JPWO2005026132A1 - CAMP substrate-specific inhibitors of phosphodiesterase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a drug containing a benzoguanamine derivative or a pharmaceutically acceptable salt thereof as an active ingredient, which has a cAMP substrate-specific inhibitory action of phosphodiesterase. The present invention provides (1) a cAMP substrate-specific inhibitor of phosphodiesterase, which contains, as an active ingredient, a benzoguanamine derivative represented by the following general formula (I) or a pharmaceutically acceptable salt thereof: R1 and R2 are the same or different and each represents hydrogen or halogen.) (2) A benzoguanamine derivative represented by the general formula (I) of the above (1) or a pharmaceutically acceptable salt thereof as an active ingredient The present invention relates to a preventive drug or a therapeutic drug for bronchial asthma including atopic asthma.

Description

（式中、Ｒ^１及びＲ^２は、同一又は異なって、水素又はハロゲンを表す。）The present invention relates to a cAMP substrate-specific inhibitor of phosphodiesterase containing as an active ingredient a benzoguanamine derivative represented by the following general formula (I) or a pharmaceutically acceptable salt thereof (hereinafter referred to as the compound of the present invention). .

(In the formula, R ¹ and R ² are the same or different and represent hydrogen or halogen.)

  The compound of the present invention has an excellent anti-ulcer action, cell defense action, and gastric mucosal blood flow increase action (see, for example, Patent Documents 1 to 3 and Non-Patent Documents 1 to 3). -Diamino-6- (2,5-dichlorophenyl) -1,3,5-triazine maleate (generic name: irsogladine maleate) (hereinafter referred to as irsogladine maleate) is trade name "Gaslon N" It is marketed as a treatment for gastric ulcer, acute gastritis, and chronic gastritis. Moreover, it is known that irsogladine maleate has an action of increasing cAMP in rat gastric mucosa cells (for example, see Non-Patent Document 4).

Many hormones and neurotransmitters regulate tissue function by increasing intracellular levels of cAMP (3 ′, 5′-cyclic adenosine monophosphate). The intracellular level of cAMP, which functions as a second messenger, is regulated by mechanisms that control synthesis and degradation.
The synthesis of cAMP is controlled by adenylate cyclase, which is directly activated by drugs such as forskolin. In addition, prostaglandins (such as PGE ₂ or PGI ₂ ) and β-adrenergic receptor agonists are specific agonists for cell surface receptors and indirectly activate adenylate cyclase to activate cells. It acts as a trigger to increase the cAMP in the inside.
On the other hand, the degradation of cAMP is controlled by several phosphodiesterase (hereinafter referred to as PDE) isozymes. These series of PDEs also control the degradation of cGMP (guanosine 3 ′, 5′-cyclic monophosphate). PDE plays an extremely important role in the degree of activation of the intracellular signal transduction mechanism and its persistence by controlling the hydrolysis of cAMP. It is also an important inactivating enzyme involved in compartments that determine the diversity of biological functions of cAMP.

PDEs are functionally classified based on their substrate specificity, enzyme activity regulation mechanism, and sensitivity of various inhibitors. To date, PDEs have 11 known families (PDE1 to PDE11) that are genetically different. Is known (see, for example, Non-Patent Document 5). Among them, PDE4, PDE7, and PDE8 are cAMP substrate-specific PDEs that hydrolyze only cAMP as a substrate. PDE5, PDE6 and PDE9 are cGMP-specific PDEs that hydrolyze only cGMP as a substrate. PDE1 to PDE3, PDE10, and PDE11 are PDEs that hydrolyze both cyclic nucleotides of cAMP and cGMP. Among them, PDE1 is controlled by calmodulin, and the activities of PDE2 and PDE3 are controlled by cGMP.
It is well known that the expression of PDE varies greatly depending on tissues. For example, PDE10 and PDE11 are both PDEs that hydrolyze both cyclic nucleotides of cAMP and cGMP. PDE10 is very distributed in the brain, while PDE11 is distributed in peripheral tissues such as skeletal muscle and prostate. PDE5, PDE6 and PDE9 are all cGMP-specific PDEs, but PDE6 is known as a PDE expressed only in the retina.

Increased intracellular cAMP due to PDE4 inhibition has been reported to cause inactivation of inflammatory cells such as neutrophils, eosinophils, T-lymphocytes, macrophages, and relaxation of tracheal smooth muscle. (For example, refer nonpatent literatures 6 and 7.). Therefore, PDE4 inhibitors are bronchial asthma including atopic asthma, chronic bronchitis, chronic obstructive pulmonary disease, adult dyspnea syndrome, systemic lupus erythematosus, allergic rhinitis, atopic dermatitis, conjunctivitis, urticaria, It can be used for the treatment of psoriasis, keloid, gingivitis, periodontitis, alveolar pyorrhea, scleroderma, rheumatoid arthritis, chronic glomerulonephritis, Crohn's disease, ulcerative colitis, esophagitis and the like.
Since PDE7 is also highly expressed in T-lymphocytes (see, for example, Non-Patent Document 8), the inhibitor can be used for the treatment of T cell-dependent diseases such as rheumatoid arthritis and psoriasis.
PDE1 is a calmodulin-dependent PDE. An inhibitor of PDE1 is useful as a therapeutic agent for urinary incontinence (see, for example, Patent Document 9).
PDE3 is a cGMP-inhibiting PDE. The increase in intracellular cAMP due to PDE3 inhibition causes, for example, relaxation of vascular smooth muscle and inhibition of platelet aggregation (see, for example, Non-Patent Document 10), and this inhibitor is useful as a therapeutic agent for thrombosis. .

Japanese Patent Laid-Open No. 50-111085 Japanese Patent Laid-Open No. 50-111086 JP 51-75083 A Ueda F, et al, Arzneim. -Forsch. / Drug Res. , 34 (I), 474 (1984) Ueda Fumio et al., Applied Pharmacology, 33 (1), 143 (1987) Hiramatsu, et al., Pharmacology and Treatment, 11 (7), 2481 (1983) Ueda Fumio et al., Applied Pharmacology, 33 (1), 157 (1987) Francis SH, et al, Prog Nucleic Acid Res Mol Biol, 65, 1-52 (2001) Barnes PJ, Nature, 402, B31-B38 (1999) Mauro M, et al, Trends Pharmacol Sci, 18, 164-170 (1997). Li L, et al, Science, 283, 848-851 (1999) Truss MC, et al, World J Urol, 18, 439-443 (2000). Sudo T, et al, Biochem Pharmacol, 59, 347-356 (2000). Banner KH, et al, Eur Respir J, 8, 996-1000 (1995). Mauro M, et al, Trends Pharmacol Sci, 18, 164-170 (1997). Ministry of Health, Labor and Welfare, Pharmaceutical Safety Bureau, “Safety Information on Drugs, etc., Summary of Side Effects of New Drugs, 1996”, October 1997, p12 Nichols MR, et al, Mol. Pharmacol. , 57, 738-45 (2000) Oka M, et al, Naunyn Schmiedebergs Arch Pharmacol, 356, 189-96 (1997). Wang P, et al, Mol. Pharmacol. , 56, 170-174 (1999)

  An object of the present invention is to provide a cAMP substrate-specific inhibitor of PDE, which contains a benzoguanamine derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

  As a result of intensive studies on the increasing action of cAMP in gastric mucosa cells of the compound of the present invention, the present inventors have found that the compound of the present invention selectively inhibits only hydrolysis of cAMP by PDE. Was completed.

The present invention
(1) a cAMP substrate-specific inhibitor of PDE containing the compound of the present invention as an active ingredient,
(2) The inhibitor according to (1), wherein the benzoguanamine derivative is 2,4-diamino-6- (2,5-dichlorophenyl) -1,3,5-triazine,
(3) The inhibitor of (1) above, wherein the pharmaceutically acceptable salt of the benzoguanamine derivative is irsogladine maleate,
(4) Bronchial asthma including atopic asthma, chronic bronchitis, chronic obstructive pulmonary disease, adult dyspnea syndrome, systemic lupus erythematosus, allergic rhinitis, atopic dermatitis, containing the compound of the present invention as an active ingredient, Conjunctivitis, urticaria, psoriasis, keloid, gingivitis, periodontitis, alveolar pyorrhea, scleroderma, rheumatoid arthritis, chronic glomerulonephritis, Crohn's disease, ulcerative colitis, esophagitis, urinary incontinence or thrombosis Preventive drugs for these or their therapeutic agents,
(5) The preventive or therapeutic agent according to (4) above, wherein the benzoguanamine derivative is 2,4-diamino-6- (2,5-dichlorophenyl) -1,3,5-triazine,
(6) The preventive or therapeutic agent according to (4) above, wherein the pharmaceutically acceptable salt of the benzoguanamine derivative is irsogladine maleate,
About.

  As shown in Test Example 1 described later, the compound of the present invention exhibits a strong inhibitory action on the hydrolysis of cAMP by PDE, but does not show an obvious inhibitory action on the hydrolysis of cGMP by PDE. As a result of further detailed investigations, as shown in Test Examples 2 to 4 described later, the PDE1, PDE2, PDE3 and PDE4 isozymes have almost the same inhibitory activity.

  It is well known that when the excellent anti-inflammatory action of a PDE4 inhibitor is applied clinically, the occurrence of side effects in the digestive system such as nausea and vomiting often becomes a problem (for example, Non-Patent Document 11). , 12). The compound of the present invention is clinically useful as a PDE4 inhibitor because it hardly causes gastrointestinal side effects such as nausea and vomiting (see Non-Patent Document 13, for example).

The present invention is described in detail below.
The “cAMP substrate-specific inhibitor of phosphodiesterase” refers to a drug that selectively inhibits only the hydrolysis of cAMP by PDE and does not have an obvious inhibitory effect on the hydrolysis of cGMP by PDE.
Examples of “halogen” include fluorine, chlorine, bromine and iodine.
Examples of the “pharmaceutically acceptable salt” include salts of mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, tartaric acid, maleic acid, succinic acid, fumaric acid, p-toluenesulfone. Mention may be made of salts of organic acids such as acids, benzenesulfonic acid and methanesulfonic acid.

It is the graph which showed the result of having examined the effect | action of the irsogladine maleate with respect to the hydrolysis of cAMP by refined PDE derived from bovine brain. The vertical axis represents the amount of cAMP (pmol / 30 min.), And the horizontal axis represents the concentration (M) of irsogladine maleate. Each column shows mean value ± standard error (n = 5). “**” in the graph represents the result of Dunnett's test ( ^** P <0.01). It is the graph which showed the result of having investigated the effect | action of the irsogladine maleate with respect to the hydrolysis of cGMP by the bovine brain origin refinement | purification PDE. The vertical axis represents the amount of cGMP (pmol / 30 min.), And the horizontal axis represents the concentration (M) of irsogladine maleate. Each column shows mean value ± standard error (n = 5). “*” In the graph represents the result of Dunnett's test ( ^* P <0.05). It is the graph which showed the result of having examined the effect | action of various PDE inhibitors with respect to the hydrolysis of cAMP by the bovine brain origin refinement | purification PDE. The vertical axis represents the amount of cAMP (pmol / 30 min.). Each column shows mean value ± standard error (n = 5). “**” in the graph represents the result of Dunnett's test ( ^** P <0.01). It is the graph which showed the result of having examined the effect | action of various PDE inhibitors with respect to the hydrolysis of cAMP by refined PDE derived from bovine heart. The vertical axis represents the amount of cAMP (pmol / 30 min.). Each column shows mean value ± standard error (n = 5). “**” in the graph represents the result of Dunnett's test ( ^** P <0.01). It is the graph which showed the result of having examined the action of irsogladine maleate with respect to hydrolysis of cAMP by bovine heart origin purified PDE in the presence of vinpocetine (5 × 10 ⁻⁵ M) or cilostamide (5 × 10 ⁻⁵ M). The vertical axis represents the amount of cAMP (pmol / 30 min.). Each column shows mean value ± standard error (n = 5). “**” in the graph represents the result of Dunnett's test ( ^** P <0.01). It is the graph which showed the result of having examined the effect | action of various PDE inhibitors with respect to cAMP production of a human neutrophil. The vertical axis represents the amount of cAMP (pmol / 30 min.). Each column shows mean value ± standard error (n = 5). “**” in the graph represents the result of Dunnett's test ( ^** P <0.01). It is the graph which showed the result of having examined the effect of irsogladine maleate on cAMP production of human neutrophils. The vertical axis shows the amount of cAMP (pmol / 30 min.). Each column shows mean value ± standard error (n = 5). “*” And “**” in the graph represent the results of Dunnett's test ( ^* P <0.05, ^** P <0.01). It is the graph which showed the result of having examined the effect | action of the irsogladine maleate in presence of IBMX with respect to cAMP production of a human neutrophil. The vertical axis represents the amount of cAMP (pmol / 30 min.). Each column shows mean value ± standard error (n = 5). “**” in the graph represents the result of Dunnett's test ( ^** P <0.01).

The compound of the present invention is a known compound and can be produced by a known method (for example, see Patent Documents 1 to 3). For example, it is produced by reacting mono- or dihalogenobenzonitrile with dicyandiamide, or reacting mono- or dihalogenobenzoic acid derivatives (carboxylic acid, acid halide, ester, amide, etc.) with biguanide. Can do.
The compound of the present invention can be used as a medicament as a free base, but can also be used in the form of a pharmaceutically acceptable salt by a known method. For example, the maleate of the compound of the present invention can be obtained by dissolving a benzoguanamine derivative in an alcohol solution or an ethyl acetate solution in which an equivalent amount of maleic acid is dissolved and concentrating.

The compound of the present invention is used as a pharmaceutical composition containing, for example, 0.01 to 99.5%, preferably 0.5 to 90% in a non-toxic and inert carrier that is pharmaceutically acceptable. Administered to the containing animal.
As the carrier, one or more solid, semi-solid or liquid diluents, fillers and other formulation aids are used. The drug is preferably administered in dosage unit form. In addition, the drug can be administered intravenously, orally, intratissue, locally (such as transdermally), or rectally, but is not limited thereto. Of course, it is administered in a dosage form suitable for these administration methods.
The dose of PDE as a cAMP substrate-specific inhibitor is preferably set in consideration of the nature and extent of the disease / injury, the patient's condition such as age and weight, the route of administration, etc. On the other hand, the amount of the active ingredient of the drug according to the present invention is generally in the range of 0.1 to 1000 mg / human, preferably in the range of 1 to 500 mg / human per day.
In some cases, this may be sufficient, and vice versa. Moreover, it can also be divided and administered in 2-4 times a day.

Oral administration should be carried out in solid or liquid dosage units such as powders, powders, tablets, dragees, capsules, granules, suspensions, solutions, syrups, drops, sublingual tablets and other dosage forms. Can do.
The powder is manufactured by making the drug fine. Powders are made by mixing the drug with appropriate fineness and then mixing it with a similarly finely divided pharmaceutical carrier such as starch, edible carbohydrates such as mannitol, and the like. A flavoring agent, a preservative, a dispersing agent, a coloring agent, a fragrance, and the like may be mixed as necessary.

  Capsules are produced by first filling a powdered powder, powder, or granulated powder as described above into a capsule shell such as a gelatin capsule. The Lubricants and fluidizing agents, such as colloidal silica, talc, magnesium stearate, calcium stearate, solid polyethylene glycol, etc., can be mixed in powder form and then filled. it can. Disintegrants and solubilizers such as carboxymethylcellulose, carboxymethylcellulose calcium, low-substituted hydroxypropylcellulose, croscarmellose sodium, carboxymethyl starch sodium, calcium carbonate, sodium carbonate The effectiveness of the medicine can be improved.

  Further, a fine powder of a drug can be suspended and dispersed in vegetable oil, polyethylene glycol, glycerin, and a surfactant, and this can be wrapped in a gelatin sheet to form a soft capsule. Tablets are manufactured by adding excipients to make a powder mixture, granulating or slugging, then adding a disintegrant or lubricant and then tableting. The powder mixture is prepared by mixing an appropriate powdered drug with the above-mentioned diluent and base, and if necessary a binder (for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, gelatin, polyvinylpyrrolidone, polyvinyl alcohol, etc.), A dissolution retardant (for example, paraffin), a resorbent (for example, quaternary salt) and an adsorbent (for example, bentonite, kaolin, dicalcium phosphate, etc.) may be used in combination. The powder mixture can be first wetted with a binder such as syrup, starch paste, gum arabic, cellulose solution or polymer solution, stirred and mixed, dried and pulverized into granules. Instead of granulating the powder in this way, it is also possible to first crush the tablet and then to crush the resulting incomplete slag into granules.

The granules thus produced can be prevented from adhering to each other by adding stearic acid, stearate, talc, mineral oil or the like as a lubricant. The mixture thus lubricated is then tableted. Film coating or sugar coating can be applied to the uncoated tablets thus produced.
Further, the drug may be directly tableted after being mixed with a fluid inert carrier without going through the steps of granulation and slag as described above. A transparent or translucent protective coating made of a shellac hermetic coating, a coating of sugar or polymer material, and a polishing coating made of wax can also be used.

Other oral dosage forms such as solutions, syrups, elixirs and the like can also be in dosage unit form so that a given quantity contains a certain amount of drug. Syrups are made by dissolving the drug in a suitable flavored aqueous solution, and elixirs are made by using a non-toxic alcoholic carrier. Suspensions are formulated by dispersing the drug in a non-toxic carrier. Solubilizers and emulsifiers (eg, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol esters), preservatives, flavoring agents (eg, peppermint oil, saccharin) and others can also be added as needed. .
If necessary, dosage unit formulations for oral administration can be microencapsulated. The formulation can also provide extended action time or sustained release by coating or embedding in polymers, waxes, and the like.

  Intra-tissue administration can be performed by using a liquid dosage unit form for subcutaneous, intramuscular or intravenous injection, for example, a solution or suspension form. These are made by suspending or dissolving a certain amount of drug in a nontoxic liquid carrier suitable for injection purposes, such as an aqueous or oily medium, and then sterilizing the suspension or solution. The Non-toxic salts and salt solutions may be added to make the injection solution isotonic. Further, stabilizers, preservatives, emulsifiers and the like can be used in combination.

  Rectal administration is a drug soluble or insoluble solid in low melting water such as polyethylene glycol, cocoa butter, semi-synthetic oils (eg, Witepsol, registered trademark), higher esters (eg, myristyl palmitate) and those It can be carried out by using a suppository or the like produced by dissolving or suspending in the mixture.

  Hereinafter, the present invention will be described in more detail with reference to test examples and formulation examples, but the present invention is not limited thereto.

Test Example 1 Examination of the Effect of Irsogladine Maleate on Bovine Brain-Derived Purified PDE The effect of irsogladine maleate on the hydrolysis of cAMP and cGMP by the purified bovine brain-derived PDE was examined.
PDE (Sigma-RBI, Natick, MA, USA) purified from bovine brain was mixed with Krebs-Ringer bicarbonate solution (120 mM NaCl, 3.3 mM KCl, 1.3 mM CaCl) in the presence of cAMP or cGMP and irsogladine maleate. ₂ , 1.2 mM MgSO ₄ , 1.2 mM KH ₂ PO ₄ , 0.03 mM EDTA, 25 mM NaHCO ₃ , 11 mM glucose, pH 7.4) (hereinafter referred to as KRB solution) for 30 minutes (37 ° C.) Performed. The reaction was stopped by adding perchloric acid (final concentration 0.2M). The reaction solution was centrifuged at 10,000 × g for 15 minutes (4 ° C.), and then the supernatant was used for quantification of cAMP and cGMP. The supernatant was neutralized by adding K ₂ CO ₃ and centrifuged at 10,000 × g for 15 minutes (4 ° C.). Quantification of cAMP and cGMP was performed using an EIA kit (Amersham, Buckinghamshire, UK) (hereinafter the same). The results are shown in FIG. 1 (effect of irsogladine maleate on hydrolysis of cAMP) and FIG. 2 (effect of irsogladine maleate on hydrolysis of cGMP).

About the influence which it has on hydrolysis of cAMP, as shown in FIG. 1, irsogladine maleate suppresses the hydrolysis of cAMP by PDE in a concentration-dependent manner almost completely, and the inhibitory action is 10 ⁻⁷ M or more. The concentration was statistically significant. On the other hand, as to the influence on the hydrolysis of cGMP, as shown in FIG. 2, irsogladine maleate only slightly suppressed the hydrolysis of cGMP only at an extremely high dose (10 ⁻⁵ M).
From this result, it is clear that irsogladine maleate selectively inhibits only the hydrolysis of cAMP by PDE and has no obvious inhibitory action on the hydrolysis of cGMP by PDE.

Test Example 2 Examination of relationship between PDE inhibitory action of irsogladine maleate and PDE isozyme Clarified isozymes contained in purified bovine brain-derived PDE, and examined the relation between cAMP hydrolysis inhibitory action of irsogladine maleate and each PDE isozyme did.
The PDE used in Test Example 1 was incubated in KRB solution for 30 minutes (37 ° C.) in the presence of cAMP and each test drug. The reaction was stopped by adding perchloric acid (final concentration 0.2M). The reaction solution was centrifuged at 10,000 × g for 15 minutes (4 ° C.), and the supernatant was used for cAMP quantification. The supernatant was neutralized by adding K ₂ CO ₃ and centrifuged at 10,000 × g for 15 minutes (4 ° C.). Quantification of cAMP was performed using an EIA kit. The result is shown in FIG.

As shown in FIG. 3, vinpocetine (5 × 10 ⁻⁵ M), which is a PDE1 selective inhibitor, and erythro-9- (2-hydroxy-3-nonyl) adenine hydrochloride (EHNA), which is a PDE2 selective inhibitor Both (5 × 10 ⁻⁵ M) and rolipram (5 × 10 ⁻⁵ M), which is a PDE4 selective inhibitor, partially suppressed the hydrolysis of cAMP by purified bovine brain-derived PDE. On the other hand, cilostamide (5 × 10 ⁻⁵ M), which is a PDE3 selective inhibitor, did not affect the hydrolysis of cAMP by purified bovine brain-derived PDE. The non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX, 10 ⁻³ M) completely suppressed PDE hydrolysis.
Comprehensively judging these results and the results of Test Example 1, the purified bovine brain-derived PDE contains at least PDE1, PDE2, and PDE4 isozymes, and irsogladine maleate inhibits PDE1, PDE2, and PDE4. It is clear that it shows an effect.

Test Example 3 Examination of the Effect of Irsogladine Maleate on Bovine Heart-Derived Purified PDE The same investigation as in Test Example 1 and Test Example 2 was performed using bovine heart-derived purified PDE instead of bovine brain-derived purified PDE.
PDE purified from bovine heart (Sigma-RBI, Natick, MA, USA) was added in KRB solution for 30 minutes (37 ° C.) in the presence of cAMP and each test drug (FIG. 4) or irsogladine maleate (FIG. 5). ) Incubation. The reaction was stopped by adding perchloric acid (final concentration 0.2M). The reaction solution was centrifuged at 10,000 × g for 15 minutes (4 ° C.), and the supernatant was used for cAMP quantification. The supernatant was neutralized by adding K ₂ CO ₃ and centrifuged at 10,000 × g for 15 minutes (4 ° C.). Quantification of cAMP was performed using an EIA kit. The results are shown in FIG. 4 (influence of various PDE selective inhibitors on hydrolysis of cAMP) and FIG. 5 (influence of irsogladine maleate on hydrolysis of cAMP).

As shown in FIG. 4, both vinpocetine (5 × 10 ⁻⁵ M), which is a PDE1 selective inhibitor, and cilostamide (5 × 10 ⁻⁵ M), which is a PDE3 selective inhibitor, are purified from bovine heart. The effect of the combined use of vinpocetine and cilostamide was partially additive while cAMP hydrolysis by PDE was partially suppressed. On the other hand, EHNA (5 × 10 ⁻⁵ M) which is a PDE2 selective inhibitor and rolipram (5 × 10 ⁻⁵ M) which is a PDE4 selective inhibitor are capable of hydrolyzing cAMP by purified PDE derived from bovine heart. It had no effect on it. IBMX (10 ⁻³ M), a non-selective PDE inhibitor, completely suppressed hydrolysis by PDE.
As shown in FIG. 5, irsogladine maleate suppressed the hydrolysis of cAMP by PDE in the presence of vinpocetine (5 × 10 ⁻⁵ M), which is a PDE1 selective inhibitor, in a concentration-dependent manner. In addition, irsogladine maleate also showed a concentration-dependent inhibitory action against cAMP hydrolysis by PDE in the presence of cilostamide (5 × 10 ⁻⁵ M), which is a PDE3 selective inhibitor.

  From these results, it is clear that purified PDE derived from bovine heart contains at least PDE1 and PDE3 subtypes. This result is consistent with a report that the bovine heart-derived PDE is mainly composed of PDE1 and PDE3 (see, for example, Non-Patent Document 14). It is clear that irsogladine maleate exhibits an inhibitory action on at least PDE1 and PDE3.

Test Example 4 Examination of the effect of irsogladine maleate on cAMP production of human neutrophils The effect of irsogladine maleate and various PDE inhibitors on cAMP production of human neutrophils was examined.
Neutrophils were collected from venous blood of healthy individuals. After heparinized blood was diluted with physiological saline, it was overlaid with a blood cell separation agent (Polymorphprep, AXIS-Shield PoC AS, Oslo, Norway), and neutrophils were separated and collected according to the blood separation agent attached manual. The collected neutrophils were adjusted to a predetermined concentration with Hank's balanced salt solution (HBSS). The measurement of the cAMP content was based on a known method (for example, see Non-Patent Document 15). The separated neutrophils (1 × 10 ⁶ per assay) were washed with HBSS and then preincubated for 10 minutes in a CO ₂ incubator at 37 ° C. Thereafter, each test drug was added and incubated at 37 ° C. for 30 minutes. The reaction was stopped by adding perchloric acid (final concentration 0.2M). The reaction solution was centrifuged at 10,000 × g for 15 minutes (4 ° C.), and the supernatant was used for cAMP quantification. The supernatant was neutralized by adding K ₂ CO ₃ and centrifuged at 10,000 × g for 15 minutes (4 ° C.). cAMP quantification was performed using an EIA kit. The results are shown in FIG. 6 (influence of various PDE selective inhibitors), FIG. 7 (influence of irsogladine maleate), and FIG. 8 (influence of irsogladine maleate, non-selective PDE inhibitor).

As shown in FIG. 6, the amount of cAMP was increased when rolipram (5 × 10 ⁻⁵ M), which is a PDE4 selective inhibitor, or IBMX (10 ⁻³ M), which is a nonselective PDE inhibitor. Increased, PDE1 selective inhibitor vinpocetine (5 × 10 ⁻⁵ M), PDE2 selective inhibitor EHNA (5 × 10 ⁻⁵ M), or PDE3 selective inhibitor cilostamide (5 × 10 ⁵ ) ^{When -5} M) was allowed to act, no increase in the amount of cAMP was observed.
As shown in FIG. 7, irsogladine maleate increased cAMP production of neutrophils in a concentration-dependent manner, and the effect was statistically significant at concentrations of 10 ⁻⁷ M or higher.
As shown in FIG. 8, the cAMP production enhancing action of irsogladine maleate was not enhanced even when IBMX (10 ⁻³ M), which is a non-selective PDE inhibitor, coexists.

  From these results, it is clear that PDE4 is mainly responsible for cAMP hydrolysis in human neutrophils. This result is consistent with a report that PDE4B is highly expressed in human neutrophils (see, for example, Non-Patent Document 16). It is also clear that irsogladine maleate exhibits an inhibitory action on at least PDE4.

Formulation Example 1
Tablet (internal tablet)
Formulation 1 tablet 80mg Irsogladine maleate 2.0mg
Corn starch 49.6mg
Crystalline cellulose 24.0mg
Methylcellulose 4.0mg
Magnesium stearate 0.4mg
This proportion of the mixed powder is formed into tablets by a conventional method.
Formulation Example 2
Tablet (internal tablet)
Formulation 1 tablet 80mg Irsogladine maleate 4.0mg
Corn starch 47.6mg
Crystalline cellulose 24.0mg
Methylcellulose 4.0mg
Magnesium stearate 0.4mg
This proportion of the mixed powder is formed into tablets by a conventional method.

  Since the compound of the present invention selectively inhibits only the hydrolysis of cAMP by PDE and has no obvious inhibitory action on the hydrolysis of cGMP by PDE, bronchial asthma including atopic asthma, chronic bronchial Inflammation, chronic obstructive pulmonary disease, adult dyspnea syndrome, systemic lupus erythematosus, allergic rhinitis, atopic dermatitis, conjunctivitis, urticaria, psoriasis, keloid, gingivitis, periodontitis, alveolar pyorrhea, scleroderma It is useful as a prophylactic or therapeutic agent for rheumatoid arthritis, rheumatoid arthritis, chronic glomerulonephritis, Crohn's disease, ulcerative colitis, esophagitis, urinary incontinence or thrombosis.

Claims (6)

A cAMP substrate-specific inhibitor of phosphodiesterase, which contains a benzoguanamine derivative represented by the following general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.

(In the formula, R ¹ and R ² are the same or different and represent hydrogen or halogen.) The inhibitor according to claim 1, wherein the benzoguanamine derivative is 2,4-diamino-6- (2,5-dichlorophenyl) -1,3,5-triazine. The inhibitor according to claim 1, wherein the pharmaceutically acceptable salt of the benzoguanamine derivative is 2,4-diamino-6- (2,5-dichlorophenyl) -1,3,5-triazine maleate. A bronchial asthma including atopic asthma, chronic bronchitis, chronic obstructive pulmonary disease, an adult comprising the benzoguanamine derivative represented by the general formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient Respiratory distress syndrome, systemic lupus erythematosus, allergic rhinitis, atopic dermatitis, conjunctivitis, urticaria, psoriasis, keloid, gingivitis, periodontitis, alveolar abscess, scleroderma, rheumatoid arthritis, chronic glomerulus Prophylactic or therapeutic agents for nephritis, Crohn's disease, ulcerative colitis, esophagitis, urinary incontinence or thrombosis. The prophylactic or therapeutic agent according to claim 4, wherein the benzoguanamine derivative is 2,4-diamino-6- (2,5-dichlorophenyl) -1,3,5-triazine. The preventive or therapeutic agent according to claim 4, wherein the pharmaceutically acceptable salt of the benzoguanamine derivative is 2,4-diamino-6- (2,5-dichlorophenyl) -1,3,5-triazine maleate.

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