interleukin-1, including, for example, IL-1β, IL-17 and IL-18; (2) antagonists family of receptors factors tumor necrosis (TNF)such as TNF-R1; (3) agonists R the receptors of interleukin 4, 10 and 13; (4) agonists of receptor superfamily TGF-βincluding, for example, BMP-2, BMP-4 and BMP-7; (5) inhibitors SOH-2; (6) inhibitors of the family of MAR-kinases, including, for example, R Markansu; (7) inhibitors of the protein family of matrix metalloproteinases (MMP), including, for example, MMP-3 and MMP-9; (8) inhibitors of protein family NF-κincluding, for example, dimeric the complex of P50/P65 with Iκ; (9) inhibitors collection nitric oxide synthase (NOS), including, for example, iNOS; (10) agonists and antagonists integranova receptors, including, for example, agonists αVβ3-integrin; (11) inhibitors of the family of protein kinase C (SW); (12) inhibitors family of tyrosine kinases, including, for example, the src subfamily; (13) modulators (protein-tyrosine-phosphatase; and (14) inhibitors domain homology 2 protein scr (SH2).

In other aspects the invention provides methods and solutions for suppressing or preventing the destruction of articular cartilage by injection directly into the joint of the patient a composition that includes one or more metabolically active for protective cartilage agents together with one or more agents to suppress pain, inflammation or the like, as previously described, or alternatively a combination of two or more metabolically active for protective cartilage agents in a pharmaceutically effective carrier for intra-articular delivery. IU is abolishes active agents include, but are not limited to compounds that act directly or indirectly to modulate or modify the biological, biochemical or biophysical state of the cells, including agents that alter the electrical potential of the plasma membrane, the activity of ligand binding or enzymatic activity of cell receptors, intracellular or extracellular enzymes, interactions protein-protein interactions, RNA-protein, or interaction of DNA-protein. In one aspect of the present invention are provided pharmaceutical compositions metabolically active for protective cartilage agents, which are based on a combination of at least two agents that act simultaneously on different molecular targets. In a typical embodiment, at least one agent is an agonist of receptors of cytokines or growth factors, which directly provide anti-inflammatory activity and/or promote anabolic processes in the cartilage, and at least a second agent is a receptor antagonist or inhibitor of the enzyme, which acts to suppress inflammatory and/or catabolic processes in the cartilage. Anti-inflammatory/anabolic cytokines, which act functionally to suppress the role of proinflammatory cytokines in the joint, FPIC is stuut the synthesis of cartilage matrix and inhibit the resolution matrix. Data receptor agonists include, for example, a specific anti-inflammatory and anabolic cytokines, such as agonists of interleukins (IL) (e.g., IL-4, IL-10 and IL-13), and specific members of the subfamily of transforming growth factor-β (for example, TGFβ and BMP-7), insulin-like growth factors (e.g., IGF-1) and fibroblast growth factors (e.g., bFGF). At least a second agent selected from the group receptor antagonists or inhibitors of enzymes, which acts to inhibit or reduce the activity or expression of Pro-inflammatory molecular targets (e.g., antagonists of IL-1 receptor antagonists of TNF-αreceptors, inhibitors of cyclooxygenase-2 inhibitors Martinez, inhibitors of nitric oxide synthase (NOS) and inhibitors of nuclear factor kappaB (NF-κ). Multiple combination agents anabolic agents and inhibitors of catabolism can be added locally by intra-articular injection or infusion, including perioperative purpose (i.e. preoperatively and/or intraoperatively and/or postoperatively) during arthroscopic surgical procedures.

Articular cartilage is a specialized extracellular matrix that is produced and stored metabolically active articular chondrocytes. Maintaining a normal, healthy extracellular matrix from Ajeet dynamic balance between the rate of biosynthesis and incorporation of matrix components and the rate of their destruction and subsequent loss of cartilage in the synovial fluid. Although the regulatory mechanisms that underlie homeostasis in the matrix, not quite well understood, they clearly change in inflammatory diseases of the joints and in response to injury of the joints so that the decay rate of the matrix exceeds the speed of the new synthesis of matrix components. Homeostasis in the matrix is usually considered as a dynamic balance between catabolic cytokines and anabolic cytokines (including growth factors). The optimum combination of therapeutic agents suitable for the protection of cartilage, shifts the dynamic equilibrium in the matrix, accelerating the rate of synthesis and simultaneously inhibiting the decay rate, thus making maximum anabolic processes and contributing to the restoration of cartilage.

Catabolic cytokines, such as IL-1β and TNF-αact on specific receptors chondrocytes inducyruya production of MMPs, which induce the collapse of the matrix, while the destruction inhibited anabolic cytokines, such as TGF-β, BMP-2 and IGF-1. Therefore, a therapeutic approach based only on the inhibition of catabolic processes (such as the composition of the MMP inhibitor and antagonist IL-1), is not optimal for recovery of cartilage as anabolic agents for induction or acceleration of biosynthesi Assembly of components for the production of the matrix. Second, many of catabolic cytokines (IL-1, TNF, IL-17, IL-18, LIF), which are involved in the degradation of cartilage matrix, indicates that it would be impractical to completely block the catabolic activity of cytokines. Conversely, an approach that relies only on the use of anabolic agents such as IGF-1, BMP-2 or BMP-7, is not optimal because it is not aimed at protivozachatocnuu role of catabolic cytokines. TGF-β, BMP-2 and IGF-1 also acts on specific receptors to the induction of chondrocytes to produce the components of the matrix, which is inhibited under the influence of IL-1β, TNF-α, IL-17 and LIF. Therefore, the optimal therapeutic combination for the protection of cartilage consists of at least one anabolic agent and one inhibitor of catabolism of cartilage.

The present invention also provides a method for obtaining a medicinal product prepared in the form of a diluted irrigating solution for use with constant irrigation space operations, usually in place of a joint of a patient during arthroscopic surgical procedures. The method comprises dissolving in physiological containing electrolytes the carrier fluid is at least one agent against the destruction of cartilage and preferably one or more agents inhibiting pain/inflammation and for some ol the change agents from and against destruction of cartilage where each agent is included at a concentration of preferably not more than 100,000 nmol, more preferably not more than about 25000 nmol, and most preferably not more than about 10,000 nmol.

The method according to the present invention delivers a diluted combination of many of the antagonists and agonists of the receptors and inhibitors and activators of enzymes directly to the wound or surgical site during a therapeutic or diagnostic procedures to suppress pain, inflammation and destruction of cartilage. Because the active ingredients in the solution are applied topically directly to tissue at the surgical site within a constant period of time, the drugs can be used effectively in very low doses compared to the doses required for the manifestation of therapeutic effect, when the same drugs delivered orally, intramuscularly, subcutaneously or intravenously. In the sense in which he used the term "local" includes the use of drugs in or around the wound or other designated operations, and excludes oral, subcutaneous, intravenous and intramuscular introduction. The term "permanent"in the sense as it is used, includes continuous use, repeated use, with frequent intervals, and application, which are continuous except for short OS is anawak, allows you to enter other drugs or agents, or equipment for the procedure, so that basically constant, predetermined concentration is maintained locally in the wound or surgical site.

The advantages of using agents in low doses are threefold. The most important is the lack of systemic side effects that often limit the usefulness of the data agents. In addition, the agents selected for special applications in the solutions of the present invention are highly specific mediators and mediating targets on which they operate. This specificity is maintained used low doses. Finally, the value of these active agents for surgical procedures is low.

The advantages of local agents are applied by spraying or other application of the liquid are as follows: (1) local application ensures the presence of a known concentration at the target site, regardless of the variability between patients in metabolism, blood flow and the like; (2) the direct method of delivering therapeutic concentrations get immediately and, thus, provides improved control of dispensing; and (3) local application of active agents directly to the wound or surgical site of the beings of the NGO reduces degradation of the agents in the system processes, for example, primary and secondary metabolism that would otherwise take place when the agents are administered orally, intravenously, subcutaneously or intramuscularly. This is especially true for those active agents, which by their nature are peptides that are rapidly metabolized. Thus, local application allows the use of compounds or agents that otherwise cannot be used for therapeutic purposes. For example, some agents in the following groups are peptides; antagonists bradykininase receptors; antagonists tachykinin receptors; opioid agonists; antagonists of CGRP receptors; and antagonists interlacing receptors; antagonists of TNF-receptors; agonists of TGF-βreceptors; agonists BMP-2 and BMP-7 receptors; agonists of the receptors IL-4, IL-10 and IL-13; agonists and antagonists integranova receptors. Local, constant delivery to the wound or surgical site minimizes the degradation or the cells metabolism and regrowth drugs, at the same time providing a permanent replacement of the portions of the agent, which can degrade to ensure that local, therapeutic concentration sufficient to maintain employment receptor or saturation of the enzyme is maintained during the entire period of the surgical procedure.

Local perioperative p is the physical alteration of the solution during surgical procedures of the present invention provides preemptive analgesic, anti-inflammatory and protective against cartilage effect. In the sense in which he used the term "perioperative" includes the application during the procedure, before and during the procedure, during and after the procedure, and before, during and after the procedure. For maximum manifestation of proactive anti-inflammatory, analgesic (for some applications) and for protective cartilage (for some applications) action solutions of the present invention is most preferably applied before, during and after surgery. Occupying the target receptors or inactivating or activating the target enzymes before significant local injury during surgery, the agents of this solution modulate specific pathways for proactive suppression target of the pathological process. If mediators or inflammatory processes proactively suppressed according to the present invention before they can cause tissue damage, the benefit is more significant than if it is after the damage has already begun.

It was shown that the inhibition of more than one mediator of pain, inflammation or destruction of the cartilage, the application of the solution of the many agents of the present invention, clearly reduced the degree of inflammation and pain, and theoretically will be provided a protective effect against the attachment of the cartilage. Irrigating solutions of the present invention include combinations of drugs, where each solution is effective on numerous receptors or enzymes. Thus, the medicinal agents are also effective against a combination of pathological processes, including pain and inflammation, and loss of homeostasis in the cartilage. It is believed that these agents is synergistic, with many receptor antagonists and agonists inhibiting of the present invention to provide in combination disproportionately increased efficiency compared to the efficiency of individual agents. The synergistic action of several agents of the present invention is discussed using the example below in the detailed description of these agents.

Used perioperative, the solution leads to clinically significant suppression of pain and inflammation at the surgical site and the destruction of cartilage in comparison with currently used irrigating fluids, thereby reducing the need for patients in anaesthetics in the post-operative period (i.e. the opiate), and accordingly, allows earlier mobilization of the place of operation in a patient. On the part of the surgeon and operating personnel does not require additional effort for the application of this solution compared to conventional irrigation W is dastani. For optimal protection of the cartilage of the solutions according to the invention are applied directly to the joint before, during and/or after the surgical procedure.

IV. BRIEF DESCRIPTION of DRAWINGS

The present invention will be described in more detail by using examples with reference to the accompanying drawings, where:

Figure 1 represents a schematic view of chondrocyte showing the molecular targets and the flow of signal information leading to production of inflammatory mediators and changes in the metabolism of cartilage. It is shown that the integration of external signals through several families of cell surface receptors, including cytokine receptors, such as the family of receptors, interleukin-1 (IL-1) family of receptors of the tumor necrosis factor (TNF)superfamily of TGF-βreceptors and integrins, converge on a common intracellular transmission of signals that include large groups of protein molecules, which are therapeutic targets for drugs included in the solutions according to the present invention (Martinez, CSWs, tyrosine kinase, SH2-proteins, SOH, PLA2 and NF-6B). Activation of these pathways signaling regulates the expression in the chondrocytes of the number of induced genes, including IL-1, TNF-α, IL-6, IL-8 and stromelysin (MMP-3) and other mediators (nitric oxide (NO) and PGE2), which can lead to inflammation and/or destruction of the cartilage is, or the synthesis of matrix molecules and proliferation of chondrocytes.

Figure 2 represents a schematic view of synoviocyte showing the molecular targets and the flow of signal information leading to production of inflammatory mediators and changes in the metabolism of cartilage. It is shown that the integration of external signals through several families of cell surface receptors, including cytokine receptors, which include the family of receptors, interleukin-1 (IL-1) family of receptors of the tumor necrosis factor (TNF)associated with G-protein receptors, which include bradykinesia, histamine and serotonin subtypes, and integrins, converge on a common intracellular transmission of signals that include large groups of protein molecules, which are therapeutic targets for drugs included in the solutions according to the present invention (MAR-kinase, CSWs, tyrosine kinase, SH2-proteins, SOH, PLA2 and NF-6B). Activation of these pathways signaling regulates the expression synoviocyte number of induced genes, including IL-1, TNF-α, IL-6, IL-8 and stromelysin (MMP-3), which can lead to inflammation and/or destruction of the cartilage.

Figure 3 represents the graph of total transmission signals in chondrocytes and synoviocytes, including key signaling proteins responsible for cross-influence between Aktivera the data GPCR-receptor pathways and proinflammatory cytokine pathways, which lead to inflammation and destruction of cartilage.

Figure 4 is a graph of total transmission signals in chondrocytes and synoviocytes, including key signaling proteins responsible for cross-influence between the activated GPCR-receptor pathways and proinflammatory cytokine pathways. Indicated specific molecular sites of action of some drugs in the preferred protective cartilage of the solution according to the present invention.

Figure 5 is a chart of molecular targets that are either chondrocytes or synoviocytes that promote an anabolic response in cartilage. Indicated specific sites of action of some drugs in the preferred protective cartilage of the solution according to the present invention.

Figure 6 is a chart of molecular targets that are either chondrocytes or synoviocytes that contribute to the catabolic response in cartilage. Indicated specific sites of action of some drugs in the preferred solution to protect the cartilage of the present invention.

Figure 7 is a graphical depiction of the production of prostaglandin E2 in cultured synoviocytes regulatory agonists of G-protein with subsequent bonus payments during the night interleukin-1 (IL-, 10 u/ml). Cultures were stimulated for the indicated periods of time with histamine (100 μm, empty columns) or bradykinin (1 μm, shaded columns) and determined released into the culture supernatant prostaglandin E2, as described in example 6. The values presented represent the average value of the±standard deviation in the typical experience and adjusted for background production of prostaglandin E2 in non-stimulated cultures.

Figure 8 is a graphical representation of the inhibition of production of prostaglandin E2 in synovial cultures under the action of Ketoprofen. Culture has primiraly during the night of IL-1 (10 u/ml) in the presence (shown as) or absence (shown as "Δ" or "▿") Ketoprofen in the indicated concentrations. After one day was determined by prostaglandin E2 in supernatant cultures treated overnight with Ketoprofen, and the remaining culture was washed, incubated for 10 min with Ketoprofen in the indicated concentrations, and then determined the production of prostaglandin E2 in response to subsequent stimulation with histamine for 3 min (100 µm, ▿) or bradykinin (1 μm; Δ) in the continued presence of Ketoprofen in the specified quantities. Data are presented normalized to the maximum response obtained for each of the CSOs agonist, respectively, and represent the average value of the ± the standard deviation obtained in the three experiments performed on different cell lines.

Figure 9 is a graphical representation of the action of Ketoprofen on the production of IL-6 in synovial cultures for 16 h in the presence of IL-1 in the indicated concentrations plus the added ligands of receptors associated with G-protein. Cultures were incubated for 16 h with IL-1 in the indicated concentrations of 0.3, and 1.0 and 3.0 PG/ml) in the absence and presence 0/75 μm Ketoprofen in the experimental culture medium with one of the following additional receptor ligands: 1) by isoproterenol (ISO) at a concentration of 1.0 μm to activate common pathways or 2) histamine (HIS) at a concentration of 100 μm for activation R/calcium pathway. Collected cultural supernatant and was replaced by aliquot of fresh medium containing the additive of the same agonists with 8-hour intervals. After processing the collected supernatant environment corresponding to the processing intervals from 8 to 16 h, and analyzed for levels of IL-6.

V. DETAILED DESCRIPTION of the PREFERRED OPTION

For carrying out the INVENTION

Irrigation and injection solutions according to the present invention are solutions of one or more agents to protect cartilage agents and optionally one or more analgesics and/or anti-inflammatory agents in f is zoologicheskom media. A medium is a liquid solution, which is designed to enable biocompatible solvents, suspensions, polymer and depolymerizes gels, pastes and ointments, components, delivery systems with constant release, such as microparticles, microspheres or nanoparticles consisting of proteins, liposomes, hydrocarbons, synthetic organic compounds or inorganic compounds. Preferably the carrier is an aqueous solution, which may include physiological electrolytes, such as normal saline or aktirovannye ringer's solution.

Agents against inflammation and/or pain selected from the group consisting of (1) antagonists, serotonin receptors; (2) agonists of serotonin receptors; (3) antagonists of histamine receptors; (4) antagonists bradykininase receptors; (5) inhibitors of kallikrein; (6) antagonists tachykinin receptors, including antagonists of receptor subtypes neirokinina1and neirokinina2; (7) receptor antagonist peptide linked to the genome of calcitonin (CGRP); (8) antagonists interlacing receptors; (9) inhibitors of enzymes active in the synthesis of metabolites of arachidonic acid, including (a) inhibitors of phospholipase, including inhibitors of the isoform PLA2and inhibitors of PLC isoforms; (b) inhibitors of cyclo is Ximenez and (C) inhibitors of lipoxygenase; (10) antagonists prostanoid receptors, including antagonists of receptor subtype eicosanoid EP-1 and EP-4 and subtype antagonists of thromboxane receptor; (11) leukotriene receptor antagonists, including antagonists subtype receptor leukotriene B4 and subtype receptor leukotriene D4; (12) opioid receptor agonists, including agonists receptor subtype μ-opioid δ-opioid and κ-opioid; (13) agonists and antagonists purinoceptors, including receptor antagonists P2Xand receptor antagonists P2Y; (14) calcium channel antagonists.

Suitable protective for cartilage agents include, for example, (1) receptor antagonists family protein interleukin-1, including, for example, IL-1β, IL-17 and IL-18; (2) antagonists family of receptors factors tumor necrosis (TNF), including, for example, TNF-R1; (3) agonists of the receptors, interleukin 4, 10 and 13; (4) agonists of receptor superfamily TGF-P, including, for example, BMP-2, BMP-4 and BMP-7; (5) COX-2 inhibitors; (6) inhibitors of the family of MAR-kinase, including, for example, R Markansu; (7) inhibitors of the protein family of matrix metalloproteinases (MMP), including, for example, MMP-3 and MMP-9; (8) inhibitors of protein family NF-KB, including, for example, dimeric complex of P50/P65 with 1kV; (9) inhibitors collection nitric oxide synthase (NOS), including, for example, iNOS; (10) agonists and antagonists integranova re is atarov, including, for example, agonists αVβ3-integrin; (11) inhibitors of the family of protein kinase C (SW); (12) inhibitors family of tyrosine kinases, including, for example, the src subfamily; (13) modulators of protein-tyrosinosis; and (14) inhibitors homology domains-2 proteins scr (SH2).

Specific preferred embodiment of the solution according to the present invention for use to protect the cartilage during arthroscopic procedures preferably include a combination of agents that act simultaneously on different molecular targets to promote cartilage anabolism and inhibiting unregulated or excessive catabolic processes in the cartilage to achieve maximum inhibition of inflammatory processes and the preservation of homeostasis in the cartilage, thereby achieving protective for cartilage action in the joint.

In each of the surgical solutions according to the present invention, the agents included in low concentrations in a liquid solution or liquid medium, and delivered locally in low doses compared to the concentrations and doses required with conventional routes of administration of drugs to achieve the desired therapeutic effect. In the same sense as it is used here, the term "liquid" or "fluid" is intended to include pharmaceutically acceptable, bi is compatible solvents, suspensions, polymerized and depolymerizing gels, pastes, and ointments. Preferably the carrier is an aqueous solution, which may include physiological electrolytes such as normal saline or aktirovannye ringer's solution. It seems impossible or impractical to obtain the same therapeutic effect upon delivery of agents to the same doses other (i.e. intravenous, subcutaneous, intramuscular or oral) routes of administration of drugs, because drugs introduced systematically subjected to primary and secondary metabolism. The concentration of each agent in part determines, based on its dissociation constant of the receptor, Kdor the inhibition constant of the enzyme, Ki. In the same sense as it is used here, the term dissociation constant is intended to include the equilibrium constant of dissociation in relation to its typical interaction of the agonist-receptor or antagonist-receptor and the equilibrium constant of inhibition with respect to its typical interaction of the activator is an enzyme or inhibitor-enzyme. Each agent is preferably included in low concentrations from 0.1 to 10,000 times from the Kdor Kiwith the exception of cyclo-oxygenase inhibitors, which may require large concentrations depending on the specific inhibitor. Preferably, each agent is included at a concentration of from 1.0 to 1000 times from the Kdor Kiand most preferably 100 times from the Kdor Ki. Data concentration was adjusted, if necessary, taking into account the dilution in the absence of metabolic transformation in place for local delivery. Specific agents selected for use in the solution, and the concentration of agents vary depending on the particular application, as described below.

The solution according to the present invention may include one or more agents to suppress pain and/or inflammation, multiple protective for cartilage agent(s), at least one of which is anabolic for protective cartilage agent, and at least one of which is an inhibitor of catabolism of cartilage, or a combination of protective cartilage agent (s) and agents for suppressing pain and/or inflammation in a low concentration. However, due to the above-mentioned synergistic actions of many agents and desire to completely block the pain, inflammation and resolution of cartilage, it is preferable that was used by many agents.

Surgical solutions represent a new therapeutic approach when combining many pharmacological agents acting on different receptor and/or enzyme molecular targets. To date, farmacologicas the e strategy focused on the development of highly specific drugs, which are selective in relation to specific receptor subtypes and isoforms of the enzymes that mediate responses to specific signaling neurotransmitters and hormones. In addition, despite inaktivirovanie one subtype of receptor or enzyme, the activation of the other subtypes of the receptor or enzyme, and the received signal transduction often can "launch" a cascading effect. This explains the significant difficulty with using one drug that is specific to a single receptor to block the pathophysiological process in which play the role of many signaling mediators (e.g. cytokines, growth factors or eicosanoids). Therefore, purposeful impact only on specific individual subtype receptor or the isotype is probably ineffective.

In contrast to the usual approach to pharmacological therapy is therapeutic approach to these surgical solutions based on a rational basis that the combination of drugs acting simultaneously on different molecular targets, is highly effective against inhibiting the full spectrum of events that underlie the development of the pathophysiological condition. In addition, instead of targeting only one specific subtype of prescriptions the Directors, surgical solutions consist of drugs, which are aimed at the General molecular mechanisms in different cell physiological processes involved in the development of pain, inflammation or destruction of the cartilage (see figure 1). In the same way a cascade of additional receptors and enzymes in nociceptors, inflammation and destruction of cartilage is reduced to a minimum surgical solutions. In these pathophysiological pathways surgical solutions inhibit the cascade effect "positive regulation and negative regulation".

An example of inhibition of the type of "positive regulation" are antagonists of cyclooxygenase in suppressing pain and inflammation. Cyclooxygenase (COX2and MOR2) catalyse the conversion of arachidonic acid to prostaglandin H, which is an intermediate product in the biosynthesis of inflammatory and nociceptive mediators, including prostaglandins, leukotrienes and thromboxanes. Cyclo-oxygenase inhibitors block "positive regulation of formation of these inflammatory mediators and nociceptors. This strategy requires blocking interactions described seven subtypes prostanoid receptors with prostanoid products on the biochemical pathway involving MOR. Such inhibitor "pological the Noah regulation", included in surgical solutions, is Aprotinin, an inhibitor of kallikrein. The enzyme kallickrein, semipretioase, cleaves high-molecular kininogen plasma with the formation of bradykinin, important mediators of pain and inflammation. Inhibiting kallickrein, Aprotinin inhibits the synthesis of bradykinin, thereby providing effective inhibition "positive regulation of inflammatory mediators.

Surgical solutions are also used inhibitors "negative regulation" for the regulation of pathophysiological pathways. In the preparations synoviocytes and chondrocytes, which were treated with different cytokines of inflammation (e.g., IL-1β and TNF-α)participating in progressive destruction of articular cartilage, inhibitors of Martinez represent a protective effect on the cartilage. R of Marginata represents the point of transmission in the transmission of signals for many of catabolic cytokines, and its inhibition prevents "positive regulation of many cellular products, mediating the destruction of cartilage. Inhibitors of Martinez, therefore, provide a significant advantage for surgical solutions in suppressing the inflammation of the joints providing a "negative regulation" protective effects on cartilage, which are independent from the physical combination of agony the tov cytokine receptors, inducyruya shift homeostasis in cartilage.

The subsequent description is appropriate drugs that fall into the above group of agents against inflammation/anti-pain and protect cartilage, and suitable concentrations for use in the solutions of the present invention. Not wanting to limit himself to theory, also provides the rationale for the selection of different groups of agents, which, as expected, makes agents applicable.

I. PAINKILLERS AND/OR anti-INFLAMMATORY AGENTS

1. ANTAGONISTS of SEROTONIN RECEPTORS

I believe that serotonin (5-HT) leads to pain by stimulating serotonin receptors2(5-HT2) and/or serotonin3(5-HT3) on the sensitive peripheral neurons. Most researchers agree that the 5-HT3receptors on peripheral nociceptors mediate instant pain sensation produced 5-HT (Richardson et al., 1985). In addition, the inhibition induced by 5-HT pain antagonists of 5-HT3receptors, inhibiting the activation of nociceptors, can also inhibit neurogenic inflammation. Barnes P.J. et al., Modulation of Neurogenic Inflammation: Novel Approaches to Inflammatory Disease, Trends in Pharmacological Sciences, 11, pp. 185-189 (1990). Research on the elbow joints of rats, however, suggests that 5-HT2the receptor responsible for the activation of nociceptors under the action of 5-HT. Grubb BD. 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-HT2receptors may also play a role in the occurrence of peripheral pain and neurogenic inflammation.

The purpose of the solution according to the present invention is blocking pain and the amount of inflammatory processes. Thus, antagonists of 5-HT2and 5-HT3receptors both are suitable for use either separately or together in the solution according to the present invention. Amitriptyline (Elavil™) is suitable as an antagonist of 5-HT2receptors for use in the present invention. Amitriptyline has been used in the clinic for many years as an antidepressant, and it was found that it has a beneficial effect in some patients with chronic pain. Metoclopramide (Reglan™) was applied in the clinic as a drug against vomiting, but he shows a moderate affinity for 5-HT3-receptor and can inhibit the action of 5-HT at this receptor, possibly by inhibiting pain by releasing 5-HT from platelets. Thus, it is also suitable for use in the present invention.

Other suitable antagonists of 5-HT2receptors include imipramine, trazodone, desipramine and ketanserin. Ketanserin was used in the clinic due to it, the antihypertensive actions. Hedner T. et al., Effects of a New Serotonin Antagonist, Ketanserin, in Experimental and Clinical Hypertension, Am J of Hypertension, pp.317s-23s (Jul. 1988). Other suitable antagonists of 5-HT3receptors include cisapride and ondasetron. Suitable antagonists of serotonin receptors1Binclude yohimbine, N-[methoxy-3-(4-methyl-1-piperazinil)phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl) [1, 1-biphenyl]-4-carboxamid ("GR127935") and methiothepin. Therapeutic and preferred concentrations for these medicines in the solution according to the present invention are shown in table 1.

TABLE 1

Therapeutic and preferred concentrations of inhibiting pain and/or anti-inflammatory agents
The group agentTherapeutic concentrations (nanomolar)The preferred concentration (nanomolar)
Antagonists of serotonin receptors2
amitriptylinof 0.1-1,00050-500
the desipramineof 0.1-1,00050-500
ketanserinof 0.1-1,00050-500
Antagonists of serotonin receptors3:
tropisetron 0,01-100of 0.05-50
metoclopramide10-10,000200-2,000
the cisaprideof 0.1-1,00020-200
ondansetronof 0.1-1,00020-200
Antagonists serotonin1B(human 1Dβ)
isomolarof 0.1-1,00050-500
GR127935of 0.1-1,00010-500
methiothepinof 0.1-5001-100
SB2166410.2 to 2,0002-200

2. AGONISTS of SEROTONIN RECEPTORS

It is known that 5-HT1A-, 5-HT1Band 5-HT1Dreceptors inhibit adenylate cyclase activity. Thus, a low dose of agonists of these receptors serotonin1A, serotonin1Band serotonin1Dthe solution is to inhibit the neurons mediating pain and inflammation. The same effect is expected from agonists of serotonin receptors1Eand serotonin1Fbecause these receptors also inhibit adenylate cyclase.

Buspirone is a suitable 1A agonist-receptor for use in the present invention. Sumatriptan is a suitable agonist 1A-, 1B-1D and 1F-receptors. Suitable agonist 1B and 1D receptors is digidroergotamin. Suitable agonist 1E receptor is ergonovine. Therapeutic and preferred concentrations data agonists of receptors is presented in table 2.

TABLE 2

Therapeutic and preferred concentrations of inhibiting pain and/or anti-inflammatory agents
The group agentTherapeutic concentrations (nanomolar)The preferred concentration (nanomolar)
Agonists serotonin1A
buspirone1-1,00010-200
sumatriptan1-1,00010-200
Agonists serotonin1B:
digidroergotaminof 0.1-1,00010-100
sumatriptan1-1,00010-200
naratriptan1-1,00010-200
rizatriptan1-1,00010-200
zolmitriptan1-1,00010-200
L-694,2471-1,00010-200
Agonists serotonin1D:
d is hydroaromatic of 0.1-1,00010-100
sumatriptan1-1,00010-200
naratriptan1-1,00010-200
rizatriptan1-1,00010-200
zolmitriptan1-1,00010-200
L-694,2471-1,00010-200
Agonists serotonin1E:
ergonovine10-2,000100-1,000
Agonists serotonin1E:1-1, 00010-200

3. ANTAGONISTS of HISTAMINE RECEPTORS

Histamine receptors are usually divided into subtypes of histamine1(H1) and histamine2(H2). Classic inflammatory response in peripheral injection of histamine is mediated by H1receptor: Douglas, 1985. Consequently, the solution according to the present invention preferably includes a receptor antagonist of the histamine H1. Promethazine (Phenergan™) is usually used against vomiting drug, which largely blocks the H1receptors and is suitable for use in the present invention. Interestingly, it was shown that this drug has a local anesthetic action is m, but the concentration required for the manifestation of this effect by several orders of magnitude higher than those needed to block H1receptors, therefore, believe that these effects are carried out by different mechanisms. The concentration of the antagonists of histamine receptors in the solution is sufficient for inhibition of H1receptors involved in the activation of nociceptors, but not to achieve a "local anesthetic" action, thereby reducing the value of systemic side effects.

Other suitable antagonists H1receptors include terfenadine, diphenhydramine, amitriptyline, mepyramine and tripolidine. Because amitriptyline is also effective as an antagonist of the serotonin receptor2it has a dual function when using the present invention. Suitable therapeutic and preferred concentrations for each of these agonists H1receptors are shown in table 3.

TABLE 3

Therapeutic and preferred concentrations of agents inhibiting pain and/or inflammation
The group agentTherapeutic concentrations (nanomolar)The preferred concentration (nanomolar)
Antagoni what you're histamine receptors 1:
promethazineof 0.1-1,00050-200
diphenhydramineof 0.1-1,00050-200
amitriptylinof 0.1-1,00050-500
terfenadineof 0.1-1,00050-500
mepyramine (pyrilamine)of 0.1-1,0005-200
tripolidine0.1 to 1005-20

4. ANTAGONISTS BRADYKININASE RECEPTORS

Bradykininase receptors usually are divided into subtypes of bradykinin1(B1and bradykinin2(In2). Studies have shown that acute peripheral pain and inflammation produced by bradykinin, mediated by a subtype of B2at that time, as induced by bradykinin pain in chronic inflammation is mediated by subtype B1: Perkins M.N. et al., Antinociceptive Activity of the Bradykinin B1and B2Receptor Antagonists, des-Arg9, [Leu8]-BK and HOE 140, in Two Models of Persistent Hyperalgesia in the Rat, Pain 53, pp.191-197 (1993); A. Dray et al., Bradykinin and Inflammatory Pain, Trends Neurosci 16, pp.99-104 (1993), each of these sources is included here for information.

Currently antagonists bradykininase receptors are not used in the clinic. Some of these drugs are peptides, and cannot be taken orally, the village is olcu they will undergo digestion. Antagonists In2receptor blocking induced by bradykinin in acute pain and inflammation: Dray et al., 1993. Antagonists of B1receptors inhibit pain in chronic inflammatory States. Perkins et al., 1993; Dray et al., 1993. Therefore, depending on the application of the solution according to the present invention preferably includes one or both of the antagonist of the bradykinin B1and In2. For example, arthroscopy performed for acute and chronic conditions, and, thus, the irrigation solution for arthroscopy may include antagonists as B1receptors and2-receptors.

Suitable antagonists bradykininase receptors for use in the present invention include antagonists bradykininase1receptors: [des-Arg10] derived D-Arg-(Hyp3Thi5D-Tic7Oic8)-BK ("[des-Arg10] derived NOAH 140", from Hoechst Pharmaceuticals); and [Leu8] des-Arg9VK. Suitable antagonists bradykininase2receptors include: [D-Phe7-BK; D-Arg-(Hyp3Thi5,8-Phe7)-BK ("NPC 349"); D-Arg-(Hyp3D-Phe7)-BK ("NPC 567"); and D-Arg-(Nur3Thi5D-Tic7-Oic8)-VK ("NOAH 140"). Suitable therapeutic and preferred concentrations are given in table 4.

TABLE 4

Therapeutic the preferred concentration of the agents, inhibiting pain and/or inflammation
The group agentTherapeutic concentrations (nanomolar)The preferred concentration (nanomolar)
Antagonists of the bradykinin receptor1:
[Leu8] des-Arg9VK1-1,00050-500
[des-AGD10] derived NOE1-1,00050-500
[leu9] [des-Arg10] kallidinof 0.1-50010-200
Antagonists of the bradykinin receptor2:
[D-Phe7]-BK100-10,000200-5,000
NPC 3491-1,00050-500
NPC 5671-1,00050-500
NOAH 1401-1,00050-500

5. INHIBITORS of KALLIKREIN

The peptide bradykinin is an important mediator of pain and inflammation, as previously noted. Bradykinin is produced as a product of splitting under the action of kallikrein on high-molecular kininogen in the plasma. Therefore, it is believed that inhibitors of kallikrein will have a therapeutic effect in the inhibition of the production of bradykinin and emerging is the result of pain and inflammation. A suitable inhibitor of kallikrein for use in the present invention is Aprotinin. Suitable concentrations for use in the solutions of the present invention are presented below in table 5.

TABLE 5

Therapeutic and preferred concentrations of agents inhibiting pain and/or inflammation
The group agentTherapeutic concentrations (nanomolar)The preferred concentration (nanomolar)
Inhibitor of kallikrein1:
Aprotinin1-1,00050-500

6. ANTAGONISTS TACHYKININ RECEPTORS

Tachykinin (DCS) represent a family of structurally close peptides, including substance P, neurokinin A (NKA) and neurokinin B (NKB). Neurons are the main source of DCS in peripheral tissues. Important General effect TKs is the stimulation of neurons, but other effects include endothelium-dependent vasodilatation, radiolabeled plasma proteins, recovery and degranulation of mast cells and stimulation of cells with inflammation: Maggi S.A., Gen. Pharmacol., Vol.22, pp.1-24 (1991). Due to a combination of the above physiological effects mediated by activation of receptors is To, purposeful impact on TC-receptors is a reasonable approach to enhance analgesia and treatment of neurogenic inflammation.

6A. ANTAGONISTS SUBTYPE NEUROKININ1RECEPTORS

Substance P activates subtype neurokinin receptors related to NK1. Substance P is undecapeptide, which is a sensory nerve endings. It is known that substance P has many effects that cause inflammation and pain in peripheral tissues after activation of C-fibers, including vasodilatation, radiolabeled plasma and mast cell degranulation. Levine J.D. et al., Peptides and the Primary Afferent Nociceptor, J. Neurosci. 13, p.2273 (1993). Suitable antagonist of the substance P is (CD-Pro9[Spiro-gamma-lactam] Leu10, Trp11] physalaemin-(1-11)) ("GR 82334"). Other suitable antagonists for use according to the present invention, which affect NK1receptors are: 1-imino-2-(2-methoxyphenyl)ethyl)-7,7-diphenyl-4-lane-hydrocondone(3R,7R) ("RP 67580"); and 2S,3S-CIS-3-(2-methoxybenzylamine)-2-benzhydrylpiperazine ("CP 96345"'). Suitable concentration of these agents is presented in table 6.

TABLE 6

Therapeutic and preferred concentrations of agents inhibiting pain and/or inflammation
The group agentTherapeutic concentrations (nanomolar)The preferred concentration (nanomolar)
Antagonists of receptor subtype neirokinina1:
GR 823341-1,00010-500
WED 96,3451-10,000100-1,000
RP 67580of 0.1-1,000100-1,000

6b. ANTAGONISTS SUBTYPE NEUROKININ2RECEPTORS

Neurokinin And is a peptide which is found in sensory neurons with substance P and which also contributes to inflammation and pain. Neurokinin And activates specific neurokinin receptors related to NKa. Edmonds-Alt S. et al., A Potent and Selective Non-Peptide Antagonist of the Neurokinin A (NK:2) Receptor, Life Sci. 50:PL101 (1992). Examples of suitable antagonists NK2include ((S)-N-methyl-N-[4-(4-acetylamino-4-phenylpiperidine)-2-(3,4-dichlorophenyl) butyl]-benzamide ("(±)-SR 48968"); Met-Asp-Trp-Phe-Dap-Leu ("MEN 10627"); and CEC(GIn-Trp-Phe-Gly-Leu-Met) ("L 659877"). Suitable concentration of these agents are shown in table 7.

TABLE 7

Therapeutic and preferred concentrations of agents inhibiting pain and/or inflammation
The group agentTherapeutic is concentratie (nanomolar) The preferred concentration (nanomolar)
Antagonists of receptor subtype neirokinina2:
MEN 10,6271-1,00010-1,000
L 659,87710-10,000100-10,000
(±)-SR 4896810-10,000100-10,000

7. Antagonists of CGRP receptors

The peptide associated with the gene calcitonin (CGRP), is a peptide, which is also in sensory neurons with substance P and which acts as a vasodilator and increases the action of the substance: R. Brain S.D. et al., Inflammatory Oedema Induced by Synergism Between Calcitonin Gene-Related Peptide (CGRP) and Mediators of Increased Vascular Permeability, Br. J. Pharmacol. 99, p.202 (1985). An example of a suitable antagonist CGRP receptors is I-CGRP-(8-37), a subset of CGRP. This polypeptide inhibits the activation of CGRP receptors. Suitable concentrations of the agent are shown in table 8.

TABLE 8

Therapeutic and preferred concentrations of agents inhibiting pain and/or inflammation
The group agentTherapeutic concentrations (nanomolar)The preferred concentration (nanomolar)
Antagonist CGRP-receptors
I-CGRP-(8-37)1-1,00010-500

8. ANTAGONIST INTERLACING RECEPTORS

Interleukins are a family of peptides called cytokines produced by leukocytes and other cells in response to inflammatory mediators. Interleukins can be strong hyperalgesia agents in peripheral tissues. Ferriera S.H. et al., Interleukin-1β as a Potent Hyperalgesic Agent Antagonized by a Tripeptide Analogue, Nature 334, p.698 (1988). An example of a suitable antagonist of IL-1β-receptor is Lys-D-Pro-Thr, which is a subset of IL-1β. This Tripeptide inhibits the activation of IL-1β-receptors. Suitable concentrations of the agent are shown in table 9.

TABLE 9

Therapeutic and preferred concentrations of agents inhibiting pain and/or inflammation
The group agentTherapeutic concentrations (nanomolar)The preferred concentration (nanomolar)
Antagonist interlacing receptors
Lys-D-Pro-Thr1-1,00010-500

9. INHIBITORS of ENZYMES INVOLVED IN the SYNTHETIC pathway ARACHIDONIC ACID METABOLISM

9a. Inhibitors of phospholipase

Products of arachidonic acid with the participation of phospholipase A2(PLA2) (cPLA2, iPLA2, sPLA2) and phospholipase C (PLC) leads to a cascade of reactions in which the formation of numerous inflammatory mediators, known as eicosanoids. There are a number of stages on the path, which can inhibit, thereby reducing the production of these inflammatory mediators. Examples of inhibition data of the various stages is given below.

Inhibition of enzyme isoforms PLA2inhibits the release of arachidonic acid from cell membranes and, therefore, inhibits the production of prostaglandins and leukotrienes, resulting in suppression of inflammation and pain: Glaser C.V., Regulation of Phospholipase A2 Enzymes: Selective Inhibitors and Their Pharmacological Potential, Adv. Pharmacol. 32, p.31 (1995). An example of a suitable inhibitor isoforms PLA2is manoalide. Suitable concentrations of this agent is included in table 10. Inhibition of isoforms of phospholipase Cγ(PLCγ) would also lead to reduced production of prostanoids and leukotrienes and, consequently, will lead to reduced pain and inflammation. An example of an inhibitor of the isoform of PLCγis 1-[6-((17β-3-petoksista-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione.

TABLE 10

Therapeutic and prepact the positive concentration agents inhibiting pain and/or inflammation
The group agentTherapeutic concentrations (nanomolar)The preferred concentration (nanomolar)
Inhibitor of phospholipase
manoalide100-100,000500-10,000
Aristolochia acid40-400,000400-40,000
ASA10-100,000100-10,000
HELSS6-6,00060-6,000

9b. Cyclo-oxygenase inhibitors

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used as anti-inflammatory, antipyretic, antitromboticeskih and analgesic agents. Lewis R.A., Prostaglandins and Leukotrienes, In: Texbook of Rheumatology, 3d ed. (N. W. Kelley et al., eds.), p.258 (1989). Molecular targets for these drugs are cyclooxygenase type I and type II (MOR-1 and MOR-2). These enzymes are also known as prostaglandin H-synthase (PGHS)-l (constitutive) and -2 (induced), and they catalyse the conversion of arachidonic acid to prostaglandin H, which is an intermediate product in the biosynthesis of prostaglandins and thromboxanes. The enzyme SOH-2 was detected in endothelial cells, macrophages and fibroblasts. This enzyme is induced by p is on the action of IL-1 and TNF-α and its expression is increased in areas of inflammation. The constitutive activity of MOR-1 and the activity-induced MOR-2 lead to the synthesis of prostaglandins, which are involved in the development of pain and inflammation.

Many commercial NSAIDs (diclofenac, naproxen, indomethacin, ibuprofen and the like) are mostly non-selective inhibitors of both isoforms of SOKH, but may show a higher selectivity for MOR-1 compared with MOR-2, although this ratio varies for different compounds. Inhibitors of MOR-1 and 2 to block the formation of prostaglandins is the best therapeutic strategy than trying to block the interaction of natural ligands with family described subtypes prostanoid receptors. Antagonists eicosanoid receptors (ER-1, ER-2, ER-3)reported, are quite rare and only in specific, it was reported about the antagonists with high affinity receptor for thromboxane A2: Wallace J. and G. Cirino Trends in Pharm. Sci., Vol. 15, pp. 405-406 (1994).

Typical therapeutic and preferred concentrations of cyclo-oxygenase inhibitors for use in the solution are given in table 11.

TABLE 11

Therapeutic and preferred concentrations of agents inhibiting pain and/or vocale is s
The group agentTherapeutic concentrations (nanomolar)The preferred concentration (nanomolar)
Cyclo-oxygenase inhibitors:
Ketorolac100-10,000500-5,000
indometacin1,000-500,00010,000-200,000

Sensibility lipoxygenase

Inhibition of the enzyme lipoxygenase inhibits the production of leukotrienes, such as leukotriene4which, as you know, is an important mediator of inflammation and pain. Lewis R.A., Prostaglandins and Leukotrienes, In: Texbook of Rheumatology, 3d ed. (N. W. Kelley et al., eds.), pp.258 (1989). An example of an antagonist of 5-lipoxygenase is 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadienal)-1,4-benzoquinone ("AA 861"), suitable concentrations as given in table 12.

TABLE 12

Therapeutic and preferred concentrations of agents inhibiting pain and/or inflammation
The group agentTherapeutic concentrations (nanomolar)The preferred concentration (nanomolar)
Inhibitor of lipoxygenase:
AA 861100-10,000500-5,00
coffee acid500-50,0002,000-20,000

10. ANTAGONISTS PROSTANOID RECEPTORS

Specific prostanoids produced as metabolites of arachidonic acid, mediate their inflammatory effects through activation prostanoid receptors. Examples of specific groups prostanoid antagonists are antagonists of receptor subtypes eicosanoid EP-1 and EP-4 and subtype antagonists of thromboxane receptor. A suitable receptor antagonist of prostaglandin E2is 8 hardy-benzo[b,f][1,4]oxazepine-10(11N)-carboxylic acid, 2-acetylhydrazide ("SC 19220"). Appropriate subtype antagonist of the thromboxane receptor is [15-[1α,2β(5Z),3β,4α]-7-[3-[2-(phenylamino)-carbonyl]hydrazino]methyl]-7-oxabicyclo-[2,2,1]-hept-2-yl]-5-heptane acid ("SO 29548"). Suitable concentration of these agents are shown in table 13.

TABLE 13

Therapeutic and preferred concentrations of agents inhibiting pain and/or inflammation
The group agentTherapeutic concentrations (nanomolar)The preferred concentration (nanomolar)
Antagonist eicosanoid EP-1:
SC 19220100-10,000500-5,000

11. LEUKOTRIENE RECEPTOR ANTAGONISTS

Leukotrienes (LTB4, LTC4and LTD4) are products of arachidonic acid metabolism with the participation of 5-lipoxygenase, which are produced by enzymatic and possess important biological properties. Leukotrienes are involved in the development of several pathological conditions, including inflammation. Many pharmaceutical companies are currently in search of specific antagonists for potential therapeutic intervention in these diseases. P.V. Halushka et al., Annu. Rev. Pharmacol. Toxicol. 29: 213-239 (1989); Ford-Hutchinson, A., Crit. Rev. Immunol. 10: 1-12 (1990). LTB4the receptor was detected in some cells of the immune system, including eosinophils and neutrophils. The LTB4 binding to these receptors leads to chemotaxis and the release of lysosomal enzymes, thereby contributing to the development of inflammation. The transmission of signals associated with activation of LTB4-receptor mediated includes G-protein stimulation of metabolism phosphatidylinositol (PI) and the increase of intracellular calcium (see figure 2).

An example of a suitable antagonist of the receptor for leukotriene B4 is SC(+)-(S)-7-(3-(2-cyclopropylethyl)-3-methoxy-4-[(methylamino)-carbonyl]phenoxy(propoxy)-3,4-dihydro-8-propyl-2H-1-benzopyran-2-propanoic acid ("SC-53228"). Concentrate the radio this agent, suitable for practice of the present invention, are shown in table 14. Other suitable receptor antagonists leukotriene 84 include [3-[-2(7-[chloro-2-chinolin)ethynyl]phenyl][[3-(dimethylamino-3-oxopropyl)thio]methyl]thiopropazate acid (MK 0571), and drugs LY 66071 and ICI 203219. MK 0571 also acts as an antagonist of the receptor subtype LTD4.

TABLE 14

Therapeutic and preferred concentrations of agents inhibiting pain and/or inflammation
The group agentTherapeutic concentrations (nanomolar)The preferred concentration (nanomolar)
The leukotriene antagonist

B4:
SC-53228100-10,000500-5,000

12. AGONISTS of OPIOID RECEPTORS

Activation of opioid receptors leads to the antinociceptive effects, and, therefore, agonists for these receptors is desired. Opioid receptors include subtypes μ-, δ- and κ-opioid receptors, μ-receptors are located in the peripheral endings of sensory neurons and activation of these receptors inhibits the activity of sensory neurons: Basbaum, A.I. et al., Opiate analgesia: How Central is a Peripheral Target?, N. Engi. J. Med., 325:116 (1991). δ- and κ-receptors are located in the sympathetic efferent endings and inhibit the release of prostaglandins, thereby inhibiting pain and inflammation. By Taiwo on COOPER et al., Kappa - and Delta-Opioids Block Sympathetically Dependent Hyperalgesia, J. Neurosci., Vol.11, page 928 (1991). Subtypes of opioid receptors are members of the superfamily associated with G-protein receptors. Therefore, all agonists of opioid receptors interact and initiate transmission of the signal through their cognate coupled with G-protein receptor. Examples of suitable agonists α-opioid receptors are fentanyl and Try-D-Ala-Gly-[N-MePhe]-NH(CH2)-HE ("DAMGO"). An example of a suitable agonist κ-opioid receptor is [D-Pen2D-Pen5]enkephalin ("DPDPE"). An example of a suitable agonist to opioid receptors is (TRANS)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzoylacetone ("U 50488"). Suitable concentrations of each of these agents are shown in table 15.

TABLE 15

Therapeutic and preferred concentrations of agents inhibiting pain and/or inflammation
The group agentTherapeutic concentrations (nanomolar)The preferred concentration (nanomolar)
Agonist μ-opioids:
DAMGO0.1 to 1000,5-20
Sufentanil0,01-501-20
fentanylof 0.1-50010-200
PL 017of 0.05-500,25-10
Agonist δ-opioids:
DPDPEof 0.1-5001,0-100
Agonist κ-opioids:
U50,488of 0.1-5001,0-100

13. ANTAGONISTS PURINOCEPTORS

Extracellular ATP acts as a signaling molecule through interaction with P2-purinoceptors. One large group of purinoceptors are R2X-purinoceptor, which are gate ligands ion channels, with internal ion channels permeable to Na+To+and CA2+. P2Xreceptors described in sensory neurons are important for primary afferent neural transmission and perception of pain. It is known that ATP depolarize sensory neurons and plays a role in the activation of nociceptors, because ATP released from damaged cells, stimulates R2Xreceptors, leading to depolarization of nociceptive endings of nerve fibers. RH3-R is the receptors have a very limited distribution (Chen S. et al., Nature, Vol.377, pp.428-431 (1995)), because they are selectively expressed in sensory nerve C-fibers, which pass into the spinal cord, and it is known that many of these C-fibers are receptors for pain stimuli. Thus, highly restricted localization of expression of subunits RH3receptors makes these subtypes are excellent targets for analgesic actions (see figures 3 and 7).

Calcium-mobilizing purine receptors, which belong to the superfamily of receptors, G-protein, is described on the surface of articular chondrocytes in mammals. It was found that ATP stimulates a dose-dependent, transient increase in the concentration of calcium ions in differentiated primary chondrocytes. In the experiments with the heterologous desensitization has been shown that chondrocytes do not show subsequent response to UTP after the start of stimulation under the action of ATP. These results are consistent with the presence of RA receptors on the cell surface of chondrocytes. Induced by purine mobilization of calcium in passirovannym the chondrocytes shows a similar pharmacological profile in relation to the sensitivity to the agonist. ATP and UTP does not alter the synthesis of cartilage matrix that was determined by the rate of incorporation of [35S]sulfate in glycosaminoglycan in cartilage explants or primary chondrocytes. Destruction full the KSA, as determined by the release of glycosaminoglycans and collagen from cartilage explants, also did not change under the action of any agonist. The presence of functional purine RU receptors on the surface of primary articular chondrocytes promotes concentrations of extracellular purines such as ATP, to activate the metabolism of the chondrocytes.

In other studies was established expression of genes of P1 - and P2-purine receptors in human articular chondrocytes and profile mediated by ligands in the release of prostaglandin E2. Agonists RU receptors ATP and UTP stimulated a minor release of PGE2, which is synergistically intensified after pre-treatment of human IL-1α. The release of PGE2 in response to the joint addition of ATP and UTP after pre-treatment of IL-1 was mymicrobalance phorbolester acetate. Function P2Y2-receptor is mediated amplification of IL-1 release of PGE2, thus resulting in pain and inflammation in the joint. Thus, the use of antagonists of P2Y the present invention is to prevent the activation of the production of inflammatory mediators, as synoviocytes and chondrocytes.

Suitable antagonists of P2X/ATF-purinoceptors for use in the present invention include, as example, suramin and pyridox the phosphate-6-zofenil-2,4-disulfonate acid (PPADS"). Suitable concentration of these agents are shown in table 16.

TABLE 16

Therapeutic and preferred concentrations of agents inhibiting pain and/or inflammation
The group agentTherapeutic concentrations (nanomolar)The preferred concentration (nanomolar)
RH and/or P2Y Antagonists:
suramin100-100,00010,000-100,000
PPADS100-100,00010,000-100,000

14. ANTAGONISTS of CA2+channels

Calcium channel antagonists represent a separate group of drugs that inhibit the transmembrane flux of calcium ions to activate cellular responses mediating neuroforamina. The entry of calcium ions in synoviocytes and chondrocytes is a key event mediating activation responses in these cells. In addition, the role bradykininase, histamine, serotonin (SHT2and neurokinin receptors (NK1and NK2in mediating the transmission signal when neurospine includes the increase of intracellular calcium, thus leading to activation of calcium channels on the plasma memb is ane. In many tissues, calcium channel antagonists such as nifedipine may reduce the release of arachidonic acid, prostaglandins and leukotrienes, which occur under the influence of various stimuli, and Moncada, S., R. Flower and Vane J. in Goodman's and Oilman's Phannacological Basis of Therapeutics, (7thed.), MacMillan Publ. Inc., pp.660-5 (1995).

Finally, calcium channel antagonists and each of the antagonists tachykinin, histamine or bradykinin show a synergistic effect in suppressing neurospine. Role installed neurokinin receptors in mediating neurospine. The transmission signal with the participation of receptors neirokinina1(NK1and neirokinina2(NK2) (members associated with G-protein superfamily includes the increase of intracellular calcium, thus leading to activation of calcium channels on the plasma membrane. Similarly, activation of the bradykinin receptor2(R2) is associated with increased intracellular calcium in synoviocytes and chondrocytes. Thus, calcium channel antagonists interact with a common mechanism causing the increase of intracellular calcium, a part of which passes through the channels of L-type. This is the basis for the synergistic interaction between calcium channel antagonists and receptor antagonists neirokinina, histamine, P 2Y and bradykinin2.

Suitable calcium channel antagonists for the practice of the present invention include nisoldipine, nifedipine, nimodipine, lacidipine, isradipine and amlodipine. Suitable concentration of these agents are shown in table 17.

TABLE 17

Therapeutic and preferred concentrations of agents that suppress spasms
The group agentTherapeutic concentrations (animalerie)The preferred concentration (nanomolar)
Calcium channel antagonists:
nisoldipine1-10,000100-1,000
nifedipine1-10,000100-5,000
nimodipine1-10,000100-5,000
lacidipine1-10,000100-5,000
isradipine1-10,000100-5,000
Antipin1-10,000100-5,000

II. AGENTS FOR inhibiting the DESTRUCTION of CARTILAGE

Recent advances in understanding the biochemistry and molecular biology of inflammation and destruction of cartilage clarify the role of numerous endogenous cytokines. Numerous proposalthe is ranked on the mediators, involved in the breakdown of cartilage in the inflamed joint, are the cytokines, TNF-α, IL-1, IL-6 and IL-8. Elevated levels of a number of data proinflammatory cytokines quickly appear in the synovial fluid of the knee in acute injury and remain elevated in patients for at least 4 weeks (Cameron, ML et al., "Synovial fluid cytokine concentrations as possible prognostic indicators in the ACL-deficient knee". Knee Sung. Sports Traumatol. Arthroscopy 2:38-44 (1994)). These cytokines are produced locally in the joint multiple active types of cells, including synovial fibroblasts, synovial macrophages and chondrocytes. Educated locally cytokines mediate pathophysiological events in acute and chronic inflammatory conditions and are an important autocrine and paracrine mediators in the catabolism of cartilage. These cytokines characterized by their ability to cause multiple effects on different cellular targets and their ability to communicate positive or negative synergistic way with other cytokines. Especially important are IL-1 and TNF-αbecause they also initiate destructive cartilage effects by an imbalance between normal metabolism and degradation of components of cartilage matrix by modulating the activity of endogenous proteins, such as matrix metalloproteinases (MMPs), and tissue Inga is forom metalloproteinases (TIMP). Regulation under the action of cytokine homeostasis in cartilage is a highly regulated balance between active mediators acting in chondrocytes, which determines going to be the destruction of the matrix or its recovery.

Damage to the joint often leads to an inflammatory response within the joint space, which involves the synovial tissue and can lead to destruction of the articular cartilage. Described illustrative shifts metabolism in synovial fluid and the cartilage of the knee in humans after damage to the joint and troscopically operations (Cameron M.L. et al. (1994), above; Cameron M.L. et al. "The natural history of the anterior cruciate ligament-deficient knee: Changes in synovial fluid cytokine and keratan sulfate concentrations", Am. J. Sports Med. 25:751-754 (1997)). The specific levels of proinflammatory cytokines clearly increased (2-4 orders of magnitude greater than normal value) in the synovial fluid of the knee joint during the acute phase of inflammation after rupture of the anterior cruciate ligament of the knee (ACL). Significant changes also occur in the concentrations of molecules of cartilage matrix due to the increased production of matrix metalloproteinases (MMPs), such as collagenase and stromelysin-1, are increased in the synovial fluid of patients after acute injury (L.S. Lohmander et al. "Temporal patterns of stromelysin-1 tissue inhibitor, and proteoglycan fragments in human knee joint fluid after injury to the cruciate ligamentor meniscus", J. Bar And Large Res. 12:21-28 (1994)). Temporal changes of cytokines and markers of cartilage matrix (e.g., proteoglycans) in the synovial fluid, which correlate with the destruction of cartilage, are maximum during acute damage, but remain elevated for an extended period of time (from 3 months to one year), slowly descending and remaining higher than background levels prior to the damage.

By itself, the injury arthroscopic surgery causes severe postoperative inflammation, which reflects additional inflammatory activation of cells in the joint, including positive regulation of cyclooxygenase-2 and other Pro-inflammatory cytokines. A significant proportion of patients (60-90%) with ACL rupture revealed changes of knee x-ray image, pointing to osteoarthritis (OA) in 10-15 years after injury (Cameron M.L. et al. (1994), above). Thus, the combined effects of the initial joint damage and surgical trauma can induce continuous inflammation and related changes in the metabolism of cartilage matrix, which are the causal factors leading to the subsequent development of degenerative changes in the articular cartilage and soon the development of osteoarthritis. The value of this health problems is significant is, since the total set number of arthroscopic procedures conducted only in the United States amounted in 1996 to 1.8 million rate of growth of about 10% per year. Thus, it is desirable to provide a pharmaceutical way to prevent destruction of the articular cartilage within the joint.

Although pain and inflammation in the postoperative period are recognized as a significant clinical problem, schemes pharmacological treatment currently focused only on acute analgesia in the postoperative period. The existing rules surgical treatment is not aimed at chronic inflammatory condition that occurs after surgery, and the need to suppress the degradation of cartilage in the joint after surgery. Therefore, there is a clear need to develop effective integrated drug therapy, which is aimed at both acute and chronic aspects of pain and inflammation, as well as pathological changes in the metabolism of cartilage in damaged or operated joint.

According to this aspect of the invention provides a method of suppressing or preventing the degradation of articular cartilage in the joint by injection directly into the joint of the patient a composition that includes one or more metabolically active protective for cartilage and the clients together with one or more analgesic and anti-inflammatory agents, as previously described, or alternatively a combination of two or more metabolically active for protective cartilage agents, at least one of which promotes the flow of anabolic processes in the cartilage and at least one of which is an inhibitor of catabolic processes in the cartilage, in a pharmaceutically effective carrier for intra-articular delivery. Metabolically active agents include, but are not limited to, all compounds that act directly or indirectly by modulation or change of a biological, biochemical or biophysical state of the cells, including agents that alter the electrical potential of the plasma membrane, ligand binding or enzymatic activity of cell receptors, intracellular and extracellular enzymes, interactions protein-protein interactions, RNA-protein, or interaction of DNA-protein. For example, such agents may include agonists of the receptors, which starts a cascade of signaling, receptor antagonists, which inhibit the transmission of signals, activators and inhibitors of intracellular and extracellular enzymes and agents that modulate the binding of transcription factors to DNA.

In particular, one aspect of the present invention provides a pharmacological method of treating damaged or operated the CSOs joint using a combination of protective cartilage agents, delivered locally to achieve maximum therapeutic effect. Applying a combination of protective cartilage agents overcomes the limitations of existing therapeutic approaches, which are based on the use of single agent for blocking the multifactorial process of destruction of cartilage in which the shift occurs between synthesis and degradation in favor of catabolic processes. This aspect of the invention exclusively uses the approach of combining agents that act simultaneously on different molecular targets to promote cartilage anabolism and inhibition unregulated or excessive catabolic processes in the cartilage to achieve maximum suppression of inflammatory processes and maintaining homeostasis in the cartilage, thereby achieving protective for cartilage effect inside the joint. Inhibition of one molecular target or biochemical mechanism known as inducing degradation (catabolism) of cartilage, such as the inhibition of the binding of interleukin-1 (IL-1) receptor, IL-1, probably will not be optimal, since, for example, the effect of TNF-α, mediated through its unique receptor, shares many overlapping proinflammatory and catabolic functions in cartilage with IL-1, and he was also recognized as one of the important mediators of the degradation of cartilage in the joint is. Similarly, the use of pharmaceutical agents, which only enhance the anabolic processes in the cartilage in the absence of suppression of catabolic processes, will not be optimally counteract catabolic factors present in the damaged joint.

In particular, one aspect of the present invention provides pharmaceutical compositions metabolically active for protective cartilage agents, which are based on a combination of at least two agents that act simultaneously on different molecular targets. In a typical embodiment, at least one agent is an agonist of receptors cytokines and growth factors, which directly provides anti-inflammatory activity and/or promotes anabolic processes in the cartilage, and at least a second agent is a receptor antagonist or inhibitor of the enzyme, which acts to suppress Pro-inflammatory and/or catabolic processes in the cartilage. A typical combination of drugs includes at least one agent selected from the group of anti-inflammatory/anabolic cytokines, which operates functionally to suppress the role of proinflammatory cytokines in the joint, contributes to the synthesis of cartilage matrix and inhibits the destruction of the matrix. These agonis the s receptor include, but not limited to, specific anti-inflammatory and anabolic cytokines, such as agonists of interleukins (IL) (e.g., IL-4, IL-10 and IL-13) and specific members of the superfamily of transforming growth factor-β (for example, TGFB-β and BMP-7), insulin-like growth factors (e.g., IGF-1) and fibroblast growth factors (e.g., bFGF). At least a second agent selected from the group receptor antagonists and enzyme inhibitors, which inhibits and reduces the activity or expression of Pro-inflammatory molecular targets (e.g., antagonists of IL-1 receptor antagonists of TNF-αreceptors, inhibitors of cyclooxygenase-2 inhibitors Martinez, inhibitors of nitric oxide synthase (NOS) and inhibitors of nuclear factor kappaB (NFκ (B). Metabolically active agents include functional agonists and antagonists of receptors located on the surfaces of cells, and inhibitors binding to membranes or secreted extracellular enzymes (for example, stromelysin and collagenase). In addition, many agents directed at new targets, which are intracellular enzymes and transcription factors that transmit and integrate signals from surface receptors, including inhibitors of NOS enzymes, COX-2 and mitogen-activated protein kinases (MARK) and inhibitors of protein interactions-D Is To, such as the transcription factor NFκB. This method allows you to preserve the integrity of the cartilage, while flow mediated by cytokines anabolic processes and inhibiting catabolic processes.

The combination of many drugs can be delivered locally intra-articular injection or infusion, including perioperative introduction (i.e. preoperatively and/or intraoperatively and/or postoperatively) during arthroscopic surgical procedures, one or in combination with postoperative continuous delivery, such as controlled delivery system with a pump or other delivery system with a constant release. Delivery system with a constant release may include, but are not limited to, microparticles, microspheres or nanoparticles consisting of proteins, liposomes, hydrocarbons, synthetic organic compounds or inorganic compounds. Thus, in some embodiments the invention provides a combination of agents for delivery by injection or infusion, either alone or together with analgesic or anti-inflammatory agents. Rapid onset of action, providing direct, local delivery for protective cartilage agents during or close to the time of the injury (for example, perioperative) has the potential to inhibit the initial processes per the D. the, as they launch the subsequent response, and thereby restricts local tissue damage and eventual destruction of cartilage.

The advantages of this aspect of the present invention include: 1) a combination therapy of drugs aimed at multifactorial causes of the cartilage destruction during acute and chronic conditions; 2) a combination of the protective cartilage agents can be combined with anti-inflammatory and analgesic agents; 3) local delivery of a drug combination is achieved instant creation of therapeutic concentrations to be protective cartilage agents inside the joint; 4) the use of irrigating solution perioperative provides a continuing levels of drugs inside the joint in a therapeutically desirable limit during arthroscopic surgical procedures; 5) local delivery can reduce the total dose of drug and frequency of dosing compared with the system delivery; 6) local, aimed at the surgical site delivery in the joint avoids systemic toxicity and to reduce side effects; and 7) direct local delivery joint allows the use of new pharmaceutically active peptides and proteins, including cytokines and growth factors, which may not be therapeutically useful, if limited to systemic way and injection.

1. RECEPTOR ANTAGONISTS of INTERLEUKIN-1 (IL-1)

Interleukin IL-1 is present in two forms, IL-1α and IL-1β, which are polypeptides, derivatives of individual gene products that share the same range of immunoregulatory and proinflammatory functions. IL-1 is a polypeptide weight of 17 kD, which affects and can be produced near the cell types in the joint, including synovial fibroblasts and macrophages, chondrocytes, endothelial cells and monocytes, and macrophages. There is substantial evidence that IL-1 plays a Central role in the inflammation of the joint and pathophysiological loss of articular cartilage, which take place in the damaged joint.

The action of both forms of this destructive cartilage cytokine mediated by two IL-1 receptors (IL-1R)receptor type I IL-1 or type II IL-1. IL-1 receptors are structurally different and specific to an individual superfamilies, characterized by the presence of the binding domains of immunoglobulins. These receptors have a strong amino acid homology with other receptors, including immunoglobulin domains. The expression of IL-1-receptor larger type I available in T-cells and fibroblasts, whereas IL-1-receptor smaller type II is available in b cells, monocytes, neutrophils and bone marrow cells.

IL-1 receptor type II is associated IL1β with high affinity, but the binding of IL-1β does not initiate intracellular signal transmission, as occurs upon binding with the IL-1 receptor type I. In contrast, receptor type II serves as a precursor of the soluble factor binding IL-1, which, as shown, extends from the cells, and this soluble receptor acts as a physiological antagonist of IL-1β. Described natural protein binding of IL-1, which corresponds to the soluble outer part of the receptor type II.

Natural secretory soluble ligand that binds to IL-1 receptors, alternative-specific antagonist IL-1 receptor (sIL-1RA, IL-1Ra, IL-1ra), was cloned, sequenced, and found that it encodes a protein mass of 22 kD. IL-1Ra competitively inhibits the binding of IL-1α and IL-1β with IL-1 receptors of both types I and II. IL-1Ra is a pure receptor antagonist, because of its binding to the receptor activates the mechanism of cell signal transmission, membrane-bound IL-1 receptor. Despite the high binding affinity of this protein with IL-1Rs required 10-100-fold molar excess for the inhibition of the biological responses of cells to IL-1, which Express the type I IL-1R. Known cells that produce IL-1Ra, include monocytes, neutrophils, macrophages, synoviocytes and x is negrocity. It was shown that IL-1Ra inhibits PGE2 synthesis, induction of proinflammatory cytokines and MMPs and production of nitric oxide. Secreted IL-1Ra released in vivo during experimentally induced inflammation. It is important that IL-1Ra is expressed in synovial tissue and is in normal synovial fluid of humans. In patients with knee damage levels of IL-1Ra in synovial fluid clearly increased in the acute phase after injury and then decrease to normal levels in the subacute and chronic phases. Thus, it was shown that IL-1Ra plays a physiological role in the response reactions of joint injury.

IL-1 is considered a major destructive cartilage cytokine that plays a Central role in joint destruction due to its ability to stimulate production of degradation enzymes and proinflammatory cytokines as chondrocytes, and synoviocytes. In addition, IL-1β is a strong inhibitor of the synthesis of proteoglycan and collagen in chondrocytes. At the cellular level, induced by IL-1β response of synovial fibroblasts include increased production of PGE2, collagenase and other neutral proteases and positive regulation of the levels of Pro-inflammatory cytokines, IL-6 and IL-8.

IL-1, which is in the joint fluid of patients with arthritic diseases, stimulates chondrocyte is to: 1) synthesis of high amounts of enzymes, such as stromelysin, collagenase fibroblasts and neutrophils and plasminogen activator and 2) inhibition of synthesis inhibitor-1 plasminogen activator and TSR. In addition, IL-1β is a strong inhibitor of the synthesis of matrix components such as collagen type II, the predominant form of collagen in the articular cartilage and proteoglycans. The imbalance between the levels of inhibitors and proteases leads to an increase in the number of active proteases. This increase, combined with the suppression of the biosynthesis of matrix leads to the destruction of cartilage. In animal experiments, the injection of IL-1 in the knee joints of rabbits causes depletion of proteoglycan of articular cartilage.

Because IL-1 is one of the key cytokines involved in the pathogenesis of chronic synovitis and destruction of cartilage, reducing its production or blocking its action is an appropriate strategy for new therapies to suppress synovial inflammation and provide for protective cartilage effect. You can use a variety of therapeutic approaches to prevent the interaction of the agonist, IL-1, with its natural associated with a membrane receptor, which include: 1) natural specific activity inhibitors of IL-1 that have been characterized to date, including IL-1Ra and soluble IL-1 receptors; 2 anti-ILAbs and 3) a small molecule antagonists, which can be peptides or dipeptide.

The ability to block the action of this key cytokine will affect many cell types in the joint (e.g., synovial fibroblasts and chondrocytes), thus inhibiting pathological effects, such as infiltration of inflammatory cells into the joint, synovial hyperplasia, activation of synovial cells, as well as the breakdown of cartilage and suppression of the synthesis of cartilage matrix. The antagonist of IL-1 receptors will block the spread of the inflammatory response to IL-1 and thus interrupt the disease process. Installed on models of inflammation and arthritis (RA and OA) in animals, therapeutic potential of a number of antagonists of IL-1 receptors. The condition of patients suffering from RA, improved clinically after subcutaneous injection of IL-1Ra or intraarticular injection of soluble IL-1R type I.

The effects of IL-1β and IL-1Ra depend on their respective local concentrations. In supernatant pieces of synovium levels of IL-1β there were three times higher than those for IL-1Ra. Thus, spontaneous local production of IL-1Ra is not sufficient for inhibiting the effects of IL-1βbecause we needed a larger (10-100-fold) molar excess of IL-1Ra for inhibition of IL-1-induced biological responses in cells that Express IL-1R type I. This is the way, high-dose IL-1Ra was used in vivo to block IL-1 in volunteers and patients with RA. IL-1Ra, located locally in the synovium, provides a negative signal suppressing at least a part mediated by IL-1 processes in synovitis, such as the accumulation of leukocytes in inflamed tissue, the production of PGE2 and production of collagenase synovial cells. Was shown to be protective cartilage effect of IL-1Ra using direct injection of IL-1Ra in the joint model ACL in dogs and using a gene therapy approach based on the transfection of the gene of IL-1Ra in human synovial fibroblasts.

The present invention discloses local delivery of soluble receptor protein IL-1, which consists of the extracellular domain of IL-1 and capable of contact with a molecule of the cytokine IL-1 in solution. In particular, in this example, there is disclosed a soluble human IL-1 receptor polypeptide (shuIL-1R), mainly comprising the amino acid sequence 1-312, as disclosed in U.S. patent No. 5319071 and U.S. patent No. 5726148, for use in combination with one or more drugs selected from anti-inflammatory group, analgesic group or for protective cartilage of the group. Alternative offers local delivery fused protein consisting of a polypeptide comprising a binding domain of sIL-1R, for the applications in order to protect the cartilage, as disclosed in U.S. patent No. 5319071. In addition, local delivery of antagonist IL-1-receptor disclosed in U.S. patent No. 5817306 is disclosed for use in the present invention. It was shown that soluble receptor shuIL-1R binds to IL-1 with nanomolar affinity. Local delivery of therapeutically effective concentrations of soluble IL-1R receptor, such as shuIL-1R, can occur by direct injection into the joint or irrigating solution (for example, during arthroscopic surgical procedures in combination with one or more protective for cartilage drug, anti-inflammatory drugs or pain medication, and is revealed here as a cover for cartilage agent, the local application on the joint tissues in various inflammatory or pathophysiological conditions. Such processing will advance to inhibit stimulation under the influence of IL-1 production of collagenase-1 and stromelysin-1. Using an entirely different method, based on gene delivery for local production of soluble receptor type I IL-1 and/or TNF-α, it was found that the presence of soluble receptors for these cytokines are capable of providing protection of the knee in rabbits during the acute phase of inflammation induced by antigen arthritis.

Antagonists-pepti the s IL-1 receptor (11-15 amino acids), which specifically bind with high affinity to human IL-1 receptor type I is suitable for use in the present invention as the protective cartilage agents. Data in small peptides provide several advantages compared to larger recombinant soluble IL-1 receptors or recombinant IL-1ra, which include the ease and cost of synthesis and the ability to penetrate biological barriers. Two of the most powerful peptides, based on data on the effectiveness of in vitro are: AC-FEWTPGWYQJYALPL-NH2(AF12198, IC50=0.5 to 2 nm) and Ac-FEWTPGWYQJY-NH2(AF11567). AF11567 is a subset of the AF12198 loss 4 C-terminal residues and exhibiting lower affinity in relation to IL-1 receptor type I, but with similar half-life from plasma of 2.3 to 2.6 hours was that the poor solubility and rapid metabolism limits the effectiveness AF12198 in vivo with the systemic administration by intravenous infusion. These restrictions are partially overcome by using methods of direct local delivery, such as injection into the intra-articular space or inclusion in surgical irrigating fluid or other infusion, as described in this invention. Examples of antagonists agents IL-1 receptors that are suitable for the present invention, is shown below. For all methods, local delivery (i.e. injection, infusion or irrigation), the optimal dose and/or concentration of each candidate agent is such that a therapeutically effective. As an example, for each of the listed agents are preferred and the most preferred concentration irrigating solution containing included in the list of the agent, it is expected that these concentrations are therapeutically effective.

TABLE 18

Therapeutic and preferred concentrations of receptor antagonists of interleukin-1
ConnectionTherapeutic concentrations (nm)The most preferred concentration (nm)
rshuIL-lR0.2 to 2000200
rhIL-Ira0.2 to 2000200
anti-IL-l-antibodies0.2 to 2000200
AF121980.2 to 2000200
AF115670.2 to 2000200

2. RECEPTOR ANTAGONISTS of TUMOR NECROSIS FACTOR (TNF)

TNF-α, a cytokine mainly produced by activated macrophages, has many biological effects, including regulation of transcription was carried by the channels at genes which are mediated by specific TNF-receptors, as well as immunoregulatory activity. Were initially cloned and characterized two different receptors, designated TNF-R1 and TNF-R2, and also found that they are produced as soluble receptors.

Receptors in this family are single transmembrane proteins with significant homology in their extracellular domains at a time, as their relatively short intracellular domains have very low homology sequences. The effect of TNF is the result of binding of the factor to cell surface receptors that are found on almost all cell types that have been studied. Have been identified and cloned two of the receptor. One type of receptor, designated as TNFR-II (or type, or 75 kDa), encodes a transmembrane protein of 439 amino acids and has an apparent molecular mass of 75 kDa. The second type of receptors, designated TNFR-I (or type In, or 55 kDa), has an apparent molecular mass of 55 kDa and encodes a transmembrane protein of 426 amino acids. TNFR1 includes intracellular domain, which initiates the transmission of a signal along the path with the participation of NF-kB.

Both TNF-receptor show a high affinity for binding of TNF-α. Were allocated soluble TNF receptors (sTNFR), and it has been proven that they are about aboutsa allocation extracellular domains, membrane-bound receptors. It was identified two types of sTNFR and they were marked as sTNFRl (TNF BRI) and sTNFRII (TNF BPII). It was shown that both these forms of soluble receptors represent truncated forms two types of TNFR described above.

TNF-α plays a Central role in the sequence of cellular and molecular events underlying the inflammatory response and destruction of cartilage. Many of the effects of TNF-α overlap with proinflammatory effects of IL-1. Among the proinflammatory effects of TNF-α it is the stimulation of the release of other proinflammatory cytokines, including IL-1, IL-6 and IL-8. TNF-α also induces release of matrix metalloproteinases from neutrophils, fibroblasts and chondrocytes, which lead to the destruction of cartilage, partly as a result of stimulation of collagenase. In addition, TNF-α increases the activity of MOR-2 in normal human articular chondrocytes and synovial fibroblasts, leading to increased production of PGE2.

It is believed that this cytokine, along with IL-1, initiates and produces pathological effects on the cartilage in the joint, including infiltration by white blood cells, synovial hyperplasia, activation of synovial cells, the breakdown of cartilage and inhibition of the synthesis of cartilage matrix. In particular, during synovial inflammation installed elevated levels of TNF-#x003B1; in the synovial fluid of joints, and there is increased production of TNF-α synovial cells. Thus, local delivery of soluble TNF-α-receptor in irrigating fluid infusion or injection will lead to binding of free TNF-α and to function as an antagonist of TNF-receptors in the surrounding tissue, thus providing a protective effect against cartilage.

The present invention describes the use of functional antagonists of TNF-αthat act to block extracellular ligand interaction with their cognate membrane receptors or disposal of available free ligand or a direct competitive interaction with the receptor, single or in combination with other agents with provision for protective cartilage effect. You can use a variety of therapeutic approaches that counteract the interaction of the agonist, TNF-αwith its natural associated with a membrane receptor, which include: 1) the use of specific natural inhibitors of the activity of TNF-αthat characterized to date, including soluble TNF-αreceptors; 2) the use of anti-TNF-α antibodies and 3) the use of small molecule antagonists that can be peptides or dipeptide.

The present invention is revealing is that the use of chimeric soluble receptor (CSR) protein, in which the extracellular domain of the TNF-receptor, which has activity to bind the molecule TNF, covalently linked to the domain of the IgG molecule. In particular, in the first example, you can use the chimeric polypeptide (recombinant Chimera), including the extracellular domain of the TNF receptor extracellular peptide linked to the CH2 and CH3 regions of the heavy chain of murine IgGI, as disclosed in U.S. patent No. 5447851. It was shown that recombinant soluble TNF-receptor (also designated as "chimeric inhibitor of TNF in U.S. patent No. 5447851) binds to TNF-α with high affinity, and it was shown that it is highly active as an inhibitor of the biological activity of TNF-α. In addition, the second example is a chimeric fused structure, which includes a ligand-binding domain of the TNF-receptor with portions of the Fc antibody (labeled fused soluble Fc receptors), which were obtained for TNF-α-receptors. The present invention also discloses the use of soluble TNF receptor: Fc merged-protein or any modified forms, as disclosed in U.S. patent No. 5605690. The molecular form of active soluble receptor fused protein can be either Monomeric or dimeric. There are studies in which it was found that soluble TNF receptor: Fc merged-protein retains high binding activity is for TNF-α .

In the context of determining the soluble receptors as pharmacological antagonists of the term soluble receptors include, but are not limited to: 1) soluble receptors, which correspond to the natural (endogenous) educated amino acid sequences or their soluble fragments comprising the extracellular domain of the full length membrane receptor; 2) recombinant soluble receptors, which are truncated or incomplete sequences from full-length natural amino acid sequences of receptors that retain the ability to bind to a cognate ligand and retain biological activity and their analogs; and (3) chimeric soluble receptors, which are recombinant soluble receptors, including truncated or incomplete sequence corresponding section of the extracellular binding domain of the full length amino acid sequences of receptors that are related through the oligomers (e.g., amino acids), with the sequence corresponding to the site of IgG-polypeptide (for example, loop IgG and Fc-domain)that retain biological activity and the ability to bind to a cognate ligand.

Soluble extracellular ligand binding domains of cytokine receptors imets is in body fluids, and I believe that they are involved in the regulation of the biological activity of cytokines. It was reported the presence of natural soluble truncated forms of a number of hematopoietic cytokine receptors (IL-1R, IL-4R, IL-6R, TNFR). For example, soluble TNFR detected at concentrations of 1-2 ng/ml in the serum and urine of healthy subjects. Losing the function of signal transmission, data cytokine binding proteins arise from alternative splicing of mRNA for full receptor sequence (associated with the membrane form) or as a result of proteolytic cleavage and release associated with the membrane form of the receptor. Although the functions of these soluble truncated receptors in vivo is not yet fully established, it appears that they act as a physiological antagonist of their complementary endogenous cytokines. This antagonism is due to the fact that 1) the disposal of the free ligand by binding to its cognate soluble receptor reduces effective free concentration available for membrane-bound receptors and 2) the effect of cytokines occurs only after subsequent binding to receptors on the cell surface.

Soluble TNF-α-receptor to function as a natural antagonist of TNF-R1 and TNF-R2, competing with on nami receptors on cell surfaces for total pool of free ligands. Pharmacologically soluble TNF-receptor to function as an antagonist to a greater extent due to its ability to reduce the bioavailability of free ligands than the mechanism of competitive inhibition (i.e. competing with the endogenous ligands for the overall place of binding to the membrane receptor). The introduction of a therapeutically effective amount of a soluble TNF-receptor in the joint will effectively neutralize the biological activity of the ligand. Experiments in which recombinant soluble receptors were injected in vivo, have shown their ability to inhibit the inflammatory response and act as antagonists.

In this invention, the agents which are suitable as protective for cartilage agents for use in combination with other protective for cartilage, analgesic and/or anti-inflammatory agents to suppress the destruction of the cartilage, include soluble TNFR, human chimeric polypeptide (recombinant Chimera), including the extracellular domain of TNF-α-receptor (P80)associated with Fc-plot of human IgGI, and anti-TNF-α antibodies. For all methods, local delivery (i.e. injection, infusion, and irrigation), the optimal dose and/or concentration of each candidate agent is such that a therapeutically effective. As an example, for each read is certain agents are preferred and the most preferred concentration of irrigating solution, containing included in the list of the agent, it is expected that these concentrations are therapeutically effective.

TABLE 19

Therapeutic and preferred concentrations of antagonists of TNF-receptors
ConnectionTherapeutic concentrations (nm)The most preferred concentration (nm)
sTNFR0,1-2000200
chimeric rhTNFR:Fc0,1-2000200
anti-TNF-α antibodies0.2 to 2000200

3. AGONISTS INTERLACING RECEPTORS

Some cytokines are signaling glycoproteins, which are important mediators of synovial inflammation and destruction of cartilage. Based on recent analysis of the mechanism of destruction of cartilage can be assumed that in the definition of the cartilage destruction is important, not only the absolute level of the main proinflammatory cytokine, IL-1, but also that the regulation of cytokine homeostasis in cartilage is determined by the balance of catabolic and anabolic regulatory cytokines and factors anabolic growth. If the balance between the production of IL-1β and IL-1Rα changes in inflammation in favor of IL-1βthen he sposobstvuyuthie chronic inflammatory conditions and destruction of cartilage, which, as is known, occurs after surgery on the knee joint. Potential therapeutic agents that will inhibit the production of proinflammatory cytokines in areas of inflammation inside the joint, include anti-inflammatory cytokines, IL-4, IL-10 and IL-13. Observed that these cytokines significantly reduce the degradation of articular cartilage in vitro and in vivo through their actions in a number of ways that reduce the level of giving impetus IL-1. Thus, anti-inflammatory cytokines, such as IL-4, IL-10 and IL-13, can be appropriate to suppress inflammation through: 1) reducing the production of proinflammatory cytokines and 2) the induction of the production of natural anti-inflammatory cytokines, such as IL-IRa, as was recently shown in vivo for IL-4.

It appeared that IL-4 attenuates the inflammatory process in the synovium of patients with rheumatoid arthritis (RA). It was shown that IL-4 in the synovium in rheumatoid arthritis inhibits the production of proinflammatory cytokines pieces of the synovium, with inhibition of cell proliferation synoviocytes and decrease bone resorption. IL-4 may have a direct protective for cartilage effect through suppression of the synthesis of matrix metalloproteinase-3 (MMP-3) in human articular chondrocytes. Was used for cell culture of human articular chondrocytes for assessment of the actions of IL-4-induced IL-1 production of MMP-3 and tissue inhibitor of metalloproteinases-1 (TIMP-1). It was found that IL-4 suppresses IL-1-stimulated MMP-3 protein and its enzymatic activity. In addition, IL-4 suppresses induced IL-1 mRNA of MMP-3. Induction of iNOS can also be inhibited IL-4, IL-10 and IL-13. Thus, IL-4 can be characterized as a protective mediator in the process of joint destruction found in inflammatory diseases of the joints.

In addition, it was found that the effects of IL-4 to the balance of the levels of regulatory IL-1 cytokines contribute to its protective role in the relationship of cartilage. It is established that IL-4 and IL-10 inhibited the production of inflammatory cytokines freshly rheumatoid synovial cells. Despite the fact that every interleukin effective individually, the combination of IL-4 and IL-10 synergistically inhibited stimulated IL-1 and TNF-α, the production of IL-6 and IL-8, without effects on cell viability. The addition of IL-4 to cultures of the synovium in RA increases the production of IL-1Ra and reduces that of IL-1β. Recently it was reported that the processing of IL-4 in vivo contributes to the suppression of experimental arthritis in rats differentially acting on the balance of IL-1β/IL-1Ra. IL-13, another cytokine that shares many properties with IL-4 also induces IL-1Ra in the synovium in RA. Therefore, local delivery of a combination of IL-4 and IL-13 can provide synergistic terapeutiche the cue effect.

IL-10 has a number of properties that indicate that it is a good potential candidate to suppress the degradation of cartilage. It inhibits the release of both IL-1 and TNF-αand stimulates the production of TIMP-1, at the same time inhibiting MMP-2. It was recently reported the production of IL-10 within the synovium in RA, and were evaluated anti-inflammatory effects of IL-10. IL-10 suppresses the production of IL-1β on the model of RA ex vivo using pieces of synovium, but to a lesser extent than IL-4.

Installed protective effect of treatment with IL-4 and IL-10 in respect of the cartilage destruction in models of arthritis in animals using non-local delivery of cytokines. On the model of collagen-induced arthritis in mice treatment with a combination of IL-4 and IL-10 led to a significant improvement. In addition to the suppression of macroscopic signs of inflammation, combined treatment of IL-4 and IL-10 were also reduced cellular infiltration in the synovial tissue and caused a pronounced protection against destruction of cartilage. In addition, the levels of mRNA for TNF-αand IL-1 were significantly reduced in the synovial tissue and articular cartilage. In contrast, the mRNA levels of antagonist IL-1 receptor (IL-IRa) remained elevated, whereby it is assumed that the protection mechanism can be associated with inhibited production of TNF-α and IL-1, along with a side the respective increase of the balance of IL-1Ra/IL-1. These results are consistent with a dominant role of IL-10 in suppression of endogenous inflammatory response and destruction of the articular cartilage, and it turns out that the combined treatment of IL-4 and IL-10 has the potential therapeutic value.

Also we investigated the role of endogenous IL-4 and IL-10 and therapeutic effect of the addition of these cytokines in joint inflammation and destruction of cartilage in the early stages of macrophage model of streptococcal arthritis in mice. It was shown that endogenous IL-10 plays a role in the regulation of SCW arthritis. Adding endogenous IL-10 was enhanced suppressive effect of endogenous IL-10. Even more pronounced effect was found with the combination of IL-4 and IL-10. The combination has led to reduced swelling and increased synthesis of proteoglycan in chondrocytes. Processing the combination of IL-4 and IL-10 significantly reduced the levels of TNF-αhow it takes place while processing one IL-10, but also resulted in significantly reduced levels of IL-1β in the synovium, which is the additional effect of potential benefit to the clinic. In General, the data are consistent with a role as IL-4 and IL-10 as a cover for cartilage agents delivered locally in the joints to prevent the destruction of cartilage, and indicates that the combination containing IL-4 and IL-10 may provide greater potential therapeutic effect, che is any one agent.

Mice with severe combined immunodeficiency was used as a model to assess the effect of injection of IL-4 or IL-10 on the degradation of cartilage or recovery of mononuclear cells in human synovium in rheumatoid arthritis in vivo. In human rheumatoid synovium and cartilage from five patients with rheumatoid arthritis were injected with recombinant human IL-4 (rhIL-4, 100 ng; rhIL-10, 100 ng), a combination of IL-4 and IL-10 or TNF-alpha (1000 F), or phosphate buffered saline twice a week for 4 weeks. It was found that the combination of human IL-4 and IL-10 inhibited the resolution of cartilage and invasion of human synovial tissue, which indicates the protective cartilage properties data agonists interleukins.

Human IL-13 was cloned and sequenced, and found that it shares many properties with IL-4. IL-13 by about 25% homologous to IL-4. Like IL-4, IL-13 reduces the production of proinflammatory cytokines, including IL-1 and TNF-α, mononuclear cells and synovial fluid. IL-13 exerts anti-inflammatory effects in vivo and, thus, has therapeutic potential in the treatment of the destruction of cartilage in the joint.

Suitable as agonists of IL-4, IL-10 and IL-13 compounds include natural human IL-4, IL-10 and IL-13, human recombinant IL-4 (rhIL-4), rhIL-10 and rhIL-13, and they are not olnie sequence or peptide sequence, which were constructed using methods of recombinant DNA "recognition" of IL-4-, IL-10 and IL-13 receptors, and which are able to activate these receptors on the cell surface. They, in particular, include multispecificity molecules containing anti-Fc-receptor site and aHTH-IL-4, anti-IL-10 and aHTH-IL-13 receptor site, where at least one plot constructed using methods of recombinant DNA. In the context of determining agonists interleukins as pharmacological agonists term agonist interleukins include, but are not limited to: 1) peptide sequences that correspond to the natural (endogenous) educated amino acid sequences or their fragments; 2) recombinant interleukins, which are truncated or incomplete sequences of the full length of the natural amino acid sequence of interleukins, which retain the ability to bind to a cognate receptor and retain biological activity and their analogues and 3) a chimeric interleukins, which are recombinant polypeptides consisting of truncated or incomplete sequences corresponding to the section from the full length amino acid sequence, linked via oligomers (e.g., amino acids), after which outlineto, the relevant section of IgG-polypeptide (for example, loop IgG and Fc-domain), which retain the ability to bind to a cognate receptor and retain biological activity.

Examples of agonists of interleukins that are suitable for the present invention below. For all methods, local delivery (i.e. injection, infusion, and irrigation), the optimal dose and/or concentration of each candidate agent is such that a therapeutically effective. As an example, for each of the listed agents are preferred and the most preferred concentration irrigating solution containing included in the list of the agent, it is expected that these concentrations are therapeutically effective.

TABLE 20

Therapeutic and preferred concentrations of agonists interleukins
ConnectionTherapeutic concentrations (nanomolar)The preferred concentration (nanomolar)
Ruman IL-40.5 to 5,0005-500
Ruman IL-100.5 to 5,0005-500
Ruman IL-130.5 to 5,0005-500

4. AGONISTS of the SUBFAMILY of the TRANSFORMING GROWTH FACTOR is-β

Members of the subfamily of the transforming growth factor-β (TGF-β) are pleiotropic multifunctional protein that has the ability to influence a variety of cellular functions, and it is known that they are involved in the restoration and reconstruction of tissues. In many cases, they enhances interaction of cells with extracellular matrix (ECM) and increase the accumulation of ECM stimulation of production and secretion of ECM proteins and protease inhibitors. It was also shown that TGF-β has synergy with other cytokines, mainly exhibiting anti-inflammatory activity. Identified many isoforms of TGF-βwho share close homology of amino acid sequences. TGF-β1, TGF-β2 and TGF-β3 were detected in human tissue, and they are active in mammalian cells, although differ in the affinity of binding.

Members of the subfamily of TGF-β are strong modulators of cell proliferation, differentiation of chondrocytes and accumulation of extracellular matrix. In organ cultures of cartilage TGF-β1 regulates the metabolism of proteoglycans and stimulates the synthesis of glycogen and fractions of rabbit articular chondrocytes. In addition, TGF-β1 increases the expression of TIMP in human articular chondrocytes and reduce the expression of IL-1 receptors in articular is Rasa.

Bone morphogenetic proteins (BMPs) are multifunctional regulators of growth, differentiation and apoptosis of cells, which belong to the superfamily of transforming growth factor-β (TGF-β). In mammals was identified more than a dozen members of the BMP protein family, which can be divided into several groups depending on their structure. BMP-2 and BMP-4 are very similar to each other. BMP-5, BMP-6, osteogenic protein (OP)-1 (also designated BMP-7) and ER-2/BMP-8 structurally close to each other. Growth factor-differentiation (GDF)-5 (also denoted morphogenetic protein-1, derived cartilage), GDF-6 (also designated morphogenetic derived cartilage protein-2) and GDF-7 form another similar group. In contrast, BMP-2, BMP-4, BMP-6 and OP-1/BMP-7, which induce the formation of bone and cartilage in vivo, GDF-5, GDF-6 and GDF-7 more effectively induce the formation of cartilage and similar tendon structures in vivo (film the wolfman year et al., 1997).

Members of the superfamily of TGF-β exert their effects through binding to two types of receptor serine/trainingin, both of which are important in the transmission signal (Massague, 1998). Receptor type II are constitutively active kinases, which Transfrigoroute receptor type I in the binding of the ligand. Receptor type I activate intracellular substrates such as the Smad tree, and it is believed that through this mechanism is shown the specificity of intracellular signal transduction. In mammals has been allocated seven different receptor type I, which were originally called activin-rizatriptan kinase (ALK)-1-ALK-7. BMP receptor type IA (BMPR-IA or ALK-3) and BMP receptor type IB (BMPR-IB or ALK-6) is structurally similar to each other and specifically bind BMPs along with receptor type II. It was shown that ALK-2 binds activin, but recent data have shown that this receptor type I for some BMPs, for example, OP-1/BMP-7 (Macias-Silva et al., 1998). ALK-1 is structurally very similar to ALK-2, but its physiological ligand is still unknown. ALK-5 and ALK-4 receptors are type I, respectively, for TGF-β TβR-1) and activin (ActR-IB). ALK-7 is structurally similar to ALK-4 and ALK-5, but its ligand has not yet been installed.

Natural agonists of TGF-β and BMP, as well as synthetic or recombinant human (rh) agonists suitable for use in the protective cartilage of the solution according to the present invention, can interact with any of the BMP receptors described above. In the same sense as it is used here, the term "agonists of TGF-β and BMP" includes fragments, deletions, insertions, substitutions of amino acids, mutations and modifications which retain the biological properties of natural human ligands agonists TGF-β and BMP. Agonists of TGF-#x003B2; or BMP may be used singly or in synergistic combinations with other members of the superfamily of TGF-β as anabolic for cartilage agents (chondrogenic or contributing to the restoration of cartilage matrix) or in combination with any abscopal agents that block the catabolism of cartilage.

Receptor type I function as a negative regulatory components for receptor type II. The specificity of intracellular signaling receptor type I is determined by the specific area in the domain serine/trionychinae called loop L45. Thus, the structure of the L45 loop in BMPR-IA/ALK-3 and BMPR-IB/ALK-6 (BMPR-I-group) identical to each other, they can transmit the same signals in cells. Similarly L45 loop in Tβ3R-I/ALK-5, ActR-IB/ALK-4 and ALK-7 (T(3R-1-group) identical to each other, and they activate the same substrates (Chen et al., 1998). Loop L45 at ALK-1 and ALK-2 (ALK-1-group) is most different from the other receptor type I, but they activate the substrates similar to those for receptors type I BMPR-I group (Armes et al., 1999).

Different proteins can transmit signals from TGF-βand BMP-receptor-series/trainingin. Among them, the most studied molecules are proteins of the Smad family. In mammals were identified eight different Smad proteins, and these proteins are divided into three subgroups, i.e. adjustable prescriptions what PR Smads (R-Smads), regular partner (Co-Smads) and inhibiting Smads. R-Smads are directly activated receptors type I, resulting in the formation of complexes with Co-Smads, and moved into the kernel. Smad-heteromera contact DNA directly or indirectly through other DNA-binding proteins and thus regulate the transcription of target genes. Smad1, Smad5 and Smad8 are activated by BMPs, while Smad2 and Smad3 are activated by TGF-β. For example, Smad2 in combination with Smad4, which functions as a Co-Smad, moves into the nucleus, where it activates the transcription of genes that mediate the biological effects of TGF. Smad6 and Smad7 structurally different from other Smads and act as inhibitory Smads. It has been shown that BMPs induce new formation of cartilage and bone in vitro and in vivo and regulate the growth and differentiation of chondrocytes. In addition, these proteins are also involved in the process of cartilage restoration. In various studies it has been shown that BMPs also promote and preserve chondrogenic phenotype that indicates their ability to stimulate the synthesis of proteoglycan in cell cultures of avian embryonic limbs and embryonic rat chondroblast, as well as in rabbit and bovine articular chondrocytes. The value of BMPs for the formation of cartilage and bone has been proven transgenic approach in which studied throwing peculiar the x BMP genes.

It turned out that one member of the BMP family, osteogenic protein (OP-1 or BMP-7) is particularly important for maintaining homeostasis in the cartilage in normal and pathological conditions, as well as during recovery of the cartilage. It turned out that the PR-1 is the only member of the BMP family along with morphogenetic proteins, derived from cartilage, which is expressed in Mature articular chondrocytes (Chubinskaya, S., J. Histochemistry and Cytochemistry 48:239-50 (2000)). EO-1 was originally isolated from bone matrix, and it has been shown that it induces the formation of cartilage and bone. The gene of human OP-1 was cloned, and were obtained biologically active recombinant homodimeric EO-1. Recombinant human OP-1 can stimulate the synthesis of aggrecan and collagen type II in human articular chondrocytes in vitro. It can also counteract the negative effects of IL-1 on the metabolism of these chondrocytes and block damage bovine cartilage, mediated by fragments of fibronectin. This effect was shown when studying the effect of recombinant human OP-1 on the production of proteoglycan, prostaglandin E2 and antagonist of IL-1 receptors in human articular chondrocytes, cultured in the presence of interleukin-beta. Treatment of human articular chondrocytes EO-1 was effective in overcoming the reduced synthesis about eagleman, induced with low doses of IL-1β. In addition, the study found that the OP-1 stimulates the synthesis of hyaluronan and CD44, and other molecules needed to build the matrix in human chondrocytes. Based on these studies on the expression and regulation of PR-1 in human adult cartilage is possible to show an assumption about the role of PR-1 in the protection and restoration of cartilage, and they indicate that the EO-1 can be used as a therapeutic agent that promotes cartilage anabolism and recovery of human articular cartilage.

The OP-1 (BMP-7) induces the formation of cartilage and bone when implanted in locations inside and outside of the skeleton in vivo. Investigated the effect of OP-1 on healing of defects of articular cartilage full-thickness by drilling two adjacent holes through the articular cartilage in the knee joint of rabbits. PR-1 was induced healing of articular cartilage and the regeneration of the surface of the joint, which consisted of cells resembling Mature articular chondrocytes.

Based on these data, we can assume that one preferred embodiment of the solution suitable for the practice of the present invention in order to prevent the destruction of cartilage and maintaining biological homeostasis in articular cartilage in humans after surgical trauma may include topical application of a member in the family of TGF-β preferably TGFβ2, BMP-7 (OP-1 or BMP-2, or equivalent agonist that acts through the same receptors used by these ligands. Local delivery may take place in combination with other medicines, or drugs that are inhibitors of catabolic processes in the cartilage (such as inhibitors of Martinez, MMP-inhibitors or inhibitors of nitric oxide synthase), and/or other analgesic or anti-inflammatory agents.

In the context of determining agonists TGF-β and BMP as pharmacological agents, the term agonist TGF-β and BMP includes, but is not limited to: (1) peptide sequences that correspond to the natural (endogenous) educated amino acid sequences or their fragments, (2) recombinant TGF-βs and BMPs that are truncated or incomplete sequences of the full length of the natural amino acid sequence of TGF-β and BMP, which retain the ability to bind to their cognate typical receptor and retain biological activity and their analogues, and (3) chimeric TGF-βs and BMPs, which are recombinant polypeptides, including truncated or incomplete sequence corresponding to the area of the full length amino acid sequence, the connection is built through the oligomers (for example, amino acids) with the sequence corresponding to the site of IgG-polypeptide (for example, loop IgG and Fc-domain), which retain the ability to bind to a cognate receptor and retain biological activity.

Examples of agonists of TGF-β and BMP, suitable for the present invention are listed below. For all methods, local delivery (i.e. injection, infusion, and irrigation), the optimal dose and/or concentration of each candidate agent is such that a therapeutically effective. As an example, for each of the listed agents are preferred and the most preferred concentration irrigating solution containing included in the list of the agent, it is expected that these concentrations are therapeutically effective.

The limits of therapeutic concentrations for delivery to the surgical solution into the joint can be set according to the values of the dissociation constants (Kd) of each of the ligands to their cognate receptor. Despite the fact that these values will vary for specific cell types and tissues, provides the following example for BMP-4. In the experiments on the evaluation of the binding125I-BMP-4 was established the presence of specific, high affinity binding sites with an apparent dissociation constant of 110 PM and about 6000 receptors on the cell. Therefore, p and 11 nm BMP-4 binding ligand is maximum, and the available receptors are fully occupied (filled). It was shown the presence of functional receptors for BMP-4 in primary articular chondrocytes.

TABLE 21

Therapeutic and preferred concentrations of agonists TGF-β and BMP receptors
ConnectionTherapeutic concentrations (nanomolar)The most preferred concentration (nanomolar)
TGF-β1of 0.05-5000.5 to 100
TGF-β2of 0.05-5000.5 to 100
BMP-20,1-20001-200
BMP-40,1-20001-200
BMP-7 (OP-1)0,1-20001-200

5. INHIBITORS of CYCLOOXYGENASE-2 (SOH-2)

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used as anti-inflammatory drugs, but they were not specifically designed or used therapeutically as a cover for cartilage agents. Direct molecular target for NSAID is the first enzyme in the synthesis of prostaglandins, related either to endoperoxide-prostaglandin synthase or cyclooxygenase fatty acids. Characterized by two relatives of formallooking, called cyclooxygenase-1 or (MOR-1) type I and cyclooxygenase-2 (SOH-2). These isozyme also known as prostaglandin G/H synthase (PGHS)-1 and PGHS-2. Both enzymes catalyze limiting the speed stage of the formation of prostanoids, which is the conversion of arachidonic acid to prostaglandin H2. MOR-1 is present in platelets and endothelial cells and exhibits constitutive activity. In contrast SOH-2 was detected in endothelial cells, macrophages, fibroblasts and other cells in the joint, and its expression is induced by proinflammatory cytokines such as IL-1 and TNF-α.

In the inflamed joint expression of MOR-2 is increased, and it was shown that a significant increase in activity SOH-2 occurs simultaneously with its positive regulation, leading to increased synthesis of prostaglandins, which are found in the synovial fluid of patients with inflammatory arthropathy. Cellular sources of prostaglandins (PGs) in the joint include activated chondrocytes, synoviocytes type a and b and macrophages infiltration. Cellular functions of importance for the metabolism of cartilage, moduliruetsya under the influence of PGs include gene expression, synthesis of extracellular matrix and proliferation. Because SOH-2 is expressed in the tissue of inflamed joints near or after the effects of inflammatory mediators (for example, as a result of injury or surgical trauma), it is assumed that the inhibitor SOH-2 will provide both anti-inflammatory and protective for cartilage action.

Destruction of cartilage in inflammatory arthropathy can "run" as a consequence of joint damage and result in arthroscopic surgical procedures. Chondrocytes are the only cell type in the articular cartilage, and it is known that they are involved in the collapse of their own matrix through the release of endogenous mediators of inflammation, including PGs. Studies have shown that gene expression of MOR-2, protein synthesis and release of PG in normal human articular chondrocytes rapidly induced under the action of cytokines, including IL-1, TNF-α and IL-6. Levels of mRNA are detected already after 2 h after induction by cytokines, achieving high levels after 6 h, and has a long duration of expression at least within 72 hours Similarly in studies with cell cultures by activation of the human synoviocytes under the action of IL-1α and TNF-α it has been shown that there is a significant increase in expression of MOR-2 and production of prostaglandin E2 (PGE2). Treatment of various NSAIDs such as Ketoprofen, eliminates induced RSE response. In cell culture chondrocytes specifications the ical inhibitor SOH-2, connection NS-398, prevented the increase in PGE2 production induced by cytokines, whereas the levels of MOR-1 remained stable (Morisset, S., 1998, J. Rheumatol. 25:1146-53). Thus, it can be concluded that blocking the production of PG activated chondrocytes, which is associated with the expression of MOR-2 may provide for protective cartilage effect.

Usually NSAIDs are used to treat patients with osteoarthritis or rheumatoid arthritis, but their effect on the metabolism of articular cartilage in the context of data arthritic diseases remains a subject for discussion. For example, the treatment of osteoarthritis and rheumatoid arthritis with NSAIDs is successful in suppressing inflammation. However, it is believed that some NSAIDs that are selective for MOR-2, mainly salicylates and indomethacin, accelerate the degradation of cartilage in osteoarthritis, disrupting the synthesis of proteoglycan in chondrocytes, while it is believed that other NSAIDs have some protective for cartilage effect, stimulating the recovery of cartilage. Many studies have shown that NSAIDs have a small effect or no effect on the cartilage. Due to the fact that this group of drugs is not used currently in the treatment of synovitis and destruction of cartilage after traumatic injury of the joint surgeons who eskay injury, you must install the unique properties of each NSAID effect on the pathophysiological mechanisms that are involved in the degradation of cartilage.

Because the two isozyme SOH are pharmacologically distinct, were developed specific regarding isozyme (selective) inhibitors of cyclooxygenase, which are suitable for the treatment of inflammation, and some of these inhibitors SOH-2 were tested in models of inflammation of the joints. However, the effects of inhibitors SOH-2 in vitro synthesis and degradation of cartilage proteoglycans, as well as the production of IL-1, IL-6 and IL-8 and prostanoids in synovial fluid indicates that some NSAIDs can greatly vary in their effects on cartilage in vivo and products in synovial fluid interleukin and eicosanoids, so that the integrated effects of these parameters can influence the output of inhibitors SOH-2 on the integrity of the cartilage. For example, some NSAIDs may accelerate joint damage in osteoarthritis by increased production of proinflammatory cytokines or the inhibition of the synthesis of proteoglycans. However, despite possible differences in clinical effect among specific inhibitors SOH-2, usually inhibition of MOR-2 leads to the suppression of synovitis and intended to reduce the risk of damage to the cartilage.

Were developed the s various biochemical, cellular tests on animals to evaluate the relative selectivity of inhibitors isoforms of MOR-1 and MOR-2. Basically, the criterion for determining the selectivity is the ratio of the inhibition constant Ki SOH-1/MOR-2 MOR-2/SOH-1)obtained in this biochemical or cellular test. The selectivity ratio is calculated for different absolute values of the IC50in relation to the inhibition of the enzymatic activity obtained in microsomal and cellular tests (for example, cell lines platelets and macrophages stably expressing recombinant human isozyme SOH). In addition, inhibition of MOR-2 resembles the inhibiting effects, "running" for protective cartilage (any abscopal) cytokines, such as IL-4 that reduce the intracellular synthesis of MOR-2. Comparison of the selectivity of more than 45 NSAIDs and selective inhibitors MOR-2 (1997, Can. J. Physiol. Pharmacol. 75:1088-95) showed the following sequence of relative selectivity for the MOR-2 compared to COX-1: Dup 697 > SC-58451 = celecoxib > nimesulide = meloxicam = piroxicam = NS-398 = RS 57067 > SC-57666 > SC-58125 > flosulide > etodolac > L-745337 > DFU-T-614, when values of the IC50between 7 nm and 17 microns.

On the basis of molecular and cellular mechanism of action set for selective inhibitors SOH-2 such as celecoxib and oteracil, the results of animal experiments, it is assumed that these compounds are protective for cartilage effect when used perioperative in the irrigating solution and when injected directly into the joint. In particular, it is assumed that inhibitors SOH-2 will be effective drug delivery in the irrigating solution during arthroscopic surgical procedures or by direct injection into the joint before, during or after a surgical procedure or other damage to the joint.

Examples of inhibitors SOH-2, suitable for the present invention are listed below. For all methods, local delivery (i.e. injection, infusion, and irrigation), the optimal dose and/or concentration of each candidate agent is such that a therapeutically effective. As an example, for each of the listed agents are preferred and the most preferred concentration irrigating solution containing included in the list of the agent, it is expected that these concentrations are therapeutically effective.

TABLE 22

Therapeutic and preferred concentrations of inhibitors of cyclooxygenase-2
ConnectionTherapeutic concentrations (nm) The most preferred concentration (nm)
rofecoksib (MK 966)0,3-30,00030-3,000
SC-584510,3-30,00030-3,000
celecoxib (SC-58125)0,3-30,00030-3,000
meloxicam0.5 to 50,00050-5,000
nimesulide0.5 to 50,00050-5,000
diclofenac0,3-30,00030-3,000
NS-3980,3-30,00030-3,000
L-745,3370,2-100,00020-10,000
RS570670,2-100,00020-10,000
SC-576660,2-100,00020-10,000
flosulide0,2-100,00020-10,000

6. INHIBITORS of Martinez

Mitogen-activated protein (MAP) kinase are a group of serine/treoninove protein kinases, which are activated in response to various extracellular signals, and function in signal transduction from cell surface to the nucleus. The cascade Martinez is one of the main intracellular pathways of signal transmission, which transmits signals from growth factors, hormones and inflammatory cytokines intermediate early genes. In combination with other transmission signals given what's active 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. For example, a member of the family of MARK (R) mediates the major biochemical pathway of signal transmission from a strong anti-inflammatory cytokines, IL-1 and TNF-α, leading to the induction of cyclooxygenase-2 (SOH-2) - stimulated macrophages through CIS-acting factors involved in the regulation of transcription of the SOH-2.

Members of the group of agents Markins include at least three families, which are known to differ in the sequence, the size of the activation loop, the activation of extracellular signals and participation in various signal transduction pathways. Known members of this family Markins include adjustable extracellular signal kinase (ERKs), ERK1 and ERK2 (RMR and RMR, respectively); the family of stress-activated protein kinases (SAPK1), which also belong to the family of JNK or N-terminal family kinases; and the family R-Markines, which is also known as stress-activated kinase 2/3 (SAPK-2/3). R-kinases are activated under the influence of stress most of proinflammatory cytokines. Within the family R there are at least four different homologue (isotypes or isoenzymes), whose standard nomenclature refers respectively to S2, SAPK2b, SAPK2d, SAPK3 or R α that β, δ (SAPK4) and γ. Inhibitors of Markins suitable for practice of the present invention can communicate with any one or a combination of the above Markins. For specific inhibitors of Markines, the inhibition constants defined in tests in vitro on purified enzymes and cellular tests can vary in a wide range of concentrations and show the applicability in this application. Activation R-Martinez is mediated by dual phosphorylation of threonine residues and tyrosine. It was shown that treatment of cells with both TNF-α and IL-1 increase rapidly (within 5 min) phosphorylation and activates R-Markansu.

In previous work it was shown that small molecule inhibitors can specific inhibition R-Markansu (J. Lee et al., Nature 372: 739-746 (1994)) and show anti-inflammatory action at the biochemical level and in various animal models. Cuenda and co-authors (Cuenda A. et al., FEBS Lett. 364: 229 (1995)) showed that the compound SB203580 [4-(4-forfinal)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole] inhibited R in vitro (IC50=0.6 μm), inhibited the activation MARK-activating protein kinase-2 and prevented the phosphorylation of heat-shock proteins in response to exposure to IL-1 and cellular stress in vivo. It was shown that the selectivity of the inhibitory effect of SB203580 in respect of kinases for R in his nespoli the ability to inhibit or at best weakly to inhibit at least 15 protein kinases in vitro, including family members of the RCC, PKA, src and receptor tyrosinekinase (Lee, J. Pharmacol. Ther. 82: 389-397 (1999)). In studies on cells, it was shown that the incubation SB203580 blocks induced by IL-1 and TNF-α phosphorylation of the enzyme and the subsequent production of IL-8. This provides the basis for proactive delivery action of inhibitors during the surgical procedure.

Investigated the role of mitogen-activated protein kinase R (MARK) in biochemical inflammatory response reactions during the degradation of cartilage using SB203580, which specifically inhibits the enzyme. The effects of IL-1, which selectively adjustable R MARK, is in the regulation of prostaglandin H synthase-2 (MOR-2), metalloproteinases and IL-6 (Ridley, A. et al., 1997, J. Immunol. 158: 3165-73). In human fibroblasts and vascular endothelial cells SB203580 inhibited (IC50=0.5 µm) induced IL-1 by phosphorylation of hsp 27 (activity indicator R MARK) in fibroblasts without affecting other well-known involving the activated IL-1 protein kinases (R42 cable line/R MARK, R MAPK/c-Jun N-terminal kinase). In addition, SB203580 significantly inhibited IL-6-stimulated IL-1 (30-50% at 1 μm), but not the production of IL-8 in human fibroblasts and endothelial cells.

It is important that SB203580 significantly inhibited the production of prostaglandin-stimulated IL-1 in fibroblasts and human endotheli the selected cells. This is due to the inhibition of the induction of protein and mRNA SOH-2. PGE2 is involved in the increased expression of matrix metalloproteinases, which are important mediators of chondrolysis. Synovial fibroblasts and chondrocytes Express a gene SOH-2 at high levels when activated by cytokines and extracellular signals. Inhibitors MARK to provide a protective cartilage activity through inhibitory activity on Markansu expressed in these and other cell types.

It is assumed that inhibitors MARK will be effective as protective agents for cartilage, when applied topically to the joint tissues in various inflammatory and pathophysiological conditions. SB203580 has been studied in several pharmacological models in vivo, and has been shown to have activity with long-term oral administration. It was found that SB 203580 inhibits the stimulation of collagenase-1 and stromelysin-1 under the action of IL-1, without affecting the synthesis of TIMP-1. In addition, SB 203580 warned increase mRNA stimulated IL-1 collagenase-1 and stromelysin-1. The destruction of cartilage SB 203580 had no effect on stimulated IL-1, short of proteoglycan resorption and inhibits synthesis of proteoglycan, while were warned long collapse of collagen. In addition, it was found that SB 203580 is the tsya effective in the inhibition induced IL-1 production of nitric oxide in bovine explants of articular cartilage and chondrocytes (Badger 1998). These observations in vitro provide a basis for protective activity against cartilage of inhibitor Markins entered directly and locally in the tissues of the joint.

R-Marginata involved in induced TNF expression of cytokines, and drugs that act as inhibitors of the activity R-Martinez blocking the production of proinflammatory cytokines (Beyaert R. et al., EMBO J. 15: 1914-23 (1996)). The cell treatment with TNF-α activated path involving R-MARK, which was shown by increased phosphorylation of R-MARK and activation of its protein substrates. Pre-treating the cells with SB203580 completely blocked induced TNF-α activation MARK-activating protein kinase-2 and phosphorylation of hsp27. In the same conditions SB203580 also completely inhibited induced TNF-α the synthesis of IL-6 and expression of reporter gene, which is controlled by a minimal promoter containing two elements NF-6B. Thus, survey data and related studies on other inhibitors R it was shown that the effect of inhibitors such as SB 203580 and FR 133605 on R-MARK, selectively inhibits induced TNF-α and IL-1 activation of various proteins associated with the destruction of cartilage. Thus, selective inhibition of transmission of signals involving Martinez data key Provos Alitalia cytokines by inhibiting negative regulation of receptor points, the inhibitors of Martinez can provide for protective cartilage effect.

SB203580 was evaluated in several animal models of inhibition of cytokines and inflammatory diseases. He proved himself as a strong inhibitor of the production of cytokines of inflammation in vivo, as mice and rats with values IC50equal to 15-25 mg/kg of SB 203580 had therapeutic activity when induced by collagen arthritis in mice DBA/LACJ dose of 50 mg/kg, resulting in a significant inhibition of inflammation of the paws. Antiaritmicescuu activity was also observed when induced adjuvant arthritis in Lewis rats by oral administration of SB 203580 at doses of 30 and 60 mg/kg/day. Received additional evidence of beneficial effects on bone resorption with IC50=0.6 microns.

As a result of various biochemical, cellular and animal studies shows that R-MARK plays an important role in the regulation of responses to IL-1 and TNF-αand that it is involved in regulation of mRNA levels of some responsible for inflammation genes, such as MOR-2. Inhibitors R blocking the production of proinflammatory cytokines and inhibit the production of MMPs, and it was shown that they inhibit the breakdown of collagen in cartilage explants.

The use of the MARK inhibitor to block the actions of key proinflammatory cytokines, such as IL-1 and TNF-α will have beneficial effects on many cell types in the joint, including synovial fibroblasts, macrophages and chondrocytes, thus inhibiting subsequent pathological effects, such as infiltration of inflammatory cells into the joint, synovial hyperplasia, activation of synovial cells and destruction of cartilage. Thus, the MARK inhibitor will block the spread of the inflammatory response to the above cytokines and thereby interrupt the disease process.

Examples of inhibitors of the MARK suitable for the present invention are listed below. For all delivery methods (i.e. injection, infusion, and irrigation), the optimal dose and/or concentration of each candidate agent is such that a therapeutically effective. As an example, for each of the listed agents are preferred and the most preferred concentration irrigating solution containing included in the list of the agent, it is expected that these concentrations are therapeutically effective.

TABLE 23

Therapeutic and preferred concentrations of the inhibitors Markins
ConnectionTherapeutic concentrations (nanomolar)Predpochtitel the major concentration (nanomolar)
SB 2035800.5 to 50,00050-5,000
SB 203580 iodo0.5 to 50,00050-5,000
SB 2021900.2 to 20,00020-2,000
SB 2422350,2-10,00020-1,000
SB 2200250,2-10,00020-1,000
RWJ 676570,3-30,00030-3,000
RWJ 683540,9-90,00090-9,000
FRI 1336051-100,00010-10,000
L-1673070.5 to 50,00050-5,000
PD 98059of 0.1-10,00010-1000
PD 1693161-100,00010-10,000

7. INHIBITORS of MATRIX METALLOPROTEINASES

Destruction of articular cartilage is a common symptom in diseases of the joints such as osteoarthritis and rheumatoid arthritis, but it also occurs after damage to the joint. In pathophysiological relationship is observed structural collapse of proteoglycans and collagen, which violates the biomechanical properties of cartilage. Maintaining normal healthy extracellular matrix reflects the balance between the rate of biosynthesis and incorporation of matrix components and the rate of their destruction and subsequent loss of cartilage in the synovial fluid. Different FR the basics have the potential to break down cartilage and participate in the process of destruction, to the greatest extent of matrix metalloproteinases.

Matrix metalloproteinases (MMPs) or MatrixOne are a family of at least 15 zinc-endopeptidase, which are extracellular and play a key role in the pathological tissue destruction. The current nomenclature and alternative names of the members of the mold are shown in table 23. A large part of MMPs largely regulated and a large part of the constitutive is not expressed in normal tissues. However, proinflammatory cytokines, such as IL-1 and TNF-αinitiate transcription and expression. The imbalance that occurs when positive regulation and activation of destructive tissue MMPs, is the primary causal factor in the process for cartilage destruction during chronic inflammatory diseases and persistent synovial inflammatory response after damage to the joint. We studied the metabolism of cartilage matrix in patients with damage to the meniscus or rupture of the anterior cruciate ligament of the knee. It was shown that the concentration stromelysin-1 (MMP-3), collagenase, tissue inhibitor of metalloproteinases (TIMP-1) and fragments of proteoglycan was increased in the synovial fluid of the knee after traumatic injury of the knee. Temporarily, these figures increased instantly compared to about CNAME levels and remained significantly elevated (10-fold increase in one year. These changes will likely lead to an increase in the concentration of fragments of proteoglycans that are found in the synovial fluid after damaging knee ligaments.

It was shown that a family of enzymes MMP are secreted from human chondrocytes and cells of the synovium, such as synovial fibroblasts. In addition, using in situ hybridization, it was shown that human synovium synthesizes stromelysin-1 and collagenase. Stromelysin-1 (MMP-3) is able to destroy all of the components of cartilage matrix. There is evidence that the chondrocytes are involved in the destruction of cartilage release destructive matrix enzyme, collagenase-3. Upon activation of Pro-inflammatory cytokines, MMPs are secreted from cells in a latent form, extracellular activated and inhibited by the tissue inhibitors of metalloproteinases (TIMPs). It is believed that the balance between the activities of MMPs and TIMPs are important for the conservation of intact cartilage matrix. In several studies it was shown that in pathological conditions such as osteoarthritis and rheumatoid arthritis, elevated levels of MMPs lead to an imbalance between MMPs and TIMPs that, as expected, is responsible for the observed degradation of cartilage.

MMPs nakhodyatsyaya control of cytokines, such as interleukin-1 (IL-1) and growth factors that act on chondrocytes and synoviocytes with increased production of their proteases. Other proinflammatory cytokines, such as IL-6, IL-8 and TNF-αalso increase the production of destructive matrix enzymes. This leads to destruction of cartilage, which is usually estimated as the loss of sulfated glycosaminoglycans (GAGs) and the decomposition of collagen. IL-1, which is in the joint fluid of patients with arthritic diseases, stimulates chondrocytes to synthesize high amounts of enzymes, such as stromelysin, collagenase fibroblasts and neutrophils, and plasminogen activator. In addition, IL-1 inhibits the synthesis of inhibitor-1 activator of plasminogen and TIMP, and also inhibits the synthesis of matrix components such as collagen. The imbalance between the levels of inhibitors and enzymes leads to an increase in the number of active proteases in combination with inhibition of the biosynthesis of matrix leads to the destruction of cartilage.

Using sliced cartilage model in vitro, it was shown that collagenase inhibitors can inhibit stimulated IL-1 or IL-8 invasion of articular cartilage rheumatoid synovial fibroblasts (RSF). Inhibitors of collagenase, 1,10-o-phenanthroline and phosphoramidon, significantly inhibited dependent on the concentration of the penetration of the cartilage cells RSF, p and the concentration of 10-150 μm. Selective action of cytokines on the secretion of proteinases shows that mediated synovial fibroblast-like cells in the destruction of articular cartilage is a highly regulated process. Thus, it is assumed that the ability to inhibit protease activity and the associated destruction of the matrix locally inside the joint, will inhibit the process of destruction. On the basis of the action of inhibitors in a limited in vitro system can be assumed that therapeutic intervention using local delivery of synthetic MMP inhibitors with appropriate pharmacokinetics will be effective as protective for cartilage agents.

Examples of MMP inhibitors suitable for the present invention include U-24522 ((R,S)-N-[2-[2-(hydroxyamino)-2-oxoethyl]-4-methyl-1-oxobutyl]-L-leucyl-L-phenylalanine), peptides, such as inhibitor-1 MMP and inhibitor of MMP-3 and larger proteins, such as TIMP-1 or fragments thereof, and they are listed in the table below. For all methods, local delivery (i.e. injection, infusion, and irrigation), the optimal dose and/or concentration of each candidate agent is such that a therapeutically effective. As an example, for each of the listed agents are preferred and the most preferred concentration irrigating solution containing the th incoming list agent, it is assumed that the concentration of therapeutically effective.

TABLE 25

Therapeutic and preferred concentrations of inhibitors of matrix metalloproteinases (MMPs)
ConnectionTherapeutic concentrations (nano-molar)The most preferred concentration (nanomolar)
U-245220.2 to 2,00020-200
minocycline30-500,000300-3,000
inhibitor I MMD 4-Abz-Gly-Pro-D-Leu-D-Ala-NHOH0.3 to 3,0003-600
inhibitor of MMP-3 AC-Arg-Cys-Gly-Val-Pro-Asp-NH20.5 to 5,0005-500
Ruman TIMP10.5 to 5,0005-500
Ruman TIMP20.3 to 3,0003-600
phosphoramidon1,000-500,0005,000-100,000

8. Inhibitors of nuclear factor Carr (NFκB)

Proinflammatory and destructive for cartilage cell pathway regulated by extracellular and intracellular signaling mechanisms that are targets for new therapeutic local drug delivery. Full molecular mechanisms of signal transmission used protopalatial the m a cytokine, interleukin-1 (IL-1), activating transcription factor, nuclear factor Carr (NFκ (B)poorly installed. However, a key molecule that is involved in intracellular signal transmission at the level of transcription of the gene is a Pro-inflammatory transcription factor (NFkB). The activity of NFkB mediated by a family of subunits of the transcription factors that bind to DNA either in the form of homodimers or heterodimers. Data subunit are usually found inside the cytoplasm of cells in an inactive form by binding with inhibitory subunit, called IkB. Activation of IL-1 receptors and other extracellular signals induces the degradation of IkB and concomitant dissociation of NFkB and inhibitors with subsequent translocation into the nucleus. It was found that NFkB is involved in induced IL-1 expression and can enhance the expression of Pro-inflammatory protein SOH-2 in synovial fibroblasts in RA.

The definition of NFkB as a key molecular target based on its role as a General negative regulatory signal element regulating gene expression of several critical inflammatory mediators associated with inflammation of the joints and damaging the cartilage ways. The response of many genes (SOH-2, collagenase, IL-6, IL-8) is controlled by promoters, which include both the promoter element of NFkB. Akti is the situation NFkB mediates the induction of many proteins, important for inflammation, such as cytokines, cell adhesion molecules, metalloproteinases, and other proteins that are involved in the production of prostaglandins and leukotrienes (SOH-2) synoviocyte. Thus, this transcription factor is physiologically important target in the treatment focused on the response of human synovial fibroblasts, human articular chondrocytes and other cells at the injury in the joint.

In particular, it was shown that treatment of human rheumatoid synovial fibroblasts (RSF) interleukin-beta (IL-Bega) leads to consistent positive regulation of phospholipase A2 (PLA2) weight 85 KD and induced cyclo-oxygenase (SOH-2). Together, these two enzyme contribute to the subsequent biosynthesis of PGE2, the main mediator in the joint. Oligonucleotide traps and antisense sequences were used to demonstrate participation (NFkB) in the regulation of meta-belisirma prostanoids enzymes. Opposition NFkB mRNA with the use of antisense oligonucleotide resulted in reduced binding of the promoter of the gene MOR.

Has recently been characterized hymeniacidon, mouse natural product, as an inhibitor of NFkB activation and stimulated IL-1 and inhibited RSF products PRGE2dependent on the concentration of the OBR is ω (IC 50=1 μm). It was shown that the specificity of molecular targets using similar adesina and inhibitor of protein kinase C, RO 32-0432, which was inactive. Shows direct action hymeniacidon on induced IL-1 activation of NFkB by a significant reduction (approximately 80%) of the binding of NFkB with classic kB-motive and inhibition of stimulated migration R65 from cytosol treated cells. As expected for an inhibitor of the transcriptional regulation of the NFkB-treated hymeniacidon RSF did not transcribable mRNA for MOR-2 or PLA2 in response to IL-1. Therefore, the observed decreased protein levels of these enzymes and decrease their ability to produce PGE2. In addition, stimulated IL-1 production of interleukin-8 (IL-8), which is known to be regulated NFkB event, also inhibited under the influence hymeniacidon, while induced IL-1 production of vascular endothelial growth factor, unregulated NFkB gene, are not exposed to hymeniacidon. Thus, hymeniacidon inhibits stimulated IL-1 formation of PGE2 by synovial fibroblasts through an inhibitory effect on the activation of NFkB. This provides the basis for its use as a new inhibitor to block the role of NFkB in joint inflammation and destruction of cartilage.

Examples of NFkB inhibitors suitable for the present invention, are listed below. For all methods, local delivery (i.e. injection, infusion, and irrigation), the optimal dose and/or concentration of each candidate agent is such that a therapeutically effective. As an example, for each of the listed agents are preferred and the most preferred concentration irrigating solution containing included in the list of the agent, it is expected that these concentrations are therapeutically effective.

9. INHIBITORS of nitric OXIDE SYNTHASE

Nitric oxide (NO) is a widespread intracellular and intercellular mediator involved in the pathophysiological mechanisms of some diseases of the connective tissue. NO is formed from L-arginine with the participation of a family of enzymes, the NO synthases, which are located inside the cells. Were cloned and sequenced three isoforms of NO-synthase. Endothelial cell NO synthase (ecNOS) and NO-synthase in the brain (bNOS) are constitutively active. Another isoform of NO-synthase, inducible NOS (iNOS), found in many cell types, including chondrocytes. It is absent in normal conditions, but positively regulated in response to Pro-inflammatory mediators, such as IL-1β and TNF-α. Recent discoveries have shown that IL-1 is a very strong stimuli for the production of what PR is of NO synthesis in chondrocytes, and that IL-1 acts through its ability to increase the level of iNOS. Inside the joint chondrocytes are the main source of NO, and it is believed that iNOS chondrocytes induced proinflammatory cytokine, mediates many of the effects of IL-1 in inflammatory arthropathy.

Drugs that specifically inhibit inducible NO synthase (iNOS) chondrocytes, may have therapeutic value in the prevention of destruction of cartilage, which is the result of joint damage (for example, surgery on the joint). The proof is similar to the useful therapeutic action is based on a sufficient number of studies that evaluated various iNOS inhibitors for their ability to inhibit the activity of inducible NO-synthase in cultured chondrocytes and cartilage explants from patients with osteoarthritis. A group of compounds called S-substituted izotiomocheviny, was characterized as strong inhibitors of the biosynthesis of NO in the cartilage. 3-methylthymidine and S-(aminoethyl)estimacion were 2-4 times more active than NG-monomethyl-L-arginine, 5-10 times more active than aminoguanidine and 300 times more active than Nω-nitro-L-arginine and methyl ester of Nω-nitro-L-arginine. Data derived estimacion are a group of strong and relative activities is however, a specific iNOS inhibitors in cartilage and, thus, are suitable for local delivery of the present invention (Jang D., 1996, Eur. J. Pharmacol. 312: 341-347).

Protective cartilage therapeutic potential of inhibitors of NO-synthase was also evaluated using vitro systems, such as isolated chondrocytes, in order to determine the effect on cartilage matrix. Inhibition of endogenous NO production under the action of Nω-nitro-L-arginine (L-NMMA), a recognized inhibitor of NO-synthase, suppresses the production gelatinase, collagenase and stromelysin in stimulated IL-1β the chondrocytes. Inhibition of NO production also partially reduces the increased production of lactate, which takes place at the effect on the chondrocytes IL-1β. Treatment of cartilage fragments of L-NMMA partially suppresses the inhibitory activity of IL-1β on the synthesis of glycose-miragliano, inhibits stimulated IL-1β MMP activity and increases the production of antagonist IL-1 receptor (IL-1ra). NO may also indirectly modulate the synthesis of proteoglycans by reducing the production of TGF-β1 chondrocytes after exposure to IL-1β. She warns autocrine-stimulated increase in TGF-β1, thus reducing the anabolic effects of this cytokine in chondrocytes.

The study compared the activity of the new aminoguanidine, S-methylisothiazoline (SMT), 3-aminoethylamino the guilt (AETU), L-NMMA and methyl ester of N-nitro-L-arginine (L-NAME), as NOS inhibitors, inhibitory effect on the responses of recombinant human IL-1 on the synthesis of proteoglycans and NO production. In different culture systems was shown the response depending on the concentration on the impact of IL-1β, reflected in the significant increase in NO production and a marked suppression of the synthesis of proteoglycans. The above inhibitors of NOS (at a concentration of 1-1000 μm) inhibited NO production by cells of the cartilage treated with IL-1βand had a noticeable effect on the recovery of the synthesis of proteoglycans in chondrocytes. Therefore, if NO production can be blocked with the use of a therapeutically effective concentration and dose, then inhibition of the synthesis of proteoglycans under the action of IL-1β be warned.

NO-synthase is expressed in cartilage obtained from the joints of patients with arthritis. In patients with rheumatoid arthritis or osteoarthritis observed elevated levels of nitrite in synovial fluid, and it was shown that a significant source of NO production in these patients is articular cartilage. In addition, it was found that continuous systemic delivery of L-NIL, a strong inhibitor of iNOS, suppresses the progression of OA in dogs (induced dissection ACL) and causes a significant reduction in PR the production of IL-1β , PGE2, NO, and DFID. Based on these discoveries, we can assume that NO is a strong regulator of the effects of IL-1β and contributes to the pathophysiology of diseases of the joints.

Thus, these results in vitro and in vivo indicate that inhibitors of NO synthases are potential new drugs for the treatment of synovial inflammation and may provide for protective cartilage effects for local delivery in combination with one or more drugs selected from anti-inflammatory, protects the cartilage and pain groups for the treatment of surgically treated joint or other damaged joint.

Examples of inhibitors of NO synthases are suitable for the present invention are listed below. For all methods, local delivery (i.e. injection, infusion, and irrigation), the optimal dose and/or concentration of each candidate agent is such that a therapeutically effective. As an example, for each of the listed agents are preferred and the most preferred concentration irrigating solution containing included in the list of the agent, it is expected that these concentrations are therapeutically effective. In one preferred embodiment the inhibitors of NO-synthase for inclusion in the solutions according to the invention are 1400 W (N-3-(and Enomatic)benzyl)acetamidine, selective, slow, tightly binding inhibitor of iNOS, diphenylbenzidine and 1,3-PBIT.

TABLE 27

Therapeutic and preferred concentrations of inhibitors of nitric oxide synthase
ConnectionTherapeutic concentrations (nanomolar)The most preferred concentration (nanomolar)
NG-monomethyl-L-arginine50-50,0003,000
1400 Wof 0.1-1,0001-20
diphenylamideof 0.1-1,0001-100
S-methylisothiazoline1-1,00010-100
S-(aminoethyl)estimacion1-1,00010-100
L-N6-(1-iminoethyl)lysine1-1,00010-100
1,3-PBITU0,5-5005-100
2-ethyl-2-thiopseudourea2-20,00020-2,000

10. MOLECULES CELL ADISHI

10A. AGONISTS AND ANTAGONISTS of INTEGRINS

Integrins are heterodimeric receptors on the plasma membrane, which include αand β-subunits, which bind ligands, which are extracellular matric is e (ECM) components, or may be other large proteins, such as collagen, laminin, vitronectin, osteopontin (OPN) and fibronectin (FN). The destruction of the cartilage matrix is governed by the chondrocytes through mechanisms that depend on the interaction of these cells with the ECM. Gene expression in chondrocytes partly regulated by cell contact involving the interaction of integrins with ECM components in the environment surrounding chondrocyte. Therefore, integrins on the chondrocytes are involved in the regulation of homeostasis in the cartilage, and this family of receptors is a group of therapeutic targets to prevent the destruction of cartilage.

Human chondrocytes Express many integranova receptors, consisting of various αand β-subunits, including α1β1that α5β1that αVβ1and smaller amounts of other. αVβ3-integrin has a special meaning, which, as you know, binds OPN. αVβ3-integrated specific blocking monoclonal antibodies act as an agonist in the same way as the ligand, OPN. They reduce the production of several proinflammatory and destructive cartilage mediators, such as IL-1, NO and PGE2. Thus, it is believed that the agonistic mAb LM609 suitable for use in the present invention.

To the ome, in phase II clinical trials have studied two peptidomimetic MK-383 (Merck) and RO 4483 (Hoffmann-LaRoche). Since both are small molecules, they have a short half-life and high activity. However, it turned out that they also have lower specificity, interacting with other very close integrins. These peptidomimetics are also suitable for use in the present invention.

TABLE 28

Therapeutic and preferred concentrations of integrins
The group agentTherapeutic concentrations (µg/ml)The preferred concentration (µg/ml)
Integrins:
αVβ3mAb LM 609of 0.05-5,0005-500
echistatinof 0.1-10,000100-1,000

11. ANTIGEMORRAGICESCOE AGENTS

Antigemorragicescoe agents prevent chemotaxis of inflammatory cells. Typical examples antigemofilichesky targets upon which the agents will act include, but are not limited to F-Met-Leu-Phe receptors, IL-8 receptors, MCP-1 receptors and MIP-1-I/RANTES receptor. Drugs in this group of agents are in the early stages of development, but theorem is automatic, they can be suitable for use in the present invention.

12. INHIBITORS of INTRACELLULAR SIGNAL transduction

12A. INHIBITORS of protein KINASES

i. Inhibitors of protein kinase C (SW)

Protein kinase C (SW) plays a crucial role in the signal transmission from the cell surface for a number of physiological processes. The isozyme RKS can be activated as inhibiting targets in the initial activation associated with G-protein receptors (e.g., serotonin, bradykinin and the like) or Pro-inflammatory cytokine receptors. Both of these groups of receptors play an important role in mediating the destruction of cartilage.

When the molecular cloning of was found that the RCC exists in the form of a large family that includes at least 8 subspecies (isozyme). These isozyme differ significantly in structure and activation mechanism linking receptors to changes in the proliferative response of specific cells. The expression of specific isozyme installed in various types of cells, including synoviocytes, chondrocytes, neutrophils, myeloid cells and smooth muscle cells. Therefore, it is likely that inhibitors of the RCC affect the path of signal transmission in several types of cells, although the inhibitor shows issimo specificity. Thus, we can assume that the inhibitors of the RCC will be effective in blocking the activation synoviocytes and chondrocytes and may also have anti-inflammatory effect by blocking the activation of neutrophils and subsequent attachment. Describes several inhibitors, and the initial work was specified IC50equal to 50 μm, for inhibitory activity calphostin C. G-6203 (also known as Go 6976) is a new, strong inhibitor of the RCC with high selectivity for certain isotypes RKS with values IC50within 2-10 μm. The concentrations of these and other drug GF H, also known as Go 6850 or bilingualised I (Warner-Lambert), deemed suitable for use in the present invention, below.

TABLE 29

Therapeutic and preferred concentrations of agents that suppress the destruction of cartilage
The group agentTherapeutic concentrations (nanomolar)The preferred concentration (nanomolar)
Inhibitors of protein kinase C:
calphostin0.5 to 50,000100-5,000
GF 109203Xof 0.1-10,0001-1,000
G-6203 (Go 6976)of 0.1-10,0001-1,000

ii. Inhibitors of tyrosine protein kinases

Despite the fact that among the numerous members of the family of receptor tyrosinekinase (RTK) there is a significant difference, the mechanisms re the ACI signal, used these receptors share many common properties. In biochemical and molecular genetic studies have shown that the binding of ligand to the extracellular domain of RTK quickly activates the inherent tyrosinekinase catalytic activity of the extracellular domain (see figure 5). Increased activity leads to specific tyrosine phosphorylation of several intracellular substrates that contain a common motif sequence. As a result, this causes activation of multiple negative regulatory signaling molecules and cascades of intracellular pathways that regulate phospholipid metabolism, metabolism arachidonate, phosphorylation of proteins (including other mechanisms than with the participation of protein kinases), mobilization of calcium and activation of transcription (see figure 2). Dependent on growth factors, tyrosine kinase activity of the cytoplasmic domain of RTK is the main mechanism of generation of intracellular signals that lead to cell proliferation. Thus, the inhibitors have the potential to block the signal transmission and thereby prevent activation of synoviocytes and chondrocytes.

Any of several close derivatives tyrphostin has potential as specific inhibitors of tyrosine kinase activity (IC50in vitro in the range of 0.5-1.0 μm), POSCO is jku they have little effect on other protein kinases and other signal transmission. To date, only a few of a large number of derivatives tyrphostin are industrially available and suitable concentration of these agents for use in the present invention are given below. In addition, it was reported that staurosporin showed strong inhibitory activity against several proteincontaining the src subfamily and the appropriate concentration of the agent for use according to the present invention also below

TABLE 30

Therapeutic and preferred concentrations inhibiting agents
The group agentTherapeutic concentrations (nanomolar)The preferred concentration (nanomolar)
Inhibitors of protein kinase
lovenduski And10-100,000100-10,000
tyrphostin AG129610-100,000100-10,000
tyrphostin AG129510-100,000100-10,000
staurosporin1-100,00010-1,000
PD 158780of 0.1-10,00010-500
PD 174265of 0.1-10,00010-500

12b. Modulators of vnutri mocnych (protein-tyrosine-phosphatase

Retransmission (protein-tyrosine-phosphatase (Rtrsy), including SH2 domains of src-homology2known and the item they refer to the SH-PTP1 and SH-PTP2. In addition, SH-PTP1 also known as RTRS, NDS or SHP. SH-PTP2 also known as PTP1D or RTRS. Similarly, SH-PTP1 is expressed in large quantities in hematopoietic cells of all generations and at all stages of differentiation, and gene SH-PTP1 identified as responsible for the parent (me) mouse phenotype, and this provides the basis for the assumption that the effect of the inhibitors will block the interaction with its cellular substrates. It is known that stimulation of neutrophil chemotactic peptides leads to activation of tyrosinekinase, which mediate the response of neutrophils (Cui et al., 1994 J. Immunol.), and RTR-asna activity modulates agonist-induced activity by the opposite change effects tyrosinekinase activated during the initial phases of stimulation of cells. Agents that can stimulate the activity RTR-AZ may have potential for therapeutic applications as anti-inflammatory mediators.

It was shown that the data RTR-basics modulate the activity of some RTKs. It turned out that they counteract the effect of activated receptor kinases and, thus, can provide important is ishani for drugs. In vitro experiments showed that injection of RTR-basics blocks insulin-stimulated phosphorylation trailing residues on endogenous proteins. Thus, activators activity RTR-basics can be used for the opposite changes in activation of RTK receptors when you stenose, and believe that they are appropriate for the solutions of the present invention. Additionally, the associated with receptors RTR-basics also function as extracellular ligands that are close to those for cell adhesion molecules. Functional consequences of the binding of ligand to the extracellular domain is not yet established, but it is reasonable to assume that the binding will serve to modulate the activity of phosphatases within cells (Fashena and Zinn, 1995, Current Biology, 5, 1367-1369). Such action may block the adhesion mediated by other molecules of the cell surface adhesion (NCAM), and provide anti-inflammatory effect. For these applications medications have not yet been developed.

12C. INHIBITORS of SH2 domains domains (src homology2)

SH2 domains, originally established in the src subfamily of tyrosine kinases (PTKs)are non-catalytic protein sequences and consist of approximately 100 amino acids that are conservative among the various proteins involved the transmission signal (Cohen et al., 1995). SH2 domains function as phosphotyrosine-binding modules and thereby mediate critical Association protein-protein in the signal transduction pathways within cells (Pawson, Nature, 573-580, 1995). In particular, was well established role of SH2 domains as critical for receptor tyrosinekinase (RTK), mediating the signal transmission, such as in the case of the receptor for platelet-derived growth factor (PDGF). Phosphotyrosine-containing sites on autophosphorylated RTKs serve as binding sites S2-proteins and thereby mediate the activation of biochemical pathways signaling (see figure 2) (Carpenter G., FASEB J. 6: 3283-3289, 1992; Sierke S. and Koland, J. Biochem. 32: 10102-10108, 1993). SH2 domains responsible for binding to activated receptors of growth factors at the cellular responses that include changes in gene expression and ultimately cell proliferation. Thus, inhibitors that will selectively block the effects of the activation of specific RTKs (excluding IGFR and FGFR), expressed on the surface of synoviocytes will be effective in blocking of chondrolysis after arthroscopic procedures.

Identified at least 20 cytosolic proteins, which include SH2 domains and function in intracellular signal transmission. Distribution of SH2 domains is not limited to a specific family of proteins and they found a few groups of proteins, the protein kinases, lebedkina, proteinprospector, phospholipases, Ras-regulatory proteins and some transcription factors. Many of SH2-containing proteins have a known enzymatic activity, while others (Grb2 and Crk) function as "linkers" and "adapters" between cell surface receptors and any abscopal effector molecules (Marengere L. et al., Nature 369: 502-505, 1994). Examples of proteins that includes SN domains with enzymatic activity that are activated in signal transduction include, but are not limited to the subfamily scr tyrosine kinases (scr (Rc-src), abl, Ick, fyn, fgr and others), phospholipasesγ (PLCγ), phosphatidylinositide-3-kinase (PI-3-kinase), p21-ras activating GTP-ABC protein (GAP) and SH2-containing protein-trointestinal (SH-Stranami) (Songyang et al., Cell 72, 767-778, 1993). Due to the Central role of data in different SH2-proteins in signaling from activated cell surface receptors in a cascade of additional molecular interactions that ultimately determine cell response inhibitors, which block the specific binding of SH2-protein (for example, c-src), are desirable as agents of potential therapeutic applications for the protection of cartilage.

In addition, the regulation of many immune/inflammatory responses OPOS udaetsya through receptors which transmit signals via preceptory tyrosine kinase, including SH2 domains. Activation of T cells through antigen-specific receptors of T cells (TCR) initiates a cascade of signal transmission, resulting in secretion of lymphokines and proliferation of T cells. One of the earliest biochemical responses following TCR activation, is increased activity tyrosinekinase. In particular, activation of neutrophils partially governed by the responses of the receptors for immunoglobulin G on cell surfaces. Activation of these receptors mediates the activation of the unidentified tyrosinekinase, which are known to have SH2 domains. Additional evidence indicates that several kinases (lck, bik, fyn), related to the src family, are involved in signaling from cytokine and integranova receptors and, therefore, can serve to integrate signals from several independent receptor structures. Thus, inhibitors of specific SH2 domains have the potential to block many of the functions of neutrophils and serve as anti-inflammatory mediators.

Currently, attempts were made to develop drugs targeted affecting SH2 domains on the biochemical in vitro and at the cellular level. If such attempts will be successful is, theoretically, we can assume that the resulting drugs are suitable for the practice of the present invention.

III. SYNERGISTIC INTERACTION IN theRAPEUTIC COMBINATIONS of AGENTS, INHIBITING PAIN AND/OR INFLAMMATION, AND OTHER AGENTS USED IN SOLUTIONS TO PROTECT CARTILAGE

Given the complexity of the disease process associated with inflammation and loss of cartilage homeostasis in after arthroscopic therapeutic procedures and multiplicity of the participating molecular targets, blockade or inhibition of one molecular targets are unlikely to provide adequate efficacy in the prevention of destruction of cartilage and the development of osteoarthritis. Indeed, a number of studies conducted on animals have shown that purposeful exposure to different individual molecular receptors or enzymes is not effective in animal models, or it has not produced to date, the effectiveness in clinical trials. Therefore, desired is a therapeutic combination of drugs acting on different molecular targets, and delivered locally for the manifestation of clinical effectiveness in therapeutic approach to the protection of cartilage. As described below, the rationale for this synergistic mo is collaroy targeted therapy derives from recent advances in understanding the fundamental biochemical mechanisms, with the help of which synoviocytes and chondrocytes in cartilage and synovium transmit and integrate the signals, the effects of which they are subjected during arthroscopic procedures.

"CROSS-IMPACT" AND the CONVERGENCE of the MOST IMPORTANT routes of TRANSMISSION SIGNALS

Molecular "switches"that are responsible for the transmission of signals in the cell, have traditionally been divided into large discrete transmission signals, each of which includes a number of different protein families, which act as transduction for a specific number of extracellular signals and mediate various cellular responses. On one such path is transmitted signals from neurotransmitters and hormones through associated with G-protein receptors (GPCRs) with the formation of the responses, which include the production of inflammatory mediators, such as PGE2. GPCRs are connected with intracellular targets via activation of trimeric G-proteins (see figure 2). Examples of signaling molecules involved in the activation of synoviocytes and chondrocytes via GPCR-way, are histamine, bradykinin, serotonin, and ATP. The second main path passes the signals from proinflammatory cytokines, such as IL-1, through a cascade of kinases and NF-6B-protein in the regulation of gene expression and production of catabolic cytokines and other catabolic factors, including the NO.

Signals transmitted from neurotransmitters and hormone is in, stimulate one of two groups of receptors: GPCRs, consisting of semispinalis transmembrane sites, or ion channels with gate ligands. Braking signals of both types of receptors converge on the regulation of cytoplasmic concentration of CA2+(see figure 3). Each transmembrane GPCR-receptor activates specific group dressing rooms G-proteins, including Gq, Gior many others. Gq-subunit activates phospholipasesγ, leading to activation of protein kinase C (CSWs) and increased levels of cytoplasmic calcium (figure 3). In turn, increased intracellular calcium leads to activation PLA2 and products of arachidonic acid (AA). AA serves as a substrate for SOH in synoviocytes and chondrocytes, leading to the production of PGE2. Activation of the RCC also leads to activation of Martinez, leading to activation of NFB, and in cells and tissues, which were initially exposed to proinflammatory cytokines, modulating the increased gene expression of proteins involved in the catabolism of cartilage.

Signaling of proinflammatory cytokines, which is mediated by IL-1 and TNF-α through their different cognate receptors, also converge on the regulation of gene expression in cells. On the transmission of signals used data from various receptors, uses a variety of the kinase, which are proximal to the receptor, but the transmission of signals in the subsequent converge at the level of Markins (figure 3 and 4). Signaling-dependent phosphorylation of residues in the cascade of kinases, including inhibiting enzymes such as R-Marginata. Activation of the IL-1-receptor and TNF-α-receptor also leads to stimulation of Martinez, shared the stage partial Gq-coupled GPCRs (see figure 3). It is now recognized that independent of ligand "cross-impact" can transactivate involving kinases in response to joint stimulation of specific GPCRs and cytokines, such as IL-1, leading to synergistic cell responses (see figure 3). Thus, the combination of selective inhibitors that block the transactivation common route of transmission of the signals (as shown in figures 1 and 2), leading to increased gene expression of proinflammatory cytokines, iNOS, COX-2 and MMPs, will act synergistically to prevent inflammation and destruction of cartilage after arthroscopic surgical procedures.

IV. CONCLUSION

Based on the molecular and cellular mechanisms of action defined for data agents for the protection of cartilage, it is assumed that the data connection will be protective for cartilage action, when applied perioperative in irrigating solution (in combination with other what they described above for protective cartilage agents or in combination with other analgesic and anti-inflammatory agents), or otherwise entered directly into the joint via infusion or injection. In particular, it is assumed that these agents will be effective drugs when delivered with irrigating solution during arthroscopic surgery. Each metabolically active for protective cartilage agent can be delivered in combination with one or more protective for cartilage agents, including small molecule drugs, peptides, proteins, recombinant chimeric proteins, antibodies, oligonucleotides or vectors for gene therapy (viral and non-viral) in the joint. For example, the drug, such as MARK inhibitor, exerts its action on any cells associated with fluid spaces of the joints and structures that make up the joint, which are involved in the normal functioning of the joint, or are present in the pathological condition. These cells and the structure include, but are not limited to: synovial cells, including fibroblasts of type a and type macrophages In; cartilaginous components of the joint, such as chondroblasts and chondrocytes; cells associated with the bone, including periosteal cells, osteocytes, osteoblasts, osteoclasts; cells of inflammation, including lymphocytes, macrophages, mast cells, monocytes, eosinophils, and other cells, including endothelial the e cells, smooth muscle cells, fibroblasts and nerve cells; and combinations of the above.

This aspect of the present invention is also the composition of the active therapeutic agent(s), which can be delivered by the composition suitable for introduction and purpose of the drug into the joint that will enhance the delivery, uptake, stability or pharmacokinetics for protective cartilage agent(s). Such a composition may include, but not be limited to, microparticles, microspheres or nanoparticles consisting of proteins, liposomes, carbohydrates, synthetic organic compounds or inorganic compounds. The present invention provides for the delivery of a combination of protective for cartilage agents, or one or more agents for the protection of the cartilage of one or more agents, any abscopal pain and/or inflammation is present either in the form of many pharmaceutically active compounds in homogeneous media (for example, a single encapsulated within the microsphere), or in the form of a discrete mixture of separate carriers for delivery (for example, a group of microspheres comprising one or more agents). Examples of molecules of the composition include, but are not limited to, hydrophilic polymers, a polycation (e.g., Protamine, spermidine, polylysine), peptide or synthetic ligands and antibodies, posebnimi purposefully to influence specific types of cells, gels, matrices for sustained release, soluble and insoluble particles, as well as elements in compositions that are not listed.

In one aspect the present invention provides local delivery of a composition of two or more agents to protect cartilage, or one or more agents to protect the cartilage in combination with one or more agents, any abscopal pain and/or inflammation of one or in combination with one or more analgesic and/or anti-inflammatory agent, with irrigating solution, infusion containing drugs that are therapeutically effective in low concentrations, and that contribute to the delivery of drugs directly into the affected tissue or joint. Infusion or irrigation solution containing the drug, can be used before and/or during surgery, and/or after surgery due to surgical procedure, or you can enter in other time periods not associated with surgical procedures. Other conventional methods used for drug delivery, you need a system (e.g., intramuscular, intravenous, subcutaneous) injection, which requires a higher concentration of drugs (and higher total dose) for the introduction of the patient is to in order to achieve significant therapeutic concentrations in the target joint. Systemic injection also leads to high concentrations in tissues to a greater extent than in the target joint, which is undesirable and, depending on dose, can lead to the manifestation of side effects. When data system how the drug undergoes secondary metabolism and rapid degradation, thereby limiting the duration of preservation of effective therapeutic concentration. Because the combination of agents to protect cartilage (with or without one or more analgesics and/or anti-inflammatory agents) injected directly into the joint infusion or irrigation, vascular perfusion is not required for delivery of a drug in the target tissue. This significant advantage allows you to apply for local delivery lower therapeutically effective dose for various protective cartilage of drugs.

V. METHOD of APPLYING

The solutions of the present invention are used in various surgical/invasive procedures, including surgical, diagnostic and therapeutic methods. The combination of protective cartilage agents according to the invention can be entered by injection or irrigation. In solutions for injection amount of the active ingredient, which can be the t to be connected with carriers to obtain a single dosage form, will vary depending on the patient being treated, the nature of the active agents in the solution and the particular route of administration. However, it is clear that the specific dose for any particular patient will depend upon a variety of factors, including the activity of the specific compound, the age, body weight, General health, sex and diet of the patient, time of administration, route of administration, rate of excretion combination of drugs and the severity of the particular disease being treated.

Injectable preparations, for example sterile injectable aqueous or oily suspensions, can be ways that are known in this field, using suitable dispersing or wetting agents or suspendida agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic acceptable for injecting diluent or solvent, for example, in the form of a solution in 1/3-propane diol. Acceptable carriers or solvents that can be used are water, ringer's solution and isotonic sodium chloride solution. In addition, as a solvent or suspendida environment can be used sterile, non-volatile oils. For this purpose you can use either the non-volatile oil, including mono - or diglycerides. In addition, fatty acids such as oleic acid find use in injecting drugs.

Solutions for injection according to the invention can be introduced in connection with arthroscopic surgical procedure, or at any time, as desired by the physician performing the treatment of the patient.

Irrigating solutions according to the invention can be applied perioperative during arthroscopic surgery for anatomical joints. In the sense in which he used the term "perioperative" includes the application during the procedure, before and during the procedure, during and after the procedure, and before, during and after the procedure. Preferably the solution is applied before and/or after the procedure and during the procedure. When such procedures are commonly used physiological irrigating fluid such as normal saline or aktirovannye ringer solution, applied to the surgical site by methods well known to specialists in this field. The method according to the present invention includes the use of analgesics/anti-inflammatory/protective cartilage irrigating solutions of the present invention, instead of the commonly used irrigating fluids. Irrigating solution applied to the wound or surgical site prior to the procedure, preferably p is ed trauma of tissues and continuously during the procedure to proactively block the pain and inflammation and destruction of cartilage. In the sense in which he used the term "irrigation" means irrigation under pressure wounds or anatomical structures of the jet of fluid. The term "application" includes irrigation and other methods of local injection solution of the present invention, such as the introduction of measurable gelation solution into the surgical site, which turned into a gel solution, then remaining in place during the procedure. In the sense in which he used the term "continuously" also includes situations in which there is a repeated or frequent irrigation of wounds with a frequency sufficient to maintain a predetermined local concentration of therapeutic agents used, and be used which can intermittent stopping of the flow of irrigating fluid that is required by the method.

Concentrations listed for each of the agents in solutions of the present invention, represent the concentrations of the agents delivered locally, in the presence of metabolic transformation, to the place of action in order to achieve a pre-determined level steps in the surgical site. Obviously, it is clear that the drug concentration in this solution may require some discussion with respect to the local dilution during delivery. Concentrations in the solutions is not summarized with regard to metabolic transformation or rasb the means by distributing throughout the body, because these circumstances are eliminated upon local delivery, as opposed to oral, intravenous, subcutaneous or intramuscular use.

Arthroscopic methods in which you can apply this solution, include as non-limiting example, incomplete meniscectomy and repair ligaments in the knee, acromioplasty shoulder, sanitation cuff rotation of the shoulder, synovectomy elbow and arthroscopy of the wrist and ankle. Irrigating solution long injected intraoperative into the joint with a flow rate sufficient to expand the joint capsule, surgical removal of residues for smooth intra-articular visualization.

Suitable arthroscopic irrigating solutions for inhibiting the destruction of cartilage and struggle with pain and inflammation during arthroscopic procedures described in examples 1-4 below.

In each of the solutions of the present invention, the agents included in low concentrations and are delivered locally in low doses compared to the concentrations and doses required for conventional methods of drug administration, in order to achieve the desired therapeutic effect. It is impossible to obtain an equivalent therapeutic effect when delivered agents in similar doses of other (i.e. intravenous, enter the author, intramuscular and oral) routes of administration of drugs, because drugs are introduced systematically subjected to primary and secondary metabolism and often quickly disappear from the systemic circulation.

The practice of the present invention must be different from the usual intra-articular injection of opiates and/or local anesthetics when performing arthroscopic or open on the joint (e.g. knee, shoulder, etc. procedures. The solution according to the present invention is used for continuous infusion during surgical procedures to provide proactive suppress pain and inflammation. In contrast to the high concentrations required to achieve therapeutic efficacy at a constant infusion of currently used local anesthetics can lead to severe systemic toxicity.

During the procedure of the present invention may be desirable to implement or apply otherwise a higher concentration of the same agent to protect cartilage (agents) and/or analgesics and/or anti-inflammatory agents than what is used in the irrigating solution in the surgical site, as an alternative or Supplement to opiates. In addition, it may be desirable direct injection of combinations for protective cartilage agents, as described in detail below. K is included for protective cartilage injection shown in example 5 below.

EXAMPLES

Presents several compositions of the present invention, suitable for some surgical procedures, followed by a brief description of the three clinical trials, which used agents of the present invention.

EXAMPLE 1

IRRIGATION SOLUTION FOR ARTHROSCOPY

The following composition is suitable for use in irrigation anatomical joint during arthroscopic procedures. Each drug solubilization in the carrier fluid containing physiological electrolytes, such as normal saline or aktirovannye ringer's solution, and other solutions described in the following examples.

The group agentMedicationConcentration (nmol)
The inhibitor MartinezSB203580200
Inhibitor of matrix metalloproteinasesU-24522200
Agonist TGF-βTGF-β2200

EXAMPLE 2

ALTERNATE IRRIGATION SOLUTION FOR ARTHROSCOPY

The following composition is an alternative composition, suitable for use in the irrigation of the anatomical joint during arthroscopic procedures.

The group agentMedicationConcentration (nmol)
The inhibitor MartinezSB203580200
The inhibitor of nitric oxide synthaseL-NIL1000
Agonist of the receptor of interleukin - IL-10100

EXAMPLE 3

An ALTERNATIVE SOLUTION FOR IRRIGATION

Subsequent drugs and the limits of the concentrations in solution in physiological liquid carrier suitable for use in the present invention.

The group agentMedicationConcentration (nmol)
The inhibitor MartinezSB242235200
The inhibitor of nitric oxide synthaseL-NIL10000
Agonist TGF-βTGF-β2100

EXAMPLE 4

An ALTERNATIVE SOLUTION FOR IRRIGATION

The following composition is also suitable for the present invention.

The group agentMedicationConcentration (nmol)
The inhibitor MartinezSB242235200
The MMP inhibitorU-24522 200

EXAMPLE 5

SOLUTION FOR INJECTION TO PROTECT CARTILAGE

The following composition is suitable for injection into the anatomical joint. Each drug solubilization in the carrier fluid containing physiological electrolytes, such as normal saline or aktirovannye ringer's solution. Dose in 20 ml of a solution suitable for administration to a patient.

The group agentMedicationConcentration (nmol)
The BMP agonist-receptorBMP-7100 ng/ml
The inhibitor of nitric oxide synthase1,3 PBIT4,4 mg/ml
Agonist TGF-βpyrrolidin-dithiocarbamate16.4 ug/ml

EXAMPLE 6

SYNERGISTIC STIMULATION of QUICK SHARP RISE in PGE2 WHEN EXPOSED to AGONISTS of IL-1 AND GPCK

Fibroblast-like synoviocyte demonstrate the properties of inflammatory cells and, probably, are critical regulators of joint inflammation and destruction of cartilage. Model was used for the culture of synoviocytes to assess the synergistic interactions between IL-1 and recitative inflammatory mediators that are important in the modulation of destruction of articular tissue, including damage which occurs as the consequence of tissue injury during arthroscopic surgery. The experiments were conducted to study the effect of agonists associated with G-protein receptors (GPCK) (histamine, bradykinin and isoproterenol) on the regulation of the production of cytokines and prostanoids in cultured human synovial fibroblasts and evaluate the activity of Ketoprofen in the system. Described the kinetics of induction of prostaglandin E2 (PGE2), interleukin-6 (IL-6) and interleukin-8 (IL-8) in response to stimulation by interleukin-1. Studied the ability of GPCR-ligands to enhance the production of cytokines after premirovany IL-1.

In examples 6-8 used the following experimental methods and materials, unless otherwise specified.

Cell culture. Synovial tissue was obtained from patients with osteoarthritis who underwent the operation for replacement of the joint clinical research center MacNeal hospital and transported to the laboratory in a modified method of Dulbecco environment Needle containing penicillin (100 units/ml), streptomycin (100 μg/ml) and Fungizone (0.25 microgram/ml). The synovium was dissected and crushed with scissors and placed in the form of explants in culture medium consisting of DMEM containing L-glutamine (2 mm), heat inactivated fetal bovine serum (10% vol/vol) plus antibiotics. Cultures were kept at 37°C in a humid atmosphere with 5% CO2. Fused synovial cells from explants grew up in during the s 2-3 weeks and were subjected to treatment with trypsin. Crops fed twice a week and when the merger was massirovala. Experiments were performed on cells from passages 3-8. Experimental culture were sown on Cup diameter 35 mm with a density of 7.5×103cells/cm2in 2 ml of culture medium. Cultural experience grew to close the merger and contained 2,3±0,3×105cells (mean ± average standard error, n=3) and 104±13 µg of protein (n=10). Culture medium was changed twice a week.

Experimental treatment. One day before the start of the experimental treatments the medium was replaced with experimental culture medium, consisting of DMEM with 2% heat inactivated fetal bovine serum plus L-glutamine and antibiotics mentioned above, to obtain cells at rest. The next day the culture was primiraly the addition of the indicated concentrations of IL-1 or more ligands to the conditioned culture medium for 12-24 h, as indicated. In some experiments, the conditioned culture medium was collected for analysis after premirovany IL-1. Acute experimental premirovany conducted after this period of treatment as follows. The culture was removed from thermostat, three times washed with 2 ml portions physiological buffer Locke (LB in mm: NaCl, 154; KCl, 2,6; KN2PO4, 2,25;2HPO to 0.85; MgCl2, 5; CaCl2, 2; D-glucose, 10; HEPES, 10; pH 7.4, BSA, 0,1% weight/volume) and then balanced by the additional volume of LB containing these ligands, for 10 min in a bath at 37°C. the solution was removed by aspiration and replaced with fresh buffer containing the indicated ligands for the indicated time periods at 37°C. Pharmacological inhibitors are usually added within 10 minutes preincubation period, and agonists plus these inhibitors were within a 3-minute period of stimulation.

Determination of prostaglandin E2. After these treatments were collected aliquots of culture supernatant (1 ml) and rapidly frozen in liquid nitrogen. The samples were stored at -80°before processing. Aliquots of culture supernatant were analyzed by radioimmunoassay, competitive-binding, as indicated by the manufacturer (Sigma Chemical Co.), using antibodies with equal reactivity towards prostaglandins E2 and E1. For the quantitative analysis of the constructed standard curve for each test using constant concentrations of [3H] prostaglandin E2 and increasing concentrations authentic competing prostaglandin E2.

Determination of IL-6. Production of the cytokine IL-6 were also determined in the aliquot of the supernatant culture medium, which kept the frosting is its at -80° C. IL-6 were analyzed by sandwich ELISA with a detection of alkaline phosphatase as described by the manufacturer (Pharmingen), and quantitatively determined using standard curves constructed with typical pure recombinant human cytokines. Experimental determination was performed with two parallel cultures.

Analysis of the inclusion of [3H]thymidine and MTT

Line synoviocytes usually used to measure the ability to proliferate in response to IL-1 activated [3H] thymidine (Kimball & Fisher, 1988). This drug is most effective concentrations of IL-1 stimulated the incorporation of [3H] thymidine 10-20 times stronger compared to cultures at rest, supported in 2% serum (data not shown).

Analysis of the data. The radioimmunoassay was performed with two parallel aliquot of each culture. Experimental determination set with two or three parallel cultures. Each experiment was repeated at least two cell lines. Construction of nonlinear regression curve and static analysis was performed using Graph-PAD Prism (San Diego, CA).

Materials. Cell culture: culture medium for cells received from Sigma Gibco/BRL. Fetal bovine serum was obtained from Atlanta Biologicals Inc. (Norcross, GA). Drugs: recombinant human interleukin - was obtained from Genzyme (Cambrige, MA). Ketoprofen was obtained from Omeros Medicak Systems, Inc. (Seattle, WA). Amitriptyline, Forskolin, 5-hydroxytryptamine, isoproterenol, bradykinin, histamine and prostaglandin E2 were obtained from Sigma. Labeled reagents: [3H] prostaglandin E2 was obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO). All other reagents of the highest purity were obtained from standard commercial suppliers.

Action GPCK-agonists, histamine and bradykinin, PGE2 production in human synoviocyte was determined with and without prior stimulation of IL-1 to assess functional interactions between agonists mediating the overall pharmacological effect through their different groups of receptors. The effects of IL-1 during the night in cultured human synovial fibroblasts (10 u/ml) resulted in lower (4 h) and a constant a significant increase in PGE2 production, which can be determined by radioimmunoassay in the form of elevated levels of PGE2 in the culture supernatant. It was shown that a progressive increase in PGE2 production during long-term treatment of IL-1 (16-24 h) occurs in skoordinirovanno increased expression of cPLA2 and COX-2 (Crofford, 1984, Hulkower et al., 1984). Culture, which were premirovany IL-1 during the night, gave the response to subsequent stimulation maximally effective concentrations of histamine (100 μm) or bradykinin (1 μm) is more rapid (minutes) and strong production of PGE2. Typical time data PGE2 production in response to stimulation by histamine or bradykinin presented in figure 7. In these conditions, histamine caused a 5-10-fold increase in PGE2 production compared to premirovanii IL-1 cells without adding a GPCR agonist. Bradykinin showed a 10-15-fold increase. The absolute amount of PGE2 produced during a short 2-minute stimulation with agonist, approaches or exceeds the number which the cumulative produced during the entire 18-hour period of premirovany IL-1. Is awesome, as shown in figure 7 that the main induced by histamine sharp rise in the production of PGE2 occurs in the first 2 min, as in the subsequent 60 min is the minimum additional accumulation. Stimulated by bradykinin response PGE2 continued to increase (2 times) during the same time period. In the absence of premirovany IL-1 is not subjected to synoviocyte did not show detectable PGE2 production in response to stimulation by any one GPCR agonist. In these terms of premirovany IL-1 both histamine and bradykinin, synergistically increased the release of PGE2.

When using cultured synovial fibroblasts from patients with osteoarthritis, the applicants have established the presence of time-dependent synergistic interactions between PR the inflammatory cytokine, IL-1, and physiologically relevant paired with G-protein receptors, product PGE2, and to assess the effect of targeted therapeutic agents. GPCR agonists acting through endogenous receptors synoviocytes that are associated with increased intracellular calcium, insiteful and RKS transmission signals quickly and clearly increase the production of PGE2 in cells previously premirovany IL-1. Inhibitors SOH-2 effectively weakened as agonist-induced a sharp rise and a long-term accumulation of PGE2. Thus, different pathways of intracellular signaling involving GPCR and IL-1 synergistically interact with obtaining or fast, or slower, long-term regulation of response by PGE2.

Synergism between IL-1 and calcium-regulatory GPCRs in synoviocyte, which leads to a rapid sharp rise PGE2 can partly be explained by the rapid strengthening of the release of arachidonic acid, an indicator of the activation of cPLA2 in many types of cells. In addition to the induced expression of MOR-2, IL-1 increases the expression of cPLA2 (Hulkower et al., 1994). These two proteins act together with maintenance-free substrate, arachidonic acid, for MOR-2. Therefore, it can be assumed that the positive regulation of key metabolizing eicosanoids enzymes induced by IL-1 in combination is about the ability of GPCR-ligands to activate the release arachidonate, will increase the overall flow of substrates in the synthesis of prostaglandins. cPLA2 is the only known PLA2, which exhibits functional properties, indicating that regulation of receptors and probably he is involved in the production of eicosanoids and intracellular signal transduction. Since cPLA2 is activated for full activity with increasing concentrations of calcium, and activation of the bradykinin receptor B2 and histamine H1 is associated with mobilization of intracellular calcium, it is likely to be the main factor regulating rapid agonist-stimulated a rapid increase in the production of PGE2. Finally, a very rapid and transient increase in cytoplasmic calcium, running activation of B2-or H1-receptors with similar kinetics, known for cPLA2 activation, release of arachidonic acid and the observed sharp rise in PGE2.

EXAMPLE 7

INHIBITION of a rapid RISE in EDUCATION UNDER the ACTION of PGE2 INHIBITORS CYCLOOXYGENASE

Determined the effect of Ketoprofen, an inhibitor of cyclooxygenase, reducing formation of PGE2 co-incubation with IL-1 during prolonged exposure (16 h) and with a short pre-incubation before the subsequent stimulation of a GPCR agonist, as shown in figure 8. The addition of the indicated concentrations of Ketoprofen during premirovany IL-1 during the night abolished the formation of PGE2 with the value of the IC 50=4,5±0.8 nm, a specific non-linear regression analysis (mean ± average standard error, n=4 line synoviocytes). Such studies (data not shown) were conducted with inhibitors cyclooxygenase etodolac (IC50=15, 2±4, 6 nm, n=4), Ketorolac (2,2±0.4 nm, n=4) and indomethacin (3,2±1.5 nm, n=2).

The figure 8 also shows dependent on the concentration inhibition of agonist-induced sharp rise in PGE2 in response to stimulation with 100 mm histamine (IC503,4±0.2 nm, n=3) or 1 μm bradykinin (IC50a 9.5±2.0 nm, n=3) synoviocyte, premirovany during the night of IL-1 (10 u/ml). These values are comparable with those established for inhibiting the Ketoprofen IL-1 induction during the night PGE2. This result shows that the onset of inhibition under the action of the inhibitor SOH occurs within 10 min pre-treatment before adding the GPCR agonist, which is consistent with a direct, reversible inhibition activity SOKH and not as a result of the mechanism associated with changes in levels of expression of regulatory enzymes of prostanoids. This instant inhibitory action also provides the basis for immediate effectiveness of this drug in the local delivery joint in the irrigating solution during arthroscopic surgery.

EXAMPLE 8

The INDUS IS TION in the production of IL-6 UNDER the ACTION of AGONISTS of IL-1 And GPCR

And INHIBITION by KETOPROFEN

Described the kinetics of induction of interleukin-6 in response to stimulation by IL-1. Culture synoviocytes were subjected to the indicated treatments of IL-1 plus or histamine to activate signaling through a path involving initreport (InsP3)/protein kinase C, or isoproterenol for activation increases intracellular camp. The production of PGE2, IL-6 and IL-8 were determined in the culture supernatant after 1-, 2-, 4-, 6 -, and 24-hour processing. In the given experiment, each treatment was conducted with a separate culture. When the above processing, the production of IL-6 sharply increased under the influence of IL-1 after 24 hours exposure, but IL-6 was not detected within the first 6 hours, the Production of IL-6 in response to IL-1 did not increase with subsequent addition of histamine, and one histamine did not stimulate the production of IL-6. IL-1 also led to a significant increase of IL-8 (2000 PG/ml), which was first detected after 6 h of treatment. The production of IL-8 was constant and was significantly increased at 24-hour exposure to IL-1.

Was to assess the effect of Ketoprofen on the induction of cytokine production under the action of agonists of IL-1 and GPCR. It was also determined dependent on the concentration of the effects of IL-1 on the induction of IL-6 at rest. Culture synoviocytes were exposed to the indicated concentrations of agonists of IL-1 and GPCR. Collected cultural superna the antes and was replaced portions of fresh medium, containing added the same agonist 8-hour periods. Determined PGE2, IL-6 and IL-8 in supernatant as described.

Data on the production of IL-6 are presented in figure 9, which shows the production of IL-6 after 16 h (corresponds to the processing period from 8 to 16 h) in the presence of the indicated concentrations of IL-1 plus the added ligand. Adding histamine or isoproterenol does not increase the production of IL-6 compared with the one of IL-1. When the concentration of IL-1 and 1.0 PG/ml Ketoprofen causes partial (≤50%) inhibition induced IL-1 production by IL-6. In addition, Ketoprofen inhibited the production of IL-6 in the samples together stimulated by histamine or isoproterenol/IL-l samples.

Model culture synoviocytes used to assess the synergistic interactions between IL-1 and recitative inflammatory mediators that are important in the modulation of destruction of articular tissue, including damage which occurs as a consequence of tissue injury during arthroscopic surgery. The results can be summarized as follows: (1) IL-1 induces a significant increase in PGE2, IL-6 and IL-8 in cultured synoviocytes at that time, as the culture at rest does not produce a detected amount of data mediators, (2) induction of PGE2 occurs most rapidly and leads to the release of PGE2 in the culture supernatant after 4 h posleduyushim IL-8 after 6 h and IL-6 after longer intervals of time, and (3) the levels of all three mediators remain elevated in the culture supernatant after 24-hour exposure to IL-1.

In contrast to their effect on the production of PGE2, GPCR agonists did not enhance IL-1 induction of IL-6 or IL-8 and increased the release of IL-6 and IL-8 after premirovanii IL-1. It turned out that the induction of IL-6 and IL-8 under the action of IL-1 increases with concomitant induction of PGE2, since Ketoprofen decreases the production of these cytokines in response to IL-1. This result indicates that Ketoprofen can provide therapeutic for protective cartilage effect upon delivery into the joint during surgical procedures.

Taken together, these results show the interaction between specific modes of transmission of signals involving G-conjugate receptors and activation of synoviocytes or proinflammatory stimulation of IL-1. It is assumed that a similar mechanism operates in chondrocytes. These interactions provide the integration and modulation of Pro-inflammatory responses of synoviocytes and chondrocytes depending on the input of other autocoids or receptor systems of neurotransmitters inside the joint. These findings emphasize the rational and potential clinical benefit of therapeutic interventions, which are aimed at the inhibition associated with G-protein receptors, which mediate the transmission of signals through the mobilization of calcium, hydrolysis of phosphoinositide and activation of the RCC, and they with the voltage with the increase of PGE2 production during arthroscopic surgery. These receptors on synoviocytes and chondrocytes include receptors for histamine H1, bradykinin, substance P, NT and purinergic RU receptors.

1. Method of inhibiting the destruction of cartilage in the joint, including the introduction of a solution comprising (a) a therapeutically effective amount of an anabolic chondroprotective agent is selected from the group consisting of agonists of interleukins (IL), which promote anabolic processes in the cartilage of the members of the superfamily of transforming growth factor-βincluding agonists TGF-β and agonists bone morphogenic proteins, which promote anabolic processes in the cartilage, insulin-like growth factors that promote anabolic processes in the cartilage, and fibroblast growth factors that promote anabolic processes in the cartilage, and (b) a therapeutically effective amount of an inhibitor of catabolism of cartilage selected from the group consisting of antagonists of IL-1 receptors, which inhibit the catabolism of cartilage, antagonists of TNF-αreceptors, which inhibit the catabolism of cartilage-specific inhibitors of cyclooxygenase-2, which inhibit the catabolism of cartilage, inhibitors of Martinez that inhibit the catabolism of cartilage, inhibitors of nitric oxide synthase, which inhibit the catabolism of cartilage, and inhibitors of nuclear factor kB, the cat is which inhibit the catabolism of cartilage, inhibitors of matrix metalloproteinases, which inhibit the catabolism of cartilage, cell adhesion molecules, including agonists of integrins and antagonist integrins, which inhibit the catabolism of cartilage, antigemofilichesky agents that inhibit the catabolism of cartilage, inhibitors of intracellular signaling, including inhibitors of protein kinase C and inhibitors of tyrosine protein kinases that inhibit the catabolism of cartilage, modulators of intracellular (protein-tyrosine-phosphatases that inhibit the catabolism of cartilage, and inhibitors S2 domains that inhibit the catabolism of cartilage, and the solution is delivered locally at the joint of the patient.

2. Method of inhibiting the destruction of cartilage in the joint of a patient, comprising introducing a solution comprising (a) a therapeutically effective amount of at least one anabolic chondroprotective agent is selected from the group consisting of agonists of interleukins (IL), which promote anabolic processes in the cartilage of the members of the superfamily of transforming growth factor-βincluding agonists TGF-β and agonists bone morphogenic proteins, which promote anabolic processes in the cartilage, insulin-like growth factors that promote anabolic processes in the cartilage, and fibroblast growth factors that promote anabolic PR is the processes in cartilage, and (b) at least one other agent, which is an inhibitor of pain or inflammation selected from the group consisting of antagonists of serotonin receptors, agonists of serotonin receptors, antagonists of histamine receptors antagonists bradykininase receptors, inhibitors of kallikrein antagonists tachykinin receptors, receptor antagonists of the peptide linked to the genome of calcitonin (CGRP)antagonists interlacing receptors, inhibitors of enzymes involved in the synthetic pathway arachidonic acid metabolism, antagonists prostanoid receptors, leukotriene receptor antagonists, agonists of opioid receptors, agonists and antagonists purinoceptors, antagonists substances revealing sensitive to adenosine triphosphate (ATP a ) potassium channels and calcium channels, in liquid media locally in the joint of the patient.

3. Method of inhibiting the destruction of cartilage in the joint, including the introduction of a solution comprising a therapeutically effective amount of (a) at least one chondroprotective agent is selected from the group consisting of agonists of interleukins (IL), which promote anabolic processes in the cartilage of the members of the superfamily of transforming growth factor-βincluding agonists TGF-β and agonists bone morphogenic proteins, is the quiet promote anabolic processes in the cartilage, insulin-like growth factors that promote anabolic processes in the cartilage, and fibroblast growth factors that promote anabolic processes in the cartilage, and (b) a therapeutically effective amount of an agent selected from the group consisting of antagonists of IL-1 receptors, which inhibit the catabolism of cartilage, antagonists of TNF-αreceptors, which inhibit the catabolism of cartilage-specific inhibitors of cyclooxygenase-2, which inhibit the catabolism of cartilage, inhibitors of Martinez that inhibit the catabolism of cartilage, inhibitors of nitric oxide synthase, which inhibit the catabolism of cartilage, and inhibitors of nuclear factor kB, which inhibit the catabolism of cartilage, inhibitors of matrix metalloproteinases, which inhibit the catabolism of cartilage, cell adhesion molecules, including agonists integrins and antagonists of integrins, which inhibit the catabolism of cartilage, antigemofilichesky agents that inhibit the catabolism of cartilage, inhibitors of intracellular signaling, including inhibitors of protein kinase C and inhibitors of tyrosine protein kinases that inhibit the catabolism of cartilage, modulators of intracellular (protein-tyrosine)phosphatases that inhibit the catabolism of cartilage, inhibitors of SH2 domains, which inhibit the catabolism of cartilage, antagonists of serotonin receptors, Ah the revisionists serotonin receptors, antagonists of histamine receptors antagonists bradykininase receptors, inhibitors of kallikrein antagonists tachykinin receptors, receptor antagonists of the peptide linked to the genome of calcitonin (CGRP)antagonists interlacing receptors, inhibitors of enzymes involved in the synthetic pathway arachidonic acid metabolism, antagonists prostanoid receptors, leukotriene receptor antagonists, agonists of opioid receptors, agonists and antagonists purinoceptors, antagonists substances revealing sensitive to adenosine-triphosphate (ATP) potassium channels and calcium channels and their mixtures, locally in the joint of the patient in the acute phase after injury of the joint.

4. The method according to claim 1 or 2, in which the solution is delivered into the joint perioperative during the surgical procedure.

5. The method according to claim 1 or 2, including irrigation of the joint solution during surgical procedures.

6. The method according to claim 5, in which the procedure is arthroscopic surgical procedure and the solution is delivered into the joint before, during or after the surgical procedure.

7. The method according to claim 5, in which the procedure is arthroscopic surgical procedure and the solution is delivered into the joint before, during and after surgical procedures.

8. The method according to claim 6, in which a sufficient number is astora is delivered into the joint after surgical procedure so what a bolus of solution remains in the joint of the patient after the surgical procedure.

9. The method according to claims 1, 2 or 3, in which the solution is delivered into the joint intra-articular injection.

10. The method according to claim 9, in which the solution includes a carrier for delivery delayed release.

11. The method according to claim 10, in which the carrier for delivery delayed release selected from the group consisting of microparticles, microspheres, nanoparticles, proteins, liposomes, carbohydrates, synthetic organic compounds and inorganic compounds.

12. The method according to claims 1, 2 or 3, in which the solution is delivered into the joint by infusion.

13. The method according to item 12, in which the solution is delivered into the joint adjustable pump system for delivery.

14. The method according to claims 1 or 2, in which the solution is delivered into the joint for the treatment of chronic degenerative condition of the joints.

15. The method according to claim 1 or 2, in which the solution is delivered into the joint before the alleged injury of tissues in the joint.

16. The method according to claims 1, 2 or 3, in which the solution is delivered into the joint when the damaged joint or immediately after the joint injury.

17. The method according to claim 1 or 2, in which the solution is delivered into the joint during the acute phase after injury of the joint.

18. The method according to 17, in which the solution is delivered during the acute phase after the operation.

19. The method according to 17, or 3, in which the solution is taking Aut in the joint during a four-week period after joint injuries.

20. The method according to claim 1 or 2, in which the solution is delivered into the joint in the subacute phase after injury of the joint.

21. The method according to claim 1 or 2, in which the solution is delivered into the joint in the chronic phase after injury of the joint.

22. The method according to claim 3, in which the chondroprotective agent is an anabolic chondroprotective agent is selected from the group consisting of agonists of interleukins (IL), which promote anabolic processes in the cartilage of the members of the superfamily of transforming growth factor-βincluding agonists TGF-β and agonists bone morphogenic proteins, which promote anabolic processes in the cartilage, insulin-like growth factors that promote anabolic processes in the cartilage, and fibroblast growth factors that promote anabolic processes in the cartilage.

23. The method according to claim 1 or 2, in which an anabolic chondroprotective agent is selected from the group consisting of agonists of interleukins, which promote anabolic processes in the cartilage of the members of the superfamily of transforming growth factor-βincluding agonists TGF-β and agonists bone morphogenic proteins, which promote anabolic processes in the cartilage, insulin-like growth factors that promote anabolic processes in the cartilage, and fibroblast growth factors that promote anabolic PR is the processes in the cartilage.

24. The method according to item 22 or 23, in which an anabolic chondroprotective agent is selected from the group consisting of IL-4, IL-10, IL-13, TGFβ1, TGFβ2, BMP-2, BMP-4, BMP-6, BMP-7, IGF-1, bFGF and fragments, deletions, additions, substitutions of amino acids, mutations and modifications that retain the biological properties of natural agents.

25. The method according to claim 3, in which the chondroprotective agent is an inhibitor of catabolism of cartilage selected from the group consisting of antagonists of IL-1 receptors, which inhibit the catabolism of cartilage, antagonists of TNF-αreceptors, which inhibit the catabolism of cartilage-specific inhibitors of cyclooxygenase-2, which inhibit the catabolism of cartilage, inhibitors of Martinez that inhibit the catabolism of cartilage, inhibitors of nitric oxide synthase, which inhibit the catabolism of cartilage, and inhibitors of nuclear factor kB, which inhibit the catabolism of cartilage.

26. The method according to claim 1, wherein the inhibitor of catabolism of cartilage selected from the group consisting of antagonists of IL-1 receptors, which inhibit the catabolism of cartilage, antagonists of TNF-αreceptors, which inhibit the catabolism of cartilage-specific inhibitors of cyclooxygenase-2, which inhibit the catabolism of cartilage, inhibitors of Martinez that inhibit the catabolism of cartilage, inhibitors of nitric oxide synthase, which inhibit the catabolism of cartilage, and inhibitors poison is REGO factor kB, which inhibit the catabolism of cartilage.

27. The method according to claim 3, in which the chondroprotective agent is an inhibitor of catabolism of cartilage selected from the group consisting of inhibitors of matrix metalloproteinases, which inhibit the catabolism of cartilage, cell adhesion molecules, including agonists integrins and antagonists of integrins, which inhibit the catabolism of cartilage, antigemofilichesky agents that inhibit the catabolism of cartilage, inhibitors of intracellular signaling, including inhibitors of protein kinase C and inhibitors of tyrosine protein kinases that inhibit the catabolism of cartilage, modulators of intracellular (protein-tyrosine)phosphatases that inhibit the catabolism of cartilage, and inhibitors of SH2 domains, which inhibit the catabolism of cartilage.

28. The method according to claim 1, wherein the inhibitor of catabolism of cartilage selected from the group consisting of inhibitors of matrix metalloproteinases, which inhibit the catabolism of cartilage, cell adhesion molecules, including agonists integrins and antagonists of integrins, which inhibit the catabolism of cartilage, antigemofilichesky agents that inhibit the catabolism of cartilage, inhibitors of intracellular signaling, including inhibitors of protein kinase C and inhibitors of tyrosine protein kinases that inhibit the catabolism of cartilage, modulators of intracellular (protein-slapshot is in-phosphatase, which inhibit the catabolism of cartilage, and inhibitors of SH2 domains, which inhibit the catabolism of cartilage.

29. The method according to claim 3, in which chondroprotective agent comprises a soluble receptor that inhibits the catabolism of cartilage.

30. The method according to claim 1, wherein the inhibitor of catabolism of cartilage consists of a soluble receptor that inhibits the catabolism of cartilage.

31. The method according to clause 29 or 30, in which soluble receptor selected from the group consisting of a soluble receptor of interleukin-1 and soluble receptors of factors tumor necrosis.

32. The method according to clause 29 or 30, in which soluble receptor selected from the group consisting of soluble receptors recombinant human IL-1 soluble receptor factors tumor necrosis and chimeric rhTNFR:Fc.

33. The method according to claim 1 or 3, wherein the solution further includes one or more agents, inhibiting pain or inflammation.

34. The method according to claim 2 or 33, in which agents inhibiting pain and inflammation selected from the group consisting of antagonists of serotonin receptors, agonists of serotonin receptors, antagonists of histamine receptors antagonists bradykininase receptors, inhibitors of kallikrein antagonists tachykinin receptors, receptor antagonists of the peptide linked to the genome of calcitonin (CGRP)antagonists interlacing Rotz is tori, inhibitors of enzymes involved in the synthetic pathway arachidonic acid metabolism, antagonists prostanoid receptors, leukotriene receptor antagonists, agonists of opioid receptors, agonists and antagonists purinoceptors, antagonists substances revealing sensitive to adenosine triphosphate (ATP) potassium channels and calcium channels.

35. The method according to claim 1 or 2, in which the solution is applied topically prophylaxis in joint replacement patients.

36. The method according to claim 4, in which perioperative shipping solution includes the delivery during the procedure, together with delivery of the solution before the procedure or after the procedure.

37. The method according to claim 4, in which perioperative shipping solution includes the delivery before the procedure together with the delivery of the solution after the procedure.

38. The method according to claim 4, in which perioperative application of the solution includes the use of a solution before the procedure, during the procedure and after the procedure.

39. The method according to claims 1, 2 or 3, further comprising the stage of identification of the patient with the risk of destruction of cartilage in the joint, with subsequent delivery of the solution into the joint identified patient.

40. The method according to claims 1, 2 or 3, in which each of the agents included in the solution in the concentration or dose that is sufficient to provide the level of inhibition or therapeutic effect is in the wound for local delivery to the wound, and which results in a plasma concentration that is less than the concentration in plasma, which is required to achieve the same level of inhibition or therapeutic effect in the wound with systemic delivery.

41. Solution for application to suppress the destruction of cartilage, comprising (a) a therapeutically effective amount of an anabolic chondroprotective agent is selected from the group consisting of agonists of interleukins (IL), which promote anabolic processes in the cartilage of the members of the superfamily of transforming growth factor-βincluding agonists TGF-β and agonists bone morphogenic proteins, which promote anabolic processes in the cartilage, insulin-like growth factors that promote anabolic processes in the cartilage, and fibroblast growth factors that promote anabolic processes in the cartilage, and (b) a therapeutically effective amount inhibitor catabolism of cartilage selected from the group consisting of antagonists of IL-1 receptors, which inhibit the catabolism of cartilage, antagonists of TNF-αreceptors, which inhibit the catabolism of cartilage-specific inhibitors of cyclooxygenase-2, which inhibit the catabolism of cartilage, inhibitors of Martinez that inhibit the catabolism of cartilage, inhibitors of nitric oxide synthase, which inhibit the catabolism of cartilage and is hibition nuclear factor kB, which inhibit the catabolism of cartilage, inhibitors of matrix metalloproteinases, which inhibit the catabolism of cartilage, cell adhesion molecules, including agonists integrins and antagonists of integrins, which inhibit the catabolism of cartilage, antigemofilichesky agents that inhibit the catabolism of cartilage, inhibitors of intracellular signaling, including inhibitors of protein kinase C and inhibitors of tyrosine protein kinases that inhibit the catabolism of cartilage, modulators of intracellular (protein-tyrosine)phosphatases that inhibit the catabolism of cartilage, and inhibitors of SH2 domains, which inhibit the catabolism of cartilage, for local delivery in the joint.

42. The solution is to apply for inhibiting the destruction of cartilage, comprising (a) a therapeutically effective amount of at least one anabolic chondroprotective agent is selected from the group consisting of agonists of interleukins (IL), which promote anabolic processes in the cartilage of the members of the superfamily of transforming growth factor-βincluding agonists TGF-β and agonists bone morphogenic proteins, which promote anabolic processes in the cartilage, insulin-like growth factors that promote anabolic processes in the cartilage, and fibroblast growth factors that promote anabolic processes in HRA is e, and (b) at least one other agent, which is an inhibitor of pain or inflammation selected from the group consisting of antagonists of serotonin receptors, agonists of serotonin receptors, antagonists of histamine receptors antagonists bradykininase receptors, inhibitors of kallikrein antagonists tachykinin receptors, receptor antagonists of the peptide linked to the genome of calcitonin (CGRP)antagonists interlacing receptors, inhibitors of enzymes involved in the synthetic pathway arachidonic acid metabolism, antagonists prostanoid receptors, leukotriene receptor antagonists, agonists of opioid receptors, agonists and antagonists purinoceptors, antagonists substances revealing sensitive to adenosine triphosphate (ATP a ) potassium channels and calcium channels, for local delivery in the joint.

43. The solution according to paragraph 41 or 42, in which an anabolic chondroprotective agent is selected from the group consisting of agonists of interleukins (IL), which promote anabolic processes in the cartilage of the members of the superfamily of transforming growth factor-βincluding agonists TGF-β and agonists bone morphogenic proteins, which promote anabolic processes in the cartilage, insulin-like growth factors that promote the anabolic process is m in the cartilage, and fibroblast growth factors that promote anabolic processes in the cartilage.

44. The solution according to item 43, in which an anabolic chondroprotective agent is selected from the group consisting of IL-4, IL-10, IL-13, TGFβ1, TGFβ2, BMP-2, BMP-4, BMP-6, BMP-7, IGF-1, bFGF and fragments, deletions, additions, substitutions of amino acids, mutations and modifications that retain the biological properties of natural agents.

45. The solution according to paragraph 41, in which the inhibitor catabolism of cartilage selected from the group consisting of antagonists of IL-1 receptors, which inhibit the catabolism of cartilage, antagonists of TNF-αreceptors, which inhibit the catabolism of cartilage-specific inhibitors of cyclooxygenase-2, which inhibit the catabolism of cartilage, inhibitors of Martinez that inhibit the catabolism of cartilage, inhibitors of nitric oxide synthase, which inhibit the catabolism of cartilage, and inhibitors of nuclear factor kB, which inhibit the catabolism of cartilage.

46. The solution according to paragraph 41, in which the inhibitor catabolism of cartilage selected from the group consisting of inhibitors of matrix metalloproteinases, which inhibit the catabolism of cartilage, cell adhesion molecules, including agonists integrins and antagonists of integrins, which inhibit the catabolism of cartilage, antigemofilichesky agents that inhibit the catabolism of cartilage, inhibitors of intracellular signaling, including inhibitory protein kinases and inhibitors of tyrosine protein kinases, which inhibit the catabolism of cartilage, modulators of intracellular (protein-tyrosine-phosphatases that inhibit the catabolism of cartilage, and inhibitors S2 domains that inhibit the catabolism of cartilage.

47. The solution according to paragraph 41, in which the inhibitor catabolism of cartilage consists of a soluble receptor that inhibits the catabolism of cartilage.

48. The solution p in which soluble receptor selected from the group consisting of a soluble receptor of interleukin-1 and soluble receptors of factors tumor necrosis.

49. The solution p in which soluble receptor selected from the group consisting of soluble receptors recombinant human IL-1 soluble receptor factors tumor necrosis and chimeric rhTNFR:Fc.

50. The solution according to paragraph 41, in which the solution further includes one or more inhibitors of pain or inflammation.

51. The solution according to § 42 or 50, in which agents that inhibit pain or inflammation selected from the group consisting of antagonists of serotonin receptors, agonists of serotonin receptors, antagonists of histamine receptors antagonists bradykininase receptors, inhibitors of kallikrein antagonists tachykinin receptors, receptor antagonists of the peptide linked to the genome of calcitonin (CGRP)antagonists interlacing receptors, inhibitors of enzymes involved in sin is eticheski way of arachidonic acid metabolism, antagonists prostanoid receptors, leukotriene receptor antagonists, agonists of opioid receptors, agonists and antagonists purinoceptors, antagonists substances revealing sensitive to adenosine triphosphate (ATP) potassium channels and calcium channels.

52. The solution according to paragraph 41 or 42, in which the solution includes a carrier for delivery delayed release.

53. The solution according to paragraph 52, in which the carrier for delivery delayed release selected from the group consisting of microparticles, microspheres, nanoparticles, proteins, liposomes, carbohydrates, synthetic organic compounds and inorganic compounds.

54. The solution according to paragraph 41 or 42, in which each of the agents in the solution is included at a concentration or dose that is sufficient to provide the level of inhibition or therapeutic effect in the wound for local delivery to the wound, and which results in a plasma concentration that is less than the concentration in plasma, which is required to achieve the same level of inhibition or therapeutic effect in the wound when sistemini delivery.



 

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FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to novel substituted 2-aryl-3-(heteroaryl)imidazo[1,2-a]-pyrimidines of the formula (I):

or to their pharmaceutically acceptable salts wherein: (a) R1 is taken among the group consisting of -NH2, C1-5-alkylamino-, di-C1-5-alkylamino-, phenylmethylamino-group; (b) Y is taken among the group consisting of hydrogen atom (H), halogen atom, piperidine, OR4, SR4, -SO2CH3, NHR4 and NR4R5 wherein R4 and R5 are taken independently among hydrogen atom (H), α-alkylphenyl-C1-5-alkyl, linear or branched alkyl substituted optionally with C3-5-carbocycle, phenyl or substituted phenyl wherein indicated phenyl can be substituted with one or some substituted taken among C1-5-alkoxy-group; (c) R2 represents from one to five members taken independently among the group including hydrogen atom (H), halogen atom, trifluoromethyl; (d) R3 represents hydrogen atom (H), or radicals R3 taken in common form aromatic ring; (e) X represents nitrogen atom (N) or -CH. Also, invention relates to methods for preparing indicated compounds and to a method for treatment based on these compounds. Invention provides preparing novel compounds that can be used in relief states by reducing the level of inflammatory cytokines, for example, the indicated state represents proliferative (rheumatic) arthritis.

EFFECT: valuable medicinal properties of compounds and compositions.

40 cl, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention proposes a method for inhibition of chorionic neovascularization. Method involves irradiation of undesirable novel vascular reticulum in combination with photosensitive agent (porphyrine) and an anti-angiogenic agent taken among antagonist of phospholipase A2, inhibitor of complex kappa B, inhibitor of the growth hormone, inhibitor of insulin-like growth factor-1, inhibitor of cyclooxygenase II, inhibitor of protein kinase C (stautosporin PKC 412) and inhibitor of angiotensin II. The claimed combined treatment provides potentiation of effect of adjunctive photodynamic therapy in combination with enhanced safety.

EFFECT: improved treatment method.

7 cl

Nutrient module // 2268069

FIELD: medicine.

SUBSTANCE: method involves adding to patient food one or several compositions via closed system in module form containing nutrient substances selected and adapted to given clinical state. The modules are most kept in dry form.

EFFECT: enhanced effectiveness in preparing patient food; avoided contamination with microorganisms; enhanced effectiveness in treating and feeding patients.

20 cl

FIELD: medicine.

SUBSTANCE: method involves administering a combination of an agent reducing cholesterol content in blood and reduced coenzyme Q10 of general formula .

EFFECT: enhanced effectiveness of treatment.

8 cl, 2 tbl

FIELD: medicine, pharmacy.

SUBSTANCE: invention proposes implant prepared by mixing a carrier material with components of the preparation antibiotic/antibiotics with delayed release of an active substance (aminoglycoside, lincosamide antibiotics, 4-quinolone antibiotics and tetracyclines), and a method for preparing the implant. Release of an active substance from implant during from some days to some weeks doesn't dependent from a carrier material and adsorption effects of a carrier-material surface.

EFFECT: improved and valuable properties of preparation.

13 cl, 1 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: the present innovation deals with composition for fertilization in vitro and the system for its delivering (device). The composition suggested contains steroid at the quantity of below 5% (weight/weight), that is: 4.4-dimethyl-5α-cholesta-8.14.24-trien-3β-ol, hemisuccinate of 4.4-dimethyl-5α-cholest-8.14.24-trien-3β-ol; 5α-cholest-8.14-dien-3β-ol; hemisuccinate of 5α-cholest-8.14-dien-3β-ol; (20S)-cholest-5-en-3, 20-diol; N-(methionine)amide of 3β-hydroxy-4.4-dimethyl-5α-chol-8.14-dien-24-oic acid or cholest-5-en-16β-ol, and, also, additive (water-soluble protein or phosphoglyceride). Delivering system has got either one foramen or one cavity that contains the composition mentioned as a solid product or solution. The composition of sterols contains no constituents negatively affecting oocytes and could be dissolved in aqueous medium without physical impact (that is, heating, mixing or ultrasound treatment).

EFFECT: higher efficiency of fertilization in vitro.

8 cl, 5 ex, 3 tbl

FIELD: medicine, narcology, pharmacy.

SUBSTANCE: invention proposes applying the following agonists of gamma-aminobutyric acid receptors of B-type: β-(4-chlorophenyl)-GABA (Baclofen), 3-aminopropyl(methyl)-phosphinic acid, 3-aminopropylphosphinic acid, Y-amino-β-4-(4-chlorophenyl)-nitropropane or their salts, esters, ethers, complexes and their corresponding isomers used in treatment of nicotine dependence. Invention provides the selective suppression of smoking addiction and effect on behavior indices causing the abuse relapse and with absence of symptoms typical in nicotine dependence.

EFFECT: valuable properties of compounds.

9 cl

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to a pharmaceutical composition used in treatment or prophylaxis of hypertension, heart diseases, vascular disorders and kidney diseases. The composition comprises compound of the formula (1) as antagonist of angiotensin II receptors and one or some diuretics. The composition shows enhanced effectiveness.

EFFECT: valuable medicinal properties of composition.

23 cl, 2 tbl, 1 ex

FIELD: medicine, chemical-pharmaceutical industry.

SUBSTANCE: invention relates to new applying EP4 receptors agonist for treatment and/or prophylaxis of diseases associated with loss of osseous mass. Agonists of EP4 receptors show high effectiveness in treatment of diseases associated with loss of osseous mass, among the, as osteoporosis of different genesis. Agonists of EP4 receptors involve prostaglandin skeleton base.

EFFECT: valuable medicinal properties of pharmaceutical composition.

16 cl, 3 tbl, 5 ex

FIELD: medicine, biochemistry.

SUBSTANCE: invention relates to a method for treatment of pathology or disorder taken among inflammatory diseases. Method involves using the simultaneous, separating or distributed by time at least a single substance inhibiting activity of NO-synthase and at least a single substance inhibiting activity of phospholipase A2. Also, invention relates to the composition comprising at least a single substance inhibiting activity of NO-synthase and at least a single substance inhibiting activity of phospholipase A2 for using in the method for treatment of pathology or disorder taken among inflammatory diseases.

EFFECT: improved method for treatment.

21 cl, 4 ex

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to a pharmaceutical composition used in treatment or prophylaxis of HIV-infection in humans that comprises effective amount of β-D-D4FC or its pharmaceutically acceptable salts or a prodrug being optionally in a pharmaceutically acceptable carrier in combination with effective amount of compound being optionally in pharmaceutically acceptable carrier that is taken among the group consisting of indinavir, nelfinavir, saquinavir, amprenavir, efaverenza, delavirdin, nevirapin and abacavir as a single preparation and to a method for treatment or prophylaxis of HIV-infection in humans that involves administration of effective amount of this composition. Pharmaceutical composition elicits the preferred or improved pharmacokinetic parameters, parameters of biological distribution, metabolic parameters, parameters of resistance and other parameters as compared with administration of β-D-D4FC only.

EFFECT: improved method for treatment and prophylaxis, valuable properties of composition.

17 cl, 6 tbl, 2 dwg

FIELD: pharmaceuticals industry, in particular new method for production of alpha-1-antitrypsin and pharmaceutical product containing the same.

SUBSTANCE: alpha-1-antitrypsin is isolated from Cohn fraction IV-1 by solubilization. Then protein admixtures and eventual viral particles are removed by polyethylene glycol, target protein is precipitated with zinc salt, viral inactivation using solvent/detergent is carried out, product is fractionated using Q-sepharose, and non-active alpha-1-antitrypsin is removed with S-sepharose to produced target product. Said product represents concentrate of human serum active alpha-1-antitrypsin having purity more than 98 % and specific activity not less than 40 IU/mg in 0.15 M sodium chloride solution.

EFFECT: increased yield of high pure active alpha-1-antitrypsin.

4 cl

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