The use of derivatives to a for the treatment of disorders of the peripheral or central nervous system and excessive secretion of the cytokine

 

(57) Abstract:

The invention relates to medicine and relates to methods of suppressing excessive secretion of tumor necrosis factor alpha and interleukin-1 beta, as well as the treatment of diseases associated with excessive synthesis of these cytokines, Alzheimer's and Parkinson's disease using the derived indolocarbazole K-252a. The invention improves the efficacy of treatment. 6 C. and 2 h.p. f-crystals, 4 tab., 5 Il.

The invention relates to substituted in the ring derivative To-a for use in the methods aimed at alleviating the damaging effects of various diseases, disorders and conditions.

BACKGROUND OF THE INVENTION

1. Indolocarbazole To-a

K-a represents a connection with indolocarbazole skeleton, [Japanese publishing a non-examination of the patent applications 41489/85 (USA 4555402)] with the stereochemistry shown in formula I:

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Earlier it was reported that K-a is a potent inhibitor of protein kinase C (CSW), which plays a Central role in the regulation of cellular functions and has various kinds of activity, such as inhibitory effect on smooth muscle contraction (Jpn. J. Pharmacol., 43 (suppl.): 284, e axons (neurons) (J. Neuroscience, 8: 715, 1988), inhibitory effect on the release of histamine (Allergy, 43: 100, 1988), inhibitory effect on smooth muscle MLCK (J. Biol. Chem. , 263: 6215, 1988), anti-inflammatory action (Acta Physiol. Hung, 80: 423, 1992) and the ability to influence the survival of cells (J. Neurochemistry, 64: 1502, 1995). In Experimental Cell Research., 193: 175-182, 1991, it was shown that K-a suppress the production of interleukin 2.

In addition, it was reported that derivatives To a inhibit the activity of the RCC, inhibit the secretion of histamine [Japanese publishing a non-examination of the patent applications 295588/88] , have antitumor activity [Japanese published a non-examination of the patent applications 168689/89 (USA 4877776), WO 88/07045 (USA 4923986), WO 94/04541 able to increase the number of platelets [WO 94/06799 (EP 630898 A)], have vasodepressor activity [Japanese published a non-examination of the patent applications 120388/87/89], can accelerate cholinergic function of neurons [WO 94/02483 (USA 5461146 and USA 5621100)] and to have a therapeutic effect in prostate cancer [WO 94/27982 (USA 5516771)]. Selected aminobenzamide trentanove compounds were obtained by the Beckmann rearrangement of the corresponding staurosporine Asimov (WO 97/05140).

Indolocarbazole generally lipophilic. C biological membranes. Also indolocarbazole in General are longer than the protein half-life in vivo.

Additionally, the self-a, its various derivatives were synthesized and tested for biological activity. It is shown that among the derivatives of K-a biological activity has a connection that is opened to Lewis et al. in U.S. patent 5461146 and 5621100 and PCT publication WO 94/02488 and referred to them as "compound II-51". It is shown that compound II-51 strengthens the function of cholinergic neurons, veins neurons and sensory neurons.

2. Neurodegenerative diseases and disorders

Parkinson's disease is a neurodegenerative disorder that causes progressive and selective loss of dopaminergic neurons in the nigro-veins path (Agid, Lancet: 337:1991). Introduction 1-methyl-4-phenyl-1, 2, 3, 5-tetrahydropyridine (MRTR) mice leads to the degeneration of dopaminergic neurons and serves as an experimental model for the loss of dopaminergic neurons and behavioral disturbances observed in Parkinson's disease. Peripheral application MRTR leads to highly selective degeneration of the dopaminergic system nigro veins neurons in humans, monkeys and mice (Heikkila et al., Science 224: the th, adequately described. System introduction MRTR causes selective selective loss of the contents of dopamine (and metabolites), the activity of tyrosine hydroxylase and sites of absorption of dopamine in the dopaminergic neurons of the striatum mice (Heikkila et al., Nature, 311: 467-469, 1984, a, b; Tipton et al., J. Neurochem., 61: 1191-1206, 1993). This effect is dose-dependent. Maximum loss occurs between the third and seventh day after the defeat (Jackson-Lewis et al., Neurodegeneration, 4: 257-269, 1955). Organelles dopaminergic cells of the black substance is less sensitive to the toxic effects MRTR than their corresponding nerve endings. At high doses MRTR or multiple injections MRTR significant loss TN-immunologically cells in a black substance occurs within 1 week(Heikkila et al., Science, 224: 1451-1453, 1984; Jackson-Lewis et al., Neurodegeneration, 4: 257-269, 1995). At lower doses MRTR or when a single injection loss black substance of tyrosine hydroxylase - positive cells occurs later (Tatton et al. , J. Neuroscience. Res., 30: 666-672, 1991). Thus, at lower doses MRTR and a short period after damage to the veins of the lesion can be observed in the absence of loss of tyrosine hydroxylase - positive cells black substance. the eh is a recognized and widely used model for studying Parkinson's disease using MRTR in mice.

Nehallenia neurons, using as neurotransmitter gamma aminobutyric acid (GABA) (the so-called GABA-ergicheskie neurons distributed throughout the brain volume. For example, they are found in both the basal node in rodents (similar region in the brain called the basal nucleus of Meynert), basal area anterior of the brain that plays an important role for functions of attention and memory. Damage GABAergic neurons in the basal anterior brain can also cause behavioral disorders in neurodegenerative diseases such as Alzheimer's disease (Dekker et ai., Neurosci and Biobeh. Rev., 15: 299-317, 1991; Gallagher et al., Seminars in Neuroscience, 6: 351-358, 1994, Torres et al., Neuroscience, 63: 95-122, 1994).

Neurons in the basal anterior brain die at the several diseases of the Central nervous system, in particular in Alzheimer's disease. (Arendt et al., Acta Neuropathol. (Berl.), 61: 101-108, 1983; Iraizoz et al. , Neuroscience, 43: 33-40, 1991; Vogels et al., Neurobiol. Aging, 11: 3-13, 1990). An important factor in the death of these neurons is associated with overstimulation of the glutamate toxicity, i.e., the hyperstimulation of neurons excess glutamate (Choi, Neuron, 1: 623-634, 1988). Accordingly, in some experimental models of the disease, the Social divisions of the forebrain, where the death of neurons, i.e., in both the basal node (Wenk, Ben. Brain Res., 72: 17-24, 1996).

First, the pathology of neurons in Alzheimer's disease observed in the cortex, and as the disease progresses, the loss of neurons in this area becomes severe (Braak et al., Acta Neuropathol, 82: 239-259, 1991; Hyman et al., Ann Neurol. 20: 472-481, 1986). Neurons in the 2nd layer entorhinal crust is projected in the dentate gyrus of the hippocampus, and this neural path plays an important role in the formation of memory (Levisohnet et al., Brain Res., 564: 230-244, 1991; Olton et al., Brain. Res., 139: 295-308, 1978; Stewart et al. , Brain. Res. Bull. , 2: 41-48, 1977). Neurons in the 2nd layer entorhinal crust, like many other neurons of the cerebral cortex, using glutamate as a neurotransmitter (Mattson et al., Neuron, 1: 865-876, 1988; White et al. , Nature, 270: 356-357, 1977). Thus, the loss of glutamatergic neurons in entorhinal cortex affects behavior disorders observed in Alzheimer's disease and other neurological disorders.

3. Peripheral neuropathy

Peripheral neuropathy is usually referred to as a disorder that affects the peripheral nerves, most often manifested as isolated or combined impaired motor, sensory, sensorimotor or avenged to be surprising due to the large variety of reasons. For example, peripheral neuropathy can be acquired genetically, can result from systemic disease or may be due to a toxic agent. Neurotoxic effects may have drugs, antineoplastic agents, impurities in foods or medications, to factors of environmental pollution and industrial pollution factors.

In particular, known chemotherapeutic agents, which cause sensory and/or motor neuropathies, including vincristine, antitumor agent used for the treatment of malignant hematological diseases and sarcomas. Neurotoxicity is dose-related and is manifested in the form of reduced motility of the intestine and peripheral neuropathy, particularly of the distal muscles of the hands and feet as postural hypotension and bladder atony. Similar violations were noted when using Taxol and cisplatin (Mollman, 1990, New Eng. Jour. Med., 322: 1266-1267), although associated with cisplatin neurotoxicity can be facilitated by nerve growth factor (NGF) (Apfel et al., 1992, Annals neurolology, 31: 76-80). Although neurotoxicity sometimes is reversible after discontinuation neurotoxic agent, recovery can nasledstvennykh peripheral neuropathies, including disease Refsum And-betalipoprotein, disease Tangier disease Crabs, the metachromatic leukodystrophy, Fabry disease, syndrome Dejerine-Sottas and others. Among all hereditary neuropathies, has long been known, the most common is the disease Charcot-Marie-Tusa (for more information on peripheral neuropathies see also U.S. patent 5420112).

4. Cytokines

As is known, tumor necrosis factor alpha(TNF-) and interleukin-1 (IL-1) are polypeptides involved in a number of inflammatory and metabolic processes in vivo. Information on the role of TNF in inflammatory diseases, including septic shock, see also Ann. Rev. Imunnol. 7: 625 (1980) and Clinical Trials for Treament of Sepsis, Sibbald W. J. and Vincent, J.-L. (Eds), Springer-Verlag, Berlin, Heidelberg, 1995. It is generally accepted that excessive or inappropriate production of TNF - responsible for a number of pathological conditions, including septic shock (Spooner et al., Clinical Immunology and Immunopathology, 62: p. S11 (1992), and various other allergic and inflammatory conditions or diseases, including, but not limited to rheumatoid arthritis, osteoarthritis, asthma, bronchitis, chronic obstructive airway disease, psoriasis, allergic rhinitis, dermatitis and inflammatory Soboleva

THE INVENTION

In General, the invention is ways to alleviate the harmful effects of various diseases, disorders and conditions of use treatment need it the subject a therapeutically effective amount of compound A.

More specifically, the invention provides a method of treatment of the harmful effects of diseases, disorders and conditions, resulting in death or which causes or leads to the suppression of certain neurons or which is caused by gain of function or survival of dopaminergic, GABAergic or glutamatergic neurons in a mammal, comprising the step of contacting the neuron with a connection A. Typically a mammal, which is detected by the neuron, is the man. Usually the function of dopaminergic, GABAergic or glutamatergic neurons exposed to the compound And disturbed or threatened by the risk of death from neurodegenerative diseases. Usually a neurodegenerative disease is Parkinson's disease or Alzheimer's disease.

More specifically, the invention also provides a method of reducing peripheral neuropathy, including the introduction melinoe connection To a reduces mortality due to the introduction of endotoxin, this ability was associated with ability To a to inhibit protein kinases, in particular the protein kinase C (lhaba et al., Jpn. J. Surg., 23: 234 (1993). It has been unexpectedly found that the compound a, which has a small inhibitory effect on RCC or even no, exhibits surprisingly high activity as an inhibitor of the production of TNF - and cytokine IL-l. Therefore, another feature of the invention is that it provides a method of inhibiting the production of TNF and IL-l in a mammal and a method of treating or alleviating inflammatory conditions or diseases, including, but not limited to septic shock, rheumatoid arthritis, osteoarthritis, asthma, bronchitis, chronic obstructive airway disease, psoriasis, allergic rhinitis, dermatitis, inflammatory bowel disease and other autoimmune diseases, and the method includes the introduction of the specified mammal an effective amount of the compound and its pharmaceutically acceptable salts in combination with a pharmaceutically acceptable carrier.

Used here, the term "compound a" refers to a compound, chemical structure of which is presented below:

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Connection And"facilitate" or "facilitate" means therapeutically to improve and/or therapeutically to reduce and/or do more therapeutically portable.

Used here, the term "hazardous" means damaging and/or dangerous and/or negative.

Used here the word "excess production", when used to indicate changes produce TNF and/or IL-l, indicates the production of TNF and/or IL-l, leading to pathological conditions such as septic shock, allergic conditions, inflammatory conditions and so on

Used here, the terms "inhibit" or "inhibiting" means that the connection exists And has a relatively greater effect on the reduction and/or termination, and/or preventing the production of material in contact with the connection Rather than the comparative material is not contacted by the connection A.

Used here, the terms "enhance" or "enhancing" when used for modification of the terms "function" or "survival" means that the connection exists And has a relatively larger effect on the function and/or survival of a particular neuron than comparative neuron, not exposed to the compound A. for Example (but not limitation), with regard to survival, for example, dopaminergic under threat of death (due to damage, pathological conditions, degenerative conditions or natural progression) when compared with the population of dopaminergic neurons, not in contact with the connection And, if subjected to the action of the population has a relatively longer period of functionality than the non-treated population.

Used here, the term "dopaminergic neuron" refers to a neuron that uses as a neurotransmitter dopamine.

Used here, the term "GABA-ergicheskie neuron" refers to a neuron that uses as a neurotransmitter gamma-aminobutyric acid.

Used here, the term "glutamatergic neuron" refers to a neuron that uses as a neurotransmitter to glutam.

Used here, the term "nbm" refers to both the basal nucleus.

In the absence of other definitions used here is the technical and scientific terms have the same meaning as the terms are usually understood by an expert in this area, which is included in this invention. Although methods and materials similar or equivalent described herein can further be used in practice or when appetency, patents and other references mentioned herein are incorporated as references. In the event of a conflict this document, including definitions, will serve as a control. In the absence of other definitions described here, the materials, methods and examples are illustrative only and are not intended to be limiting. Various characteristics and advantages of the invention will become apparent from the following detailed description and from the claims.

BRIEF DESCRIPTION OF FIGURES

Fig.1 is a graphical representation of data showing that the compound is not an inhibitor of monoamine oxidase A. IR50for clorgyline amounted to 21 nmol. Triangles up for the top - clorgyline; inverted triangles - connection A; squares - L-deprenyl.

Fig.2 is a graphical representation of data showing that the compound is not an inhibitor of monoamine oxidase Century IR50for clorgyline amounted to 21 nmol. Triangles up for the top - clorgyline; inverted triangles - connection A; squares - L-deprenyl.

Fig. 3 is a bar chart showing that animals treated with compound A (light bars) had significantly is idene), compared with the damage to animals receiving only the vehicle (dark bars). *=P<0.05 criterion Newman-Keuls.

Fig. 4 is a bar chart showing the total number of errors committed when performing varying tasks in the T-shaped maze rewarded alternation, compared with the control group, falsely operated animals, animals with surgical damage, received only the carrier (the"damage.") and animals with surgical lesions treated with compound a at a dose of 0.1 mg/kg 1 p/d ("0.1"). The number of animals in each group is shown in parentheses *=P<0.05 compared to falsely operated 11= P<0.01 compared with (damaged.) using the criteria Newman-Keuls.

Fig. 5 shows the influence of the connection And caused by acrylamide peripheral neuropathy.

DETAILED DESCRIPTION OF THE INVENTION

In General, the invention describes methods of alleviating the harmful effects of various diseases, disorders and conditions of use treatment need it the subject a therapeutically effective amount of compound A.

This invention provides a method of treatment of the harmful effects of diseases, disorders and SOS is due to such diseases and disorders by enhancing the function or survival of specific types of neurons of the Central nervous system of mammals. More specifically, the invention provides a method to enhance the function or survival of dopaminergic neurons, GABAergic neurons and glutamatergic neurons in a mammal by administering to the mammal the compound, substituted in the ring derivative To-a with the following chemical structure:

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Dopaminergic neurons, GABA-ergicheskie neurons, glutamatergic neurons are widely distributed in the Central nervous system of mammals. Each of these three types of neurons undergoes a disturbance of function or even death when one or more neurodegenerative diseases of the Central nervous system. In Parkinson's disease is a progressive loss of dopaminergic neurons nigrostriarnae. In Alzheimer's disease is the loss of different types of neurons, including GABA-ergicheskie neurons in the basal nucleus of Meynert basal anterior brain. In Alzheimer's disease is loss of glutamatergic neurons in entorhinal region of the cerebral cortex.

One method of treating Parkinson's disease or Alzheimer's disease is the introduction of a compound that enhances the function or survival of topalski actively in biological quantitative definitions and models in vivo enhanced the function or survival of dopaminergic neurons, GABAergic neurons and glutamatergic neurons. Thus, the present invention is applicable for the treatment of Parkinson's disease or Alzheimer's disease. The invention, however, is not limited to the treatment of these diseases. The use of the compounds And to enhance function or survival of dopaminergic neurons, GABAergic neurons, or glutamatergic neurons, whose function is compromised or the risk of death occurs as a result of other causes than Parkinson's disease or Alzheimer's disease, is also in the scope of claims of the present invention.

The invention also discloses a method of reducing peripheral neuropathy. The method comprises the administration to a mammal to reduce neuropathy amount of compound A. In various preferred embodiments of the mammal is a human or agricultural or domestic mammals, which develops neuropathy, for example, in the treatment of the tumor with a chemotherapeutic agent. Connection And can be entered by way considered to be effective, the person skilled in the art, the preferred method of administration is subcutaneous injection.

Used here telmisartane includes the use of compounds And to reduce neurotoxicity, including, but not limited to, distal sensory-motor neuropathy or autonomic neuropathy, such as reducing the contractility of the gastrointestinal tract or atony of the urinary bladder.

Preferred neuropathy, which can be effectively cured by the connection And include neuropathies associated with systemic disease, such as post-polio syndrome; genetically acquired neuropathies, such as disease Charcot-Marie-Tusa; and neuropathies caused by a toxic agent, such as acrylamide, or a chemotherapeutic agent such as vincristine.

When the connection is used for the treatment of neuropathies caused by a toxic agent, it can be administered before, simultaneously with or after exposure to a toxic agent, or before, during or after administration of a chemotherapeutic drug. Preferably, the compound a and the chemotherapeutic agent is administered through the effective time intervals during the overlapping period of treatment. The compound a may be administered to the mammal after exposure to a neurotoxic agent, or after chemotherapy to restore at least part of the neurological functions, Narechenski agent, providing neurotoxic effects, such as vincristine, Taxol, dideoxyinosine or cisplatin.

Under "toxic agent" or "neurotoxic agent" means a substance that through its chemical action of damages, disrupts or inhibits the activity of a component of the nervous system. There is a long list of neurotoxic agents that cause neuropathy, including but not limited to, antineoplastic agents, such as vincristine, vinblastine, cisplatin, Taxol, or dideoxy compounds, such as dideoxyinosine; alcohol; metals; industrial toxins, contact with which the industrial or the environment; impurities in food or drugs or overdosing of vitamins or therapeutic drugs, such as antibiotics, such as penicillin or chloramphenicol, or megadose vitamins A, D, or B6. Extensive, though not the full list of chemical compounds with neurotoxic side effects are presented in table 1. Although this list provides examples of neurotoxic compounds, it is intended to serve as an example and not to limit the claims of the invention. Other toxic agents can cause the influence of a toxic agent" means, that a toxic agent becomes available or comes into contact with the mammal of the invention. Exposure to a toxic agent may occur by direct injection, for example by ingestion or administration with food, drugs or therapeutic agents, such as chemotherapeutic agents, accidental contamination or contact with the environment, for example through contact with air or water.

Despite the disparate morphology and causes peripheral neuropathy in vivo, the applicants suggested that the compound may be effective in the prevention or treatment of neuropathies in a mammal.

It was also shown that compound a inhibits the production of the cytokine TNF-despite the absence or low inhibitory activity against RCC. Connection And also suppresses the production of cytokine IL-1. Production of TNF is associated with many diseases and disorders, so its suppression by using the connection And may have a beneficial effect on a subject in need of suppressing production of TNF-.

Thus, the connection And can also be used for inhibiting the production of TNF in a mammal and/or in the way lcom, rheumatoid arthritis, osteoarthritis, asthma, bronchitis, chronic obstructive airway disease, psoriasis, allergic rhinitis, dermatitis and inflammatory bowel disease and other autoimmune diseases, and the method includes the introduction of the specified mammal a therapeutically effective amount of compound A.

For use in the present invention the compound And can be prepared in a pharmaceutical composition by mixing with a pharmaceutically acceptable non-toxic excipients and carriers. Such a composition can be obtained for the introduction of any of various ways. Route of administration, and the preferred dosage forms include the following: parenteral, preferably in the form of liquid solutions or suspensions; for oral, preferably in the form of tablets or capsules; intranasal, preferably in the form of powders, nose drops, or aerosols; and dermal, for example, using transdermal systems.

Connection And may for convenience be administered in a standard dose of the drug by using any of the methods well known in pharmacy, for example, as described in Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, PA, 1980). Compo is howling solution the polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. In particular, for controlling release of the active compounds can be used: biocompatible, biodegradable lacheny polymer, lactide /glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers. Other systems that could potentially be used to deliver compounds And include particles ethylenevinylacetate copolymer, osmotic pumps, implantable infusion systems, and liposomes. Compositions for inhalation as fillers include, for example, lactose, or may be aqueous solutions containing, for example, lactose, or may be aqueous solutions containing, for example, a simple polyoxyethylene-9-lauric ether, glycocholate and dezoksiholatom, or oily solutions for administration in the form of nose drops or in the form of a gel for intranasal administration. Compositions for parenteral administration may also include glycocholate for transbukkalno introduction, salicylate for rectal injection or citric acid for vaginal administration. Composition for transdermal systems are the way what about the active ingredient in the pharmaceutical composition. Alternatively, it can be used in combination with other active ingredients, such as growth factors that promote neuronal survival or axonal regeneration in diseases or disorders.

The concentration of compounds And used in therapeutic composition in the implementation of this invention, can vary. The concentration will depend on such factors as the total dose of the drug, which is supposed to be, and route of administration. Usually the connection And should be provided in an aqueous physiological buffer solution for parenteral administration containing from about 0.1 to 10 wt./vol.%. The usual dose ranges are from about 1 μg/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.01 mg/kg to 100 mg/kg of body weight per day. Probably, the preferred dose of the compound, which is supposed to enter depends on such factors as the type and severity of the disease or disorder, the overall health status of the particular patient, the relative efficiency of the connection And when subject to treatment of a particular disease or disorder, and the route of administration.

Joint in U.S. patent 5461146, issued to Lewis et al.

Hereinafter the present invention will be illustrated in the following examples. These examples should not be construed neither as limiting the range of the claims of the invention, nor as limiting the range of the claims attached claims.

Example 1

Pharmacological activity of the action on the dopaminergic neurons

Experiments carried out by using connections And damaged MRTR dopaminergic neurons in mice model using MRTR in mice. Single dose MRTR (20 mg/kg), introduced subcutaneously black mice S, caused a loss of activity by approximately 60% hydroxylase striatum. Introduction connections And the daily dose in the range of from 0.01 to 10 mg/kg (subcutaneously) in the mice treated with MRTR, reduced the loss of activity of tyrosine hydroxylase striatum. These data are shown in table 2.

Mice injected MRTR (20 mg/kg subcutaneously) after 4-6 hours after the first injection of compound A. the Connection And then enter daily until the end of the experiment, which lasted 7 days. Assessing the activity of the enzyme tyrosine hydroxylase striatum. The asterisk indicates a statistically significant once the Oia And estimate the model with the introduction of mice to high doses MRTR. A single injection of 40 mg/kg MRTR caused 7 days after injection of the reducing activity of the enzyme tyrosine hydroxylase striatum 95%. Introduction connections in the dose of 0.03 to 3.0 mg/kg per day dose-dependent manner reduced the size of the lesion. These data are presented in table 3.

Mice injected MRTR (40 mg/kg subcutaneously) after 4-6 hours after the first injection of compound A. the Connection And then enter daily until the end of the experiment, which lasted 7 days. Assessing the activity of the enzyme tyrosine hydroxylase striatum. The asterisk indicates a statistically significant difference (p<0.05) from animals treated MRTR carrier.

Dopaminergic toxicity mediated MRTR depends on indirect MAO-transformation MRTR in RAM+(ion, 1-methyl-4-phenylpyridine) and capture RAM+in dopaminergic neurons. Connection with the detected activity on the model of mice treated with MRTR should not be defined as MAO inhibitors or capture of dopamine. As has been established, the connection is not inhibited monoamine oxidase a and b in vitro (Fig.1 and 2) or does not block the capture of catecholamines in nerve endings, which indicates that this compound does not prevent metabol and data confirming the effectiveness of the compounds And on the model damage to dopaminergic neurons MRTR in mice suggest the possibility of using the compounds And in the treatment of neurodegenerative disorders such as Parkinson's disease.

Example 2

Pharmacological activity of the action on the GABA-ergicheskie neurons

Connection And examined for its ability to prevent depletion or increase the survival of GABAergic neurons in both the basal nucleus, using a well-studied model of lesion botanova acid. Botanova acid (excitotoxin), as is well known, reduces the number of GABA-expressing neurons in the area of nbm (Lindefors et al., Neurosci. Lett., 135: 262 to 264, 1992; Shaugnessy et al., Brain Res., 637: 12-26, 1994).

Adult male rats Spraque-Dawley produce injection botanova acid (5.0 g) in the nbm, on the one hand. Rats then by subcutaneous injection administered compound a (0.03 mg/kg), starting at 18 h after injury and continuing the injection of 1 day to 18 days after the defeat. Then throughout the Rostral-caudal extent nbm make the cuts and processed for detection of glutamic acid decarboxylase, an enzyme required for the biosynthesis of GABA. The number of neurons, expr is of nbm in the field, where the nbm neurons expressing the glutamate decarboxylase, easily distinguishable from the neurons of adjacent structures, expressing the glutamate decarboxylase (white matter medial and intraline from the globus pallidus). The results are expressed as the percentage of neurons with the affected side, expressing the glutamic acid decarboxylase, the number on the opposite, unaffected side. The results also calculated separately for the Rostral division nbm, middle Department of the nbm and caudal division nbm.

On average, according to estimates in the Rostral division nbm animals treated with the compound And find 7816% of neurons containing the glutamic acid decarboxylase, in comparison with 3419% in animals receiving only the media that, according to student's t-test is statistically significant difference (p<0.05). In the middle or caudal departments nbm not observe statistically significant differences between animals treated with the compound a, and animals receiving only the vehicle (control).

These results show that the connection And can protect against loss of GABAergic neurons in excitotoxic lesions and, therefore, exerts neurotrophic effects on the ass who nternet study the ability of the compound And to prevent excitotoxicity the death of neurons in the nbm neurons of adult male rats Spraque-Dawley first mark a permanent marker. This is carried out by injecting versolato (FZ), a marker of neurons, captured nerve endings and is transported in the reverse direction to the body cells (Book et al., J. Neuropath. Exp. Neurology 53: 95-102, 1994), in the area of the target of nbm neurons in frontal and parietal cortex. After 7-10 days in animals by administering 5 mg botanova acid cause unilateral damage to the nbm. After 18 h after damage by subcutaneous injection, the animals begin introducing the compound A (0.03 mg/kg) and produce 1 every 2 days up to 18 days after injury. Throughout the Rostral-caudal extent nbm on both sides of the brain to make the cuts fabric and count the number of nbm neurons labeled FZ. Using standard techniques make the correction calculations on differences in size and the results expressed as the percentage of labeled neurons in the nbm on the side of damage each animal relative to the number of labeled neurons in the nbm on the opposite, is not damaged side of the brain.

In comparison with animals with damage and received only the carrier, animals treated with compound a, in the Rostral part of the damaged nbm (more than 400 μm from the average point of damage) was significantly more neurons, mehanny who are direct evidence, that connection And can prevent the loss of pre-labeled neurons in the nbm in excitotoxicity destruction.

Example 4

Long-term functional improvement after a short introduction connections AND

First adult male rats Spraque-Dawley teach alternating reactions in a standard task in the T-shaped maze rewarded alternation (Helper et al., J. Neurosci., 5: 866-873, 1985). Then, for violations of the tasks in the T-shaped maze in animals cause bilateral damage to both basal nuclei (see, for example, Salamone et al., Behav. Brain Res., 13: 63-70, 1984). After 18 h after damage by subcutaneous injection, the animals begin introducing the compound A (0.1 mg/kg) and produce 1 every 2 days to 12 days after injury. At 24 h after the last injection all animals once a day put in a T-shaped maze to until their alternating execution reaches the preoperative level, which takes approximately 3 to 10 days. After reaching criterion animals 8-10 weeks is not subjected to further research. At this point the animals again once per day placed in a T-shaped maze until then, until they reach the p shown in Fig.4. Animals with damage to receiving only the media, has made significantly more errors than not operated or falsely operated animals, whereas animals with damage, for 10-12 weeks before receiving the connection And did not differ from normal animals.

These data show that compound a is a persistent improvement in the behaviour, reflected in the improvement in attention or memory in a few months after cessation of administration.

Example 5

The efficiency of the connection And the damage model interinale crust

Containing glutamate neurons in layer 2 interinale crust projected into the molecular layer of the dentate gyrus, where they form synapses on the dendrites of nerve cells-grains dentate gyrus. Damage in the 2nd layer entorhinal crust may be caused by using stereotaxic injections excitotoxin N-methyl-D-aspartate (NMDA). In terms of anesthesia pentobarbital sodium rats produce an injection of NMDA (15 nm/plot) in each of 2 sites in entorhinal cortex. After 2 weeks, rats hammer and perfusion a solution containing 50 mm of sodium sulfide. The brain is removed, producing a slice thickness of 40 μm in the horizontal plane and paint kresila Fioletovo purple, while the integrity of the endings of their axons in the molecular layer of the dentate gyrus was measured in alternating sections stained with dye Timm.

Injection of NMDA destroyed 6211% 2 layer entorhinal crust and reduced the area of the middle molecular layer of the dentate gyrus by 196%. Daily introduction of compound A (1 mg/kg subcutaneously) for the first injection, produced immediately after the injection of NMDA, reduced neuronal loss 2 entrialgo layer to 2210% (p<0.05 by the t student test) and prevented the reduction of the area of the middle molecular layer (p<0.05 by the t student test). Therefore, the connection And protected the neurons in entorhinal bark from excitotoxic damage and maintain the integrity of the endings of their axons in the dentate gyrus.

These data show the effectiveness of the compounds And in the protection of glutamatergic neurons interinale crust. In experimental models it has been shown that damage interinale crust cause memory impairment and in Alzheimer's disease occur severe degenerative changes in these neurons. This confirms the view that the connection And can be used in the treatment of neurological disorders associated with loss or p is before and traumatic brain injury, but not limited to.

Example 6

The influence of the connection And the peripheral neuropathy caused by acrylamide

Acrylamide causes people and animals of the Central-peripheral distal neuropathy with "withering away in the opposite direction". Damage is a mixed sensory-motor neuropathy, characterized in humans weakness, tremor and ataxia. Animals acrylamide causes changes in behavior (sensory, motor, and proprioceptive), paralogical changes in the structure and electrophysiological characteristics of tissue and loss of body weight. On the periphery of acrylamide mainly affects the fiber AB large diameter long axoneme. By measuring the increase of positioning legs when landing (RLP), which is a measure of proprioception, rats introduction acrylamide causes axonopathy.

Neuropathy caused by acrylamide, is a model of toxic effects of chemical compounds. It differs from other models of the chemical compounds (such as chemotherapeutic model) because of its relatively short duration (3 weeks) and dogs feel good. Acrylamide is not wyzwanie Spraque-Dawley weighing 250 g Acrylamide (50 mg/kg intraperitoneally) administered 3 times per week for three weeks. Animals are thrown from a height of 30 cm and record the distance between the traces of hind limbs (Edwards P. M. and Parker V. H. (1977): "a Simple, sensitive and objective method for early assessment acrylamide neuropathy in rats" (Toxicol Appl Pharmacol, 40: 589-591). Compound A (0.1, 0.3 or 1.0 mg/kg subcutaneously in 5% solutol) is administered once a day during the whole experiment. Animals treated with acrylamide, the distance RLP was greater than that of animals receiving carrier. Compound A (0.3 mg/kg/day) reduced the rate of increase RLP caused by acrylamide (Fig.5).

These data show that compound a can be used for the treatment of peripheral neuropathies.

Example 7

The inability of the connection And to inhibit the protein kinase in vitro

Quantitative determination of protein kinase C and suppression under the influence of protein kinase C-a disclosed in U.S. patent 4923968 and in the work of H. Kase et al. (eds.), Japan Scientific Societies Press, Tokyo, pp. 93-296 (1988). By essentially the same quantitative determination, it was found that K-a inhibits protein kinase C PC50component 0.028 mm, while PC50connection amounts to 16.0 μm, i.e., it ActiveStor production of IL-l value PC50is 261 nm. This is the concentration at which the connection is not active as an inhibitor of protein kinase C.

Example 8

Suppression of in vitro production of TNF - a and IL-l connection AND

The ability of compounds And to suppress the induction of TNF - has been shown using methods standard pharmacological studies in vitro, as described below. Royal solutions, consisting of 4 mm connection And in 100% dimethyl sulfoxide (DMSO), stored at 4oC. Environment RMPI 1640 cell culture (Media Tech, Herndon, Va) and fetal calf serum (Hyclone, Logan, UT) constitute the environment for research. Use the lipopolysaccharide (LPS) (Sigma, St. Louis, MO) serotype 0111:B4 (E. coli, extracted trichloroacetic acid. Royal LPS solutions are prepared and stored at 4oWith in saline phosphate buffer (PBS). Sets enzyme immunoassay for the quantitative determination of tumor necrosis factor alpha (TNF - and IL-1) purchased from the company Boehringer-Mannheim (Indianapolis, IN) and used in accordance with manufacturer's instructions. Cells TNR-1, a cell line derived from human monocytes, obtained from American Type Culture Collection (ATCC TIB 202). Cells grown in RMPI 1640 containing 10% fetal calf serum (Wednesday) in wetted atmospherehandler 5105cells TNR-1 in 1 ml of medium. For the induction of TNF-cells incubated with LPS at a concentration of 2 µg/ml After 3-hour incubation period, cell and environment centrifuged at 1000g for 5 min and either immediately conduct a quantitative determination of the resulting supernatant on TNF-, or store it at -70oWith and quantification spend later. Using quantitative enzyme-linked immunosorbent definitions measure the content of TNF - and IL-1 in the samples of the environment.

To study the connections And the ability to suppress the induction of TNF - cells before the addition of LPS incubated for 1 h with varying concentrations of compound A. Compound is diluted so that in the medium for cell culture never DMSO in concentrations above 0.1%. Investigation and quantification of compounds And conduct, as described above, the ability to suppress the production of TNF - and IL-1.

Example 9

Suppression of TNF - and IL-1 induced by LPS in the serum of mice

60 female mice C57BL (age 4-6 weeks) divided into 6 groups, each containing 10 animals. Three groups (1, 3, 5) intraperitoneally (b) impose only medium (PBS containing 0.1% TEA (tetraethylammonium hydrochloride)) - 200 µl per animal. Of the other three is rocketeering acid; solutol). After 2 h the animals in b/W administered 0.1 mg/kg LPS (group 1 and 2), 0.5 mg/kg LPS (groups 3 and 4) and 1.0 mg/kg LPS (groups 5 and 6). After 2 h after LPS injection all animals were slaughtered, get plasma samples and conduct quantitative determination of the content of TNF - a and IL-l. Quantitative determination carried out as described in example 8.

The data summarized in table 4. Numbers in parentheses represent the number of the above groups. N indicates the number of animals in the plasma which have definitions. ND - determination was not conducted.

As shown in table 4, the introduction of LPS to mice caused the production of TNF in these mice, and the number of produced TNF - a was significantly decreased in the preliminary introduction connections And for 2 h before the study. The pretreatment connection And significantly suppressed the production of IL-l in response to the introduction of LPS at a dose of 1.0 mg/kg

Example 10

To prevent death of mice caused by LPS

45 mice divided into 3 groups, two of which consist of 10 mice and one group of 20 mice. Media, LPS and connection And injected intraperitoneally. Group 1 (n=10) initial (time 0) was injected media (saline phosphate buffer for LPS, 200 µl per mouse), and after 2 h the medium for connection And (200 µl per mouse).CL on the mouse). Group 3 (n=20) initial (time 0) is administered compound a (30 mg/kg in 200 μl of media), and after 2 h of LPS (2 mg in 200 µl per mouse).

The introduction of LPS caused 90% mortality within 20 hours and 100% mortality after 42 hours In the group of animals for 2 hours before the administration of LPS treated with the compound a, 20 h after injection of LPS mortality was 25% through 42 h after injection of LPS mortality was 75% and after 1 week after administration of LPS mortality was 80%. Introduction media was well tolerated and did not cause the death of the mice.

Specialists in this field it will be understood that in preferred implementations of the invention may be made of numerous changes and modifications and such changes and modifications may be made without departing from the invention. Therefore, it is assumed that the appended claims cover all equivalent variations within the true essence and breadth of the claims of the invention. The materials contained in the entire description of this patent application, are incorporated into it by reference.

1. The way to suppress excessive secretion of tumor necrosis factor alpha in a mammal, comprising the stage of introduction needs it the mammal a therapeutically-effective collarbone of tumor necrosis factor alpha in a mammal, including the stage of introduction needs it the mammal a therapeutically - effective amount of a compound As represented by formula

< / BR>
3. The way to suppress excessive secretion of interleukin-1 beta in a mammal, comprising the stage of introduction needs it the mammal a therapeutically-effective amount of a compound As represented by formula

< / BR>
4. A way of alleviating the detrimental effects of excessive secretion of interleukin-1 beta in a mammal, comprising the stage of introduction needs it the mammal a therapeutically-effective amount of a compound As represented by formula

< / BR>
5. The method according to p. 2, where these harmful effects selected from the group including septic shock, rheumatoid arthritis, osteoarthritis, asthma, bronchitis, chronic obstructive airway disease, psoriasis, allergic rhinitis, dermatitis and inflammatory bowel disease.

6. The method according to p. 2, wherein said adverse effect is an autoimmune disease.

7. A method of reducing the damaging effects of Alzheimer's disease neurons selected from the group consisting of GABAergic neurons and glutamatergicescuu connection And, represented by the formula

< / BR>
8. A method of reducing the damaging effects of Parkinson's disease on dopaminergic neurons, including the stage of introduction needs it the mammal a therapeutically-effective amount of a compound As represented by formula



 

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