KR960008235B1 - Method of inhibiting the activity of leukocyte derived cytokines for animal - Google Patents

Abstract

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Description

[Name of invention]

How to Inhibit Leukocyte-Induced Cytokine Activity

[Brief Description of Drawings]

The invention will be more fully explained by the following figures.

FIG. 1 shows the effect of interleukin-1 (IL-1) on n-formyl methionyl leusil phenylalanine (FMLP) directed at multinucleated leukocytes (PMN). 1,3-dibutyl 7- ( Is a graph showing relaxation by 2-oxopropyl) xanthine (DBOPX),

Figure 2 shows that the effect of the media of mononuclear leukocytes stimulated with lipopolysaccharide (LPS) on PMN directed migration to FMLP is alleviated by DBPOX.

3 shows that the effects of tumor necrosis factor (TNF) on PMN-directed migration to FMLP are alleviated by DBOPX.

4 shows that the effect of LPS-stimulated mononuclear leukocyte media on PMN fixation to nylon is alleviated by DBOPX.

5 shows that the effects of IL-1 on the release of excess oxide by PMLP stimulated by FMLP are alleviated by DBOPX,

FIG. 6 is a graph showing that the effect of LPS-stimulated mononuclear leukocyte media on the production of superoxide by PMN-stimulated PMN was alleviated by DBOPX.

FIG. 7 is a graph showing that the effect of LPS-stimulated mononuclear leukocyte media on the production of lysozyme by FMLP stimulated PMN is alleviated by DBOPX.

Detailed description of the invention

The present invention relates to a method for inhibiting the activity of leukocyte-induced cytokines such as interleukin-1 and tumor necrosis factor in humans and stray animals. More specifically, the present invention provides a method of treating or alleviating certain diseases and infections by inhibiting the activity of cytokines.

Interleukin-1 (IL-1) and tumor necrosis factor (TNP) are biological substances produced by monocytes and other macrophages in mammals. IL-1 and TNF affect a variety of cells and tissues, both in vitro and in vivo. According to the findings, IL-1, TNF and other leukocyte-induced cytokines have been reported to play an important coordinator, albeit dangerously, in various infectious diseases. Thus, inhibition of IL-1, TNF and other leukocyte-induced cytokines is advantageous for controlling, healing and alleviating such conditions.

Detection and inhibition of IL-1, TNF, and other leukocyte-induced cytokines can be readily identified by analyzing the morphology of multinuclear neutrophils in vitro. Other activities caused by IL-1 and other leukocyte-induced cytokines are the promotion of leukocyte fixation and the inhibition of neutrophil chemotaxis, all of which directly affect disease and infection symptoms.

However, although there is an urgent need for inhibition of IL-1 and TNF activity and other leukocyte-induced cytokine activity and such inhibition is readily detected in vitro, there remains a need for techniques for inhibitors that can be administered in vivo. have.

[Overview of invention]

The present invention seeks to meet this need by defining a class of compounds that can be used successfully to mitigate the disease states modulated by or caused by IL-1, TNF and other leukocyte-induced cytokines. Such compounds have been shown to significantly inhibit cytokine activity even when the modulator is present at low concentrations, as demonstrated by in vitro testing.

More specifically, the present invention provides IL-1 in mammals by administering at least one or more of 7- (oxoalkyl) 1,3-dialkyl xanthines represented by the following general formula (I) to stray animals: And methods of inhibiting the activity of TNF and other leukocyte-induced cytokines.

Wherein R ₁ and R ₂ may be the same or different, each of which is a straight chain or branched alkyl radical having 2 to 6 carbon atoms, a cyclonuclear chamber, a straight chain or branched alkyl radical, a cyclonuclear chamber, a straight chain or a ground alkoxyalkyl, and Is selected from hydroxyalkyl radicals other than 3-beta-3-hydroxybutyl lycal and 3-methyl-3-hydroxypentyl radicals and 4-methyl-4-hydroxypentyl radicals, where A represents at least 4 carbon atoms It is a hydrocarbon radical having, and may be substituted by a methyl group.

Such xanthines are administered in an amount effective to inhibit the activity of IL-1, TNF and other leukocyte induced cytokines in mammals.

[Description of preferred implementation]

The activity of IL-1, TNF and other leukocyte induced cytokines can be inhibited by administering 7- (oxoalkyl) 1,3-dialkyl xanthine to mammals.

As used herein, the expression leukocyte-induced cytokines has a wide range. In particular, the term leukocytes as used herein refers to those of the granulocyte and lymphocyte lineages of stunt cells. Such leukocyte cells include, for example, multinuclear leukocytes such as neutrophils and monocytes such as monocytes, macrophages and lymphocytes.

The word cytokine as used herein refers to a secreted product of leukocytes, especially non-antibody proteins that are secreted by leukocytes upon contact with antigens, which function as intracellular modulators in the immune response. Examples of cytokines within the scope of the present invention include chemotactic factors, factors that promote the replication of lymphocytes, factors that inhibit lymphocyte replication, factors that affect macrophage adhesion, factors that affect macrophage secretion, Factors controlling the secretion of oxidizing substances such as oxygen, superoxide, hydrogen peroxide and hydroxyl radicals.

7- (oxoalkyl) 1,3-dialkyl xanthine used in the present invention has the following structural formula (I).

In the above general formula (I), the substituents R ₁ and R ₂ may be the same or different, each selected from a straight chain or a branched alkyl radical having 2 to 6 carbon atoms, a cyclonuclear chamber, an alkoxyalkyl and a hydroxyalkyl radical. In other words, 3-butyl-3-hydroxy butyl lycal, 3-methyl-3-hydroxy pentyl radical or 4-methyl-4-hydroxy pentyl radical are excluded. Substituent A is a hydrocarbon radical having 4 or more carbon atoms, which may be substituted by a methyl group.

Among such compounds, 1,3-dibutyl 7- (2-oxopropyl) xanthine, which has been found to be particularly effective in inhibiting the action of IL-1 and other leukocyte induced cytokines on multinuclear leukocytes and monocytes. This compound, abbreviated as DBOPX, has the following structural formula (II).

The compound of formula (II) has the ability to mitigate the inhibitory action of IL-1 and other leukocyte-induced cytokines on the adhesion of multinuclear leukocytes and monocytes, chemotactic reactions of cells, hyperventilation (metabolism), and reduced granules. This has been demonstrated below.

Phagocytic cells of high importance in immunology include multinuclear leukocytes (eg, neutrophils) and mononuclear phagocytes (eg, monocytes and macrophages), and the decline in their function leads to recurrent purulent infection. To combat purulent infection, neutrophils and monocytes move towards the source in response to chemotactic factors, which ingest and kill microorganisms.

More specifically, the main function of multinuclear leukocytes and monocytes is to kill bacteria and other infectious agents by phagocytosis. The first step in the ingestion and digestion of particulate matter by these cells is to keep the particulates of the cells close together, usually through a chemotactic reaction. This response is an essential part of the host's defense against infection. The widespread migration and activity of these cells may prove to be inflamed at the wound or invasion site of the host.

IL-1 and TNF have been known to inhibit chemotactic responses of granulocytes, monocytes and macrophages. The inventors have found that the 7- (2-oxoalkyl) 1,3-dialkyl xanthine of formula (I) can mitigate the inhibitory action of IL-1 and TNF on such chemotactic reactions. This is described below.

In the form of a multinuclear leukocyte migration in response to the known chemotactic factor n-formyl methionyl louisyl phenylalanine (FMLP), the chemotaxis reaction under agarose conditions is a known analytical method for chemotactic reaction of cells. Assay through. See J. of Immunol., 115, 6, 1650-1686 (1975), this assay was performed in the absence of IL-1 and was repeated in the presence of IL-1. In each case, the analysis was also performed when DBOPX in the absence of DBOPX was present at concentrations of 0.1, 1 and 10 μg / ml. The results are shown in FIG.

As shown in FIG. 1, the cell's directed movement is about 2.08 mm in the condition that IL-1 and TNF are not present and the concentration of DBOPX is 0 μg / ml (CONT in FIG. 1), whereas IL-1 and TNF are In the presence of DBOPX concentration of 0μg / ml, the cell's directed migration dropped to about 1.5mm, inhibiting the chemotactic response of the cells toward IL-1 and TNF.

In addition, FIG. 1 shows the degree of inhibitory effect of chemotactic response by IL-1 when the concentration of DBOPX is increased. DBOPX mitigates the action of IL-1, which inhibits directed migration to FMLP. Able to know. In particular, FIG. 1 shows that DBOPX increased cell migration and alleviated the inhibition of IL-1 at all concentrations tested. Figure 1 also shows that DBOPX was effective in inhibiting chemotaxis even at very low concentrations. Thus, the compounds used in the methods of the present invention can be used very effectively to mitigate the inhibitory action of IL-1 on the chemotactic response of cells.

DBOPX can have a similar effect even when the multinuclear leukocytes are left under conditions in which a product secreted by mononuclear leukocytes stimulated with lipopolysaccharide (LPS) is present. The monochromatic cells then produce IL-1, TNF and other infectious cytokines. PMN directed migration to FMLP in this case was assayed by chemotaxis under agarose. This analysis was performed for the absence of DBOPX and for concentrations of 0.1, 1.0, 10 and 100 μg / ml. The results are shown in FIG.

According to FIG. 2, the direction of PMN in conditioned medium containing infectious cultures was 2.25 mm when no DBOPX was present, and the increase in cell directivity at all concentrations tested when DBOPX was added to the medium. It became. In other words, DBOPX had an effect of increasing chemotaxis even at low concentrations. Furthermore, when the concentration of DBOPX was 10 µg / ml, the orientation shift was about 2.6 mm. For reference, the shift in the medium but not LPS containing conditions was 2.60 ± 0.5 mm (not shown in FIG. 2). The probability that DBOPX increased directed migration inhibited by conditioned media containing infectious cultures was 95%.

DBOPX may exhibit a similar effect when PMN is left under the condition that rh-TNF (α) is present. To determine this, the PMN-directed shift of FMLP was analyzed by chemotactic reaction under agarose. This analysis was performed for the case where DBOPX was present at 0.01 mM (3.2 μg / ml) and 1 mM (320 μg / ml) without DBOPX. The results are shown in FIG.

According to FIG. 3, the directed shift of PMN in medium containing rh-TNF was increased at all concentrations tested when DBOPX was added. In other words, DBOPX was effective in increasing chemotaxis even at very low concentrations. For reference, the shift in the medium without TNF was 2.75 mm. The probability that DBOPX increased the directed shift suppressed by TNF was over 95%.

Thus, 7- (oxoalkyl) 1,3-dialkyl xanthine used in the methods of the present invention can increase the directional motility of multinuclear leukocytes and can be administered to a patient to cause chemotactic or plasma activity of bacterial or viral strains. It may increase the components assimilated by components involved in the system or cells involved in the immune system.

Under acute infectious stimulation, leukocytes produce a complex series of reactions, as part of which white blood cells adhere to the endothelium near the stimulus. Inhibiting the fixation of these leukocytes can be expected to reduce the degree of infection seen in disease states such as septic shock and adult respiratory distress syndrome. It was found that the 7- (oxoalkyl) 1,3-dialkyl xanthine used in the present invention is effective in preventing the fixation of such multinuclear leukocytes.

In particular, multinuclear leukocyte (PMN) fixation to nylon has been described by MacGregor et al., New Engl. I. Med., 13: assayed according to the method of 642-646 (1974). Purified PMN cells were left in mononuclear leukocyte conditioned medium stimulated with lipopolysaccharide containing infectious cytokines, and in nylon under conditions where DBOPX was not present and DBOPX was present at concentrations of 0.1, 1.0, and 10 μg / ml. PMN fixation was determined. The% PMN fixation to nylon at each condition was determined and the results are summarized in FIG.

According to FIG. 4, the PMN fixation to nylon was found to be about 87% in the absence of DBOPX. On the other hand, when the concentration of DBOPX was determined to be about 0.1 μg / ml, the% fixation rate was reduced. PMN fixation to nylon was suppressed. In addition, the% PMN fixation rate decreased to about 70% under the condition that the concentration of DBOPX was 10 µg / ml. The probability that DBOPX reduced PMN fixation in the conditioned medium was 99.7%. Thus, the compounds used in the methods of the present invention can be used very effectively to block the fixation of leukocytes and alleviate the extent of infection.

Mature macrophages are metabolically at rest. However, if any object and substrate are transferred to the macrophages, for example, when digestible particulates adhere to the cell surface, the macrophages will go out of the resting state and enter a state in which metabolic activity such as hypermetabolic or hyperventilation is rapidly increased. This transition of states involves a series of characteristic transformations, as part of which produces superoxide anions. Cytokines such as IL-1 and TNF cause similar effects. The production of these oxides means that metabolic cells, besides functioning in ingesting and inactivating microorganisms, are useful as an indirect measure of the intake process itself.

Arrangements for the direct determination of superoxide anions of hydrogen peroxide separated by medium are known. It has been found that the compounds used in the present invention can alleviate hyperventilation in stimulated multinuclear leukocytes as determined by the above method.

More specifically, the production of superoxide is described by Babior et al., J. Clin. Investigation, 52: 741-744 (1973), modified the method and analyzed accordingly. Purified PMN was left under conditions in which there was no oxidative stimulus and no IL-1, and then each medium was analyzed to assay the production of superoxide. In this case, the analysis was also performed when DBOPX was not present and when DBOPX was present at 0.1, 1.0, 10, and 100 µg / ml. The results are shown in FIG.

According to FIG. 5, FMLP-stimulated PMN produced 1.8 nmole / 10 min of excess oxide per million PMN in the absence of IL-1, TNF and DBOPX. Pretreatment with IL-11 (5Units / 20ml), known as its promoter, was observed to secrete 4.4 nmole / 10 min excess oxide per million PMN, significantly increasing the amount.

Indeed, when DBOPX was added to the assay, the production of excess oxide was significantly reduced, as shown in FIG. In particular, DBOPX was effective at mitigating the action of IL-1 on FMLP-stimulated PMN at all concentrations tested, and DBOPX was effective at low concentrations of 0.1 μg / ml. The probability that DBOPX is stimulated with FMLP and reduces the excess oxide production of PMN promoted by IL-1, TNF is 95%.

DBOPX can also reduce the superoxide production of PMN promoted with LPS-stimulated mononuclear leukocyte conditioned medium containing infectious cytokines. This is shown in FIG. In particular, when PMN was left in LPS-stimulated mononuclear leukocyte composition medium containing infectious cytokines and stimulated with FMLP, the yield of superoxide was 7.4 nmoles / 10 min / 10 ⁶ PMN in the absence of DBOPX. Indeed, when DBOPX was added to the assay, the production of excess oxide was observed lower than above at all tested concentrations, and furthermore, there was some effect even at concentrations lower than 1.0 μg / ml. When the concentration of DBOPX was 10 μg / ml, the amount of excess oxide was 1.5 nmoles / 10nim / ⁶ PMN. The probability that DBOPX reduced the superoxide production of PMN stimulated with FMLP and promoted with conditioned medium was 99.5%.

From these results, it was demonstrated that the compounds used in the method of the present invention can reduce the production of superoxide and alleviate the hyperrespiration of macrophages such as multinuclear leukocytes and monocytes.

During ingestion, the granules in the intracellular cytoplasm fuse to the fear formed around the foreign body, releasing their contents in fear. Some of these material layers reach the medium surrounding the macrophages. Thus, the granules become extinct during this process, which is called granulation reduction. The contents of the granules include hydrolase, lysozyme, and antimicrobial protein. In the case of neutrophils, bone marrow peroxidase is included.

Granulation reduction can be assayed by measuring the prevalence of granule-associated enzymes in the medium of the cell. In the case of multinuclear leukocytes (PMN), granule reduction can be analyzed by assaying the secretion of lysozyme. It has been found that the compounds used in the method of the present invention can mitigate the secretion of lysozyme from stimulated PMN.

More specifically, multinuclear leukocytes (PMN) were left in LPS-stimulated mononuclear leukocyte conditioned medium containing infectious psychokines. Then, after stimulating PMN with FMLP and left for a certain period of time, the lysozyme content in the cell suspension was measured using a known assay method. See J. Bacteriol., 58, 731-736 (1949), PMN was allowed to stand in the absence of DBOPX and 0.1, 1, 10 and 100 μg / ml of DBOPX. The results are shown in FIG.

According to FIG. 7, lysozyme secretion of PMN stimulated with LPS-stimulated mononuclear leukocytes conditioned medium (containing infectious cytokines) and stimulated with FMLP was 2.1 μg / ml under conditions without DBOPX. On the other hand, the addition of DBOPX reduced the release of lysozyme and this decrease was observed at all concentrations tested. Moreover, DBOPX was effective in relieving lysozyme secretion even at very low concentrations of 0.1 μg / ml. Under the condition that the concentration of DBOPX was 100 μg / ml, lysozyme secreted only 1.04 μg / ml. The probability of suppressing lysozyme secretion from PMN stimulated by DBOPX and FMLP stimulated was 95%.

From these results, it was confirmed that the compounds used in the method of the present invention can inhibit lysozyme secretion from PMN stimulated with LPS-stimulated mononuclear leukocyte conditioned medium and FMLP stimulated.

In summary, the compound of formula (I) used in the method of the present invention can mitigate the effects of leukocyte-induced cytokines such as interleukin-I and tumor necrosis factor on macrophages such as multinuclear leukocytes. These compounds actually help chemotactic reactions and prevent cell sticking. In addition, the compound can reduce oxidative damage of host tissues by macrophages, as demonstrated to mitigate hyperrespiration in stimulated multinuclear leukocytes. Finally, these compounds can mitigate the effects of cytokines on the granule reduction mechanisms of stimulated macrophages. As demonstrated above, the inhibitory action of the compounds on IL-1, TNF and other cytokines is believed to have a clinical effect on at least the following fields and disease states.

Since IL-1, TNF, and other leukocyte-induced cytokines have been linked to disease states of many mammals, the present invention is likewise widely applicable. Disease conditions that can be treated or alleviated by inhibiting IL-1, TNF and other cytokines include sepsis, septic shock, toxic shock, Gram-negative sepsis, toxic shock syndrome, adult respiratory distress, infection (eg influenza) Fever and myalgia due to infection, secondary cachexia due to infection or malignancy, secondary cachexia due to AIDS, rheumatoid arthritis, gouty arthritis, osteoporosis, cyanosis formation, scar tissue formation, loss of appetite, Crohn's disease, ulcerative colitis, central nervous system bleeding Fever, glomerulonephritis, multiple sclerosis, Creutzfeldt-Jacob disease and side effects for dialysis, diabetes mellitus and psoriasis.

When referring to a specific cause of the disease trauma (trauma) can be used as a more general term, the term is used to represent a wide range of cell infiltration and physical damage of the cell by foreign matter. Foreign substances include microorganisms, particulate matter and chemicals, and physical damages include mechanical damage such as peeling, overheating, wounds and wounds, and thermal damage such as overheating or overcooling, and contact with a power source. Electrical damage, such as caused by, and radiation damage due to overexposure in infrared, ultraviolet, or ionizing radiation.

Microorganisms that can be included in a foreign body that induces a biological response include visilis, fungi and yeast, viruses and pathogens. Examples of Bacillus are Actinomyces spp .: Bacteroldes spp .: Corynebacterium spp .: Enterobacteriacea: Enterococcus: Enterococcus: Haemophilus spp. : Micrococcus spp.: Neisseria spp.: Staphylococcus aureus: Streptococcus pneumoniae: Streptococcus pneumoniae: Clostridium spp. Streptococcus agalactiae: Bacillus spp.: H. influenzae: Moraxella spp.: Mycobacteria spp.: Pseutodomonas aeruginosa: Vibrio sp. ) And Mycobacteria spp.

Examples of fungi and yeast include Microsporum: Blastomyces: Histoplasma: Aspergillus: Cryptococcus: Candidia: Cock Coccidioides and: Candida albicans.

Examples of viruses include rhinovirus: Parainfluenza: Enterovirus: Influenza: Chlamydiae: smallpox and vaccinia: Herpes simplex: undiagnosed (Measles): Rubella: Arthropod-borne virus (Sibu, eastern and Venezuela gray gray encephalitis and California encephalitis) (Arbovirus (Western, Eastern and Venezuelan equine encephalitis, and California encephalitis): Rabies: Colony Colorado tick fever: Yellow fever: Dengue fever (Dengue): Hepatitis virus B (HB Ag): Hepatitis virus A (HAV) and: human immunodeficiency virus (HIV).

Examples of parasitic bacteria that cause a response include Trypanosoma cruzi: Entamoeba histolytica: Leishmania brasiliensis: Leishmania tropica: Lenomania donovani: Toxican Plasmodium gondii: Tropical malaria parasite (Plasmodium falcipraum): Todesia tripanosoma (Trypanosoma rhodesiense): Lia loa (Trichomonas hominis): Schistosoma japonicum: Manzos (Schistosoma mansoni) and: Fasciola hepatica.

On the other hand, microparticles that cause biological reactions include silicon, asbestos, uric acid monosodium salt, cotton yarn, dust, and beryllium.

Chemicals include thick metals such as lead, chromium, mercury and arsenic, organic solvents such as trichloroethylene, herbicides such as trichlorophenoxyacetic acid, and insecticides such as mirex.

In addition, the inhibition of IL-1, TNF and other leukocyte-induced cytokines increases the activity of macrophages in stored blood and blood products.

Hereinafter, the compounds used in the present invention will be described in more detail, and a method for preparing such compounds will be presented.

In the method of the present invention, 7- (oxoalkyl) 1,3-dialkyl xanthines of the above structural formula (I) are used. For example, the xanthines of the formula (I) may be substituted with other alkyl groups or alkoxy or hydroxyalkyl groups. Such suitable alkyl groups include milling and straight chain groups such as methyl, propyl, isopropyl, butyl, secondary butyl, tertiary butyl, amyl and nuclear chamber and the like. Alkoxy-substituted alkyls are crushed and straight chain groups having 2 to 6 carbon atoms in which alkoxy and an alkyl group are combined, and include methoxymethyl, amylooxymethyl, methoxyethyl, butoxyethyl and propoxypropyl. The hydroxy alkyl groups are those containing 1 to 6 carbon atoms, and may include hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxynucleus, and the like, but 3-methyl-3-hydroxy butyl group and 3-methyl The 3-hydroxy pentyl group and the 4-methyl-4-hydroxy pentyl group are excluded.

The hydrocarbon group represented by A in Structural Formula (I) is a bifunctional saturated aliphatic hydrocarbon group, for example, methylene, ethylene, trimethylene and tetramethylene, and methyl may be substituted for carbon recognized in the carbonyl group thereof. . Such methyl substituted groups include ethylidine, 1,2-propylene and 1,3-butylene.

The compounds used in the present invention can be synthesized by known techniques, for example at elevated temperatures, optionally in the presence of a solvent, substituted 1,3-dialkyl xanthines of the following formula (III) The compound can be prepared by reacting with the α, β-unsaturated methyl ketone of (IV).

Wherein R ₁ and R ₂ are as defined above.

R is hydrogen or a methyl group here. This reaction can be carried out in alkaline media.

As another manufacturing method, an alkali metal salt of 1,3-dialkyl xanthine derivative represented by the general formula (II), wherein R ₁ and R ₄ are as defined above, is substituted with oxoalkyl of the following formula (V) React with halides.

Where A is as defined above and Hal represents a halogen atom, preferably chlorine or bromine.

The reaction is carried out at a temperature of 40 ° C to 80 ° C, and under some circumstances, pressurized or reduced pressure conditions may be employed, but is usually performed at atmospheric pressure. Each starting material may be used in stoichiometric amounts or in excess. The alkali salt may be prepared beforehand or may be prepared in a self reaction.

The compounds of formula (I) have been shown to have a significant effect on increasing the flow of blood through the bone root and have low toxicity. The most effective of the above compounds for use according to the invention is 1,3-dibutyl 7- (2-oxopropyl) xanthine, ie DBOPX.

A more detailed description of the compounds used in the present invention and methods of making such compounds is given in US Pat. No. 4,242,345.

The effective amount of xanthine can be determined by one of a variety of methods, depending on the subject, such as oral administration in the form of a capsule or tablet, or parenteral administration in the form of a sterile solution. Xanthines are effective on their own, but can also be administered in the form of their pharmacologically acceptable addition salts for their stability, convenience of crystallization, soluble vapors, and the like.

Preferred pharmacologically acceptable addition salts include salts of inorganic acids such as hydrochloric acid, sulfuric acid and nitric acid, etc .; salts of monobasic carboxylic acids such as acetic acid and propionic acid: salts of dibasic carboxylic acids such as maleic acid, fumaric acid, and bathalic acid; Salts of tribasic carboxylic acids such as citric acid and citric acid.

Xanthine can be administered orally, such as by inert diluent or edible carrier. They can be wrapped in gelatin capsules or compressed into tablets. For oral therapeutic administration, the compounds can be used in combination with excipients in the form of tablets, troches, capsules, elixirs, dispersions, syrups, wafers and chewing gum. Such preparations should contain at least 0.5% active ingredient. The amount of xanthine in such a composition is such that a suitable dosage can be obtained, but preferred compositions and preparations according to the invention are prepared to contain 1.0-300 mg of the active compound per oral dosage unit.

Tablets, pills, capsules and troches may include the following components: a combination such as microcrystalline cellulose, trigitant rubber or cellatine: excipients such as starch or lactose: alkyne acid, primogel and corn Degrading agents such as starch, etc .: Lubricating agents, such as magnesium stearic acid or spherot; Glidants, such as colloidal silicone didomic acid; and sweeteners, such as spices such as sucrose, saccharin or peppermint, methyl salicylate or orange flavor. . When the dosage unit form is a capsule, a liquid carrier such as fatty oil may be included with the substance of the above type.

Other dosage unit forms may also include various materials, such as coatings, that change the physical form of the dosage unit. Thus tablets or pills may be coated with sugar, shellac or other enteric coatings. Syrups can contain sucrose and some preservatives, dyes, pigments and flavorings as preservatives along with the active compounds. The materials used to prepare these various compositions are pharmacologically pure and should be nontoxic at their use.

For parenteral treatment, xanthine can be combined in solution or dispersion. Such preparations should contain at least 0.1% or more of the compound but may be used in a variety between 0.5% and about 50% of its weight. The amount of effective compound in such a composition is such that a suitable dosage is obtained. Preferred compositions and preparations according to the invention are prepared to contain from 0.5 to 100 mg of an effective combination per parenteral dosage unit.

In addition, the solution or dispersion may contain the following components: sterile diluents such as distilled water for injection, saline solution, nonvolatile oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents, such as benzyl alcohol or methylpyribene. Antibacterial agents: Antioxidants such as ascorbic acid or sodium bisulfite: Chelating agents such as ethylenediaminetetraacetic acid: Buffers such as acetate, citrate or phosphate and hepatic regulators such as sodium chloride or dextrose. Parenteral agents can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

Dosage values will vary depending on the particular disease state to be treated, but when treatments such as oral, parenteral or intra-sperm administration are effective, when the compound of formula (I) is administered at 0.10 to 25.0 mg / kg body weight per day You can get good results. A particularly preferred amount is to administer 1.0 mg / kg body weight per day. In general, the daily dosage will range from 10 to 1000 mg, preferably 100 to 600 mg per day. However, it is contemplated that a particular dosage regimen for a particular subject may be determined by the professional judgment of each individual in need of administration of the compound and the person administering the administration. In addition, the dosages mentioned herein are exemplary only and do not limit the scope and practice of the present invention.

Hereinafter, the present invention will be described in more detail based on the following examples.

EXAMPLE

To demonstrate the utility of the present invention, it is demonstrated that the compound of general structure (I) inhibits its activity against both human IL-1 and other leukocyte-induced cytokines formed in vitro and human IL-1 obtained by purification. The test was conducted. Although all of the various compounds included within the scope of the general formula (I) show the effect of inhibiting the activity of IL-1 and other leukocyte-induced cytokines, in this embodiment 1,3-dibutyl is a particularly preferred form of the present invention. Illustrated in connection with 7- (2-oxopropyl) xanthine.

material

Compound 1,3-dibutyl 7- (2-oxopropyl) xanthine DBOPX was prepared according to the method described in US Pat. No. 4,242,345.

Interleukin-1: Purified human monocytes IL-1 (IL-1β) and diluents were purchased from Criston Biotechnology (Pine Brook, NY). Human IL-1 used in this experiment is human monocyte interleukin-1 obtained by purification. Diluent is PBS-0.1% bovine serum albumin. IL-1 contains 50 pg / μg of LPS as a result of Limulus amoeba cell lysate analysis. IL-1 activity of 1 LAF Unit is half that of ³ H-thyminine in the presence of large cannavaline A [0.5 μg / ml], which is half of the maximum binding to mouse [C ³ H] thymocytes. Is defined as the quantity.

Human tumor necrosis factor to be prepared: rh-TNF by Genzyme Corp. (Boston, Mass.). It is produced by E. coli and purified by phenylsepirose chromatography and FPLC to have a final purity of 99%. Purity was assayed by SPS polyacrylamide gel analysis developed by Coomassie Brilliant Blue R250 and Sylvie staining. Its molecular weight was 36,000 daltons as a result of gel filtration (FPLC) in Superose 12 and consisted of two subunits each having a molecular weight of 17,000 daltons. It is sterilized in phosphate-buffered saline solution containing 0.1% bovine serum albumin as carrier protein. Immediately before use, rh-TNF was diluted with Hanks' controlled saline solution containing 0.1% human serum albumin.

Other materials were purchased and used as follows. Dimethyl sulfoxide (DMSO), n-formyl methionyl louisyl phenylalanine (FMLP: source solution was dissolved in DMSO 10 mM concentration and stored in 20μl aliquots at -70 ° C), hapanil, cytok extracted from the heart of horse Molecular Variation Enzyme extracted from type Cl and bovine liver (SOD: the raw material solution was dissolved in Hanks' adjusted saline solution at a concentration of 5 µg / ml and stored at -70 ° C in 100 μl aliquots) (Sigma Chemical, St. Louis, Mo.): Neutrophil Separation Medium (NIM: Los Alamos Diagnostics, Inc., Los Alamos, NM): Hanks Controlled Saline Solution (HBSS), Minimum Essential Medium (MEM) and Medium 199 (M199) ( Whittaker, MA Bioproducts, Walkerville, Md.): Dulbecco's phosphate buffered saline solution (PBS: GIBCO Laboratories, Grand Island, NY): Limulis Amoeba Cell Lysis Homogenization Test (LAL: Associates of Cape Cod, Inc., Woods Hole, Ma.): Nylon yarn (3 denier200) (Fenwal Laboratories, Deerfie) ld, Ill.): Litex and agarose type HSA (Accurate Chemical and Scientific Corp. Hicksville, N.Y.)

Production of PMN: PMN (98% PMN and 95% viability due to exclusion of trypans) obtained by purification containing less than 1 platelet per 5PMN and less than 50pg / ml LPS (LAL analysis) Extraction was performed by a one-stage ficollhypague separation method from normal heparinized (10 Units / ml) semen (NIM). The extracted PMN was washed three times with HBSS or MEM. The remaining RBCs were lysed in hypertonic solution to analyze the peroxidation of PMN.

Mononuclear leukocyte condition medium: After washing and mixing the mononuclear leukocytes obtained from the NIM isolate, they were suspended in a medium of 199 (M199) containing 10% fresh magnetic serum at a concentration of 3 × 10 ⁶ / ml and Lab-Tek Flaskettes (Miles Inc.). , Naperille, Ill) with or without addition of (10% CO ₂ ) LPS at 37 ° C. for 18 hours. The suspension was centrifuged at 150 g for 10 minutes, then the suspension was filtered (pore size: 0.45 μ) and frozen (−70 ° C.).

Statistics: Results are expressed as ± SEM. P-values were obtained using a 2-tailed student T-test.

Example 1

Chemotaxis of Cells

Chemotaxis under agarose was quantified according to Nalson et al., J. Immunol., 115, 1650-1656 (1975). Purified PMN (5 × 10 ⁶ PMN) DBOPX with or without total volume of 40 μl, 60 μl and 90 μl of HBSS was maintained for 15 minutes at 37 ° C., followed by LPS-stimulated mononuclear leukocyte conditioned medium (40 μl ), IL-1 (15 Units / 60 μl), Diluent (60 μl) or rh-TNF (100 Units / 10 μl) with or without 0.1 ml of total volume at a temperature of 37 ° C. for 30 minutes It was left. Orientation shift to PMLP (100mM) was measured after standing for 2 hours at 37 ℃.

DBOPX, as shown in FIGS. 1, 2 and 3, increased the chemotactic response that was inhibited by mononuclear leukocyte conditioned media stimulated with IL-1, TNF or LPS.

Example 2

PMN adhesion to nylon

PMN fixation was tested by modifying MacGregor's method. Purified PMN was allowed to stand for 30 minutes in LPS or LPS stimulated mononuclear leukocytes conditioned medium at 1.0 ° C M199 with 37 ° C. with or without DBOPX. After standing, HBSS (0.9 ml) and magnetic serum (10 ml) were added to the cell suspension. The cell suspension was applied to the top of a 60 mg nylon column (packed up to 0.3 ml mark in a 1 ml plastic syringe) previously covered with 37 ° C. The column was eluted at 37 ° C. for 30 minutes. The number of PMNs was calculated for both pre-column and post-column samples. The results are expressed as% PMN fixation to nylon.

DBOPX (10 μg / ml) reduced PMN fixation to nylon by LPS-stimulated mononuclear leukocyte conditioned medium as shown in FIG. 4.

Example 3

PMN Peroxide

Cytochrome c Reduction: Purified PMN (2-4 × 10 ⁶ ) is suspended in 80 μl of HBSS with or without DBOPX and maintained at 37 ° C. in the presence or absence of SOD (200 Units / sample). It was left standing for 15 minutes. Next, add IL-1- (5Units / 20μl), LPS (0.1ng / 20μl), LPS stimulated mononuclear leukocyte conditioned medium (20μl) or IL-1 diluent, and cells for 30 minutes above 37 ° C. It was left.

HBSS (0.4 ml) and cytochrome c (50 μl: final concentration 120 μM) were added to all samples and FMLP (100 nM) was added. The sample was left at a temperature of 37 ° C. or higher for 10 minutes, then cooled with ice and centrifuged (2000 × g, for 10 minutes). The absorbance of the suspension was measured at a wavelength of 550 nm, and the nmole number of the SOD-inhibiting superoxide per 10 ⁶ PMN was calculated with an extinction coefficient of 2.11 × 10 ⁴ cm 2 / nmole (reduction-oxidation).

DBOPX reduced PMN superoxide production when PMN was stimulated with IL-1, TNF and stimulated with FMLP, as demonstrated in FIG. DBOPX also reduced the excess oxide production of PMN even when it was promoted with LPS stimulated mononuclear leukocyte conditioned medium, as shown in FIG.

Example 4

PMN Granule Reduction (Lysozyme Secretion)

PMN (4 × 10 ⁶ ) was suspended in HBSS (0.08 ml) with or without DBOPX and left at 37 ° C. for 15 minutes. Then, LPS (0.1 ng / 0.02 ml) or LPS stimulated mononuclear leukocyte conditioned medium (0.02 ml) was added to the sample and left for at least 30 minutes. HBSS (0.9 ml) and FMLP (10 μl: final concentration 10 ⁻⁷ M) were added to all samples. The sample was left for 10 minutes, then cooled with ice and centrifuged (2000 x g, for 10 minutes). The lysozyme content was determined by pouring the suspension, adding the suspension using the method described in J. Bacteriol., 58: 731-736 and measuring the absorbance change of the Micrococcus Iysodeikticus suspension.

DBOPX was promoted with LPS stimulated mononuclear leukocyte conditioned media and reduced lysozyme secretion from PMN stimulated with FMLP, as can be seen from FIG.

Many modifications and variations are possible in light of the above teaching. Therefore, it is contemplated that the invention may be practiced in other ways than those specifically specified herein in the appended claims.

Claims (6)

Interleukin-1 activity and tumor necrosis factor activity in the mammal by administering a predetermined amount of at least one of 7- (oxoalkyl) 1,3-dialkyl xanthines of the following formula (I) to a mammal other than a human And other leukocyte-induced cytokine activity.

Wherein R ₁ and R ₂ may be the same or different, each of which is a straight or branched chain alkyl radical having 2 to 6 carbon atoms, a cyclohexyl group, a straight chain or a branched chain alkoxyalkyl group, and hydroxyalkyl lycal; Selected from: A is a hydrocarbon radical having up to 4 carbon atoms, which may be substituted by a methyl group, provided that R ₂ is an alkyl group of 2 to 4 carbon atoms and R ₁ is a group represented by the following structural formula Or A ₁ in the formula (I) is not 2 to 4 when the aromatic hydrocarbon in which R ₁ has 6 or less carbon atoms and an oxygen atom is inserted into the carbon chain or a hydroxyl group is substituted is R ₅ .

(Wherein R ₄ is an alkyl group having 1 to 3 carbon atoms and n is an integer from 2 to 5). The method of claim 1 wherein the compound is 1,3-dibutyl 7- (2-oxopropyl) xanthine. Administration of a certain amount of 7- (oxoalkyl) 1,3-dialkyl xanthine of formula (I) mitigates disease conditions in mammals other than humans controlled by interleukin-1, tumor necrosis factor, and other leukocyte-induced cytokines How to let it.

Wherein R ₁ and R ₂ may be the same or different, each of which is a straight or branched chain alkyl radical having 2 to 6 carbon atoms, a cyclohexyl group, a straight chain or a branched chain alkoxyalkyl group, and hydroxyalkyl lycal; Selected from: A is a hydrocarbon radical having up to 4 carbon atoms, which may be substituted by a methyl group, provided that R ₂ is an alkyl group of 2 to 4 carbon atoms and R ₁ is a group represented by the following structural formula Or, when R ₁ is 6 or less carbon atoms and an aliphatic hydrocarbon in which an oxygen atom is inserted or a hydroxy group is substituted in R ₅ is R _{5, the} number of carbon atoms in A of Structural Formula (I) is not 2 to 4.

(Wherein R ₄ is an alkyl group having 1 to 3 carbon atoms and n is an integer from 2 to 5). The method of claim 3, wherein the compound is 1,3-dibutyl 7- (2-oxopropyl) xanthine. One or more compounds of the following structural formula (I) are administered to mammals other than humans in an amount sufficient to inhibit the activity of other leukocyte-induced cytokines in interleukin-1, tumor necrosis factor or polymorphonuclear leukocytes or monocytes. A method of inhibiting tissue damage accompanied by inflammation due to leukocyte activity induced by cytokines produced by the method.

Wherein R ₁ and R ₂ may be the same or different, each of which is a straight or branched chain alkyl radical having 2 to 6 carbon atoms, a cyclonuclear group, a straight chain or branched chain alkoxyalkyl group, and hydroxyalkyl lycal; Selected from: A is a hydrocarbon radical having up to 4 carbon atoms, which may be substituted by a methyl group, provided that R ₂ is an alkyl group of 2 to 4 carbon atoms and R ₁ is a group represented by the following structural formula Or, when R ₁ is 6 or less carbon atoms and an aliphatic hydrocarbon in which an oxygen atom is inserted or a hydroxy group is substituted in the carbon chain is R ₅ , the number of carbon atoms in the formula (I) is not 2 to 4.

(Wherein R ₄ is an alkyl group having 1 to 3 carbon atoms and n is an integer from 2 to 5). 6. The method of claim 5 wherein the compound is 1,3-dibutyl 7- (2-oxopropyl) xanthine.

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