WO2021177332A1 - Nlrp3 inflammasome inhibitor and pharmaceutical composition for preventing or treating inflammatory disease - Google Patents

Abstract

An NLRP3 inflammasome inhibitor containing a cephem compound as an active ingredient.

Description

NLRP3 inflammasome inhibitor and pharmaceutical composition for the prevention or treatment of inflammatory diseases

The present invention relates to NLRP3 inflammasome inhibitors and pharmaceutical compositions for the prevention or treatment of inflammatory diseases.

Inframasome consists of proteins involved in signal recognition (NLRP1, NLRP3, NLRP4, AIM2), adapter proteins ASC (apoptosis-associated speck-like protein containing caspase recruitment domain; apoptosis-related spec-like protein), and caspase-1. Caspase-1, which is a protein complex activated by a specific activation stimulus, induces the inflammatory cytokines IL-1β and IL-18 from precursors to mature bodies, causing inflammation. Among the inflammasomes, the NLRP3 inflammasome is known to be activated by various extrinsic and endogenous stimuli, and excessive activation of the NLRP3 inflammasome causes hyperuricemia, gout, and joints. Since it is involved in the development of a wide variety of inflammatory diseases such as rheumatism and chronic inflammation in diabetes, the NLRP3 inflammasome has recently attracted attention as an effective therapeutic target for these diseases, and research for the development of its inhibitors. Is being promoted all over the world (Non-Patent Document 1).

For example, existing NLRP3 inflammasome inhibitors include 1) microtubule synthesis inhibitors such as colchicine, 2) canakinumab, 3) ibrutinib, and 4) MCC950. Colchicine is used to relieve the symptoms of gout and cryopyrin-associated periodic fever syndrome, but the problem is that if it is not taken immediately after a gout attack, sufficient efficacy cannot be obtained. In addition, long-term administration causes various side effects. Canakinumab, an antibody that inhibits the IL-1 receptor, is a type of molecular-targeted drug and can be expected to have a high effect on blocking IL-1β, but it has no effect on IL-18 and has a partial anti-inflammatory effect. Become a target. In addition, there are many unclear points about the side effects that occur when long-term administration is performed, and the safety as a drug has not been established. Further, since the manufacturing process of the polymer compound is complicated, the manufacturing cost is high and the treatment cost is high. Ibrutinib is an anticancer drug used to treat B-cell tumors, but has recently been found to inhibit the activation of the NLRP3 inflammasome. However, ibrutinib has an almost 100% incidence of side effects at the time of clinical trials, and some of them are serious, so its application as an anti-inflammatory drug has not been approved. The drug price is also high. Although MCC950 is a recently identified NLRP3 inflammasome-specific inhibitor, its safety when administered to humans has not been established and its administration to humans has not been approved. If approved as a therapeutic drug in the future, the drug price is expected to be high.

An excellent NLRP3 inflammasome inhibitor has not yet been developed, and a highly safe NLRP3 inflammasome inhibitor is required.

On the other hand, cefotaxime, which is a kind of cephem antibacterial drug, is one of the essential medicines defined by the World Health Organization (WHO), and is regarded as a safe medicine without side effects, and is widely used all over the world. In the process of analyzing the mechanism of inflammatory diseases, the present inventors unexpectedly discovered that a specific cephem compound strongly suppresses the activation of the NLRP3 inflammasome, and completes the present invention. It came to.

The present invention includes the embodiments described below.
Item 1. An NLRP3 inflammasome inhibitor containing a cephem compound as an active ingredient.
Item 2. The cephem compound has the following formula (I).

(In the formula, Z is sulfur or oxygen, R ¹ is hydrogen or methoxy, R ² is an acyl, CO ₂ R ³ is a carboxyl group or a carboxylate anion, R ⁴ is hydrogen, halogen, -CH ₂ -R ⁵ or -CH ₂ OCONH ₂ , where R ⁵ is hydroxy, optionally substituted C1-6 alkoxy, optionally substituted aminocarbonyloxy, optionally substituted C1- 6 Alkoxycarbonyloxy, —S— (optionally substituted heteroaryl), or nitrogen-containing cyclic group)
Item 3. The NLRP3 inflammasome inhibitor according to Item 1, which is a compound represented by, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.
Item 3. The cephem compound has the following formula (Ia).

(In the formula, R ¹ is hydrogen or methoxy, R ² is an acyl, CO ₂ R ³ is a carboxyl group or carboxylate anion, and R ⁴ is hydrogen, halogen, -CH _2- R ⁵ . , R ⁵ is hydroxy, optionally substituted C1-6 alkoxy, optionally substituted aminocarbonyloxy, optionally substituted C1-6 alkylcarbonyloxy, —S— (may be substituted) Heteroaryl) or nitrogen-containing cyclic group)
Item 3. The NLRP3 inflammasome inhibitor according to Item 1, which is a compound represented by, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.
Item 4. The cephem compounds consist of cefoxitin, cefamandole, cefarotin, cefacrol, cefoniside, cefloxim, cefotaxime, ceftazidime, cefmenoxime, cefazolin, cefpyrom, cefepime, ceftoriazone, ceftizoxime, cefoperazone, ceftizoxime, cefoperazone, cefotetan, cefotetan, cefoperazone, cefotetan. NLRP3 Inframasome Inhibitor according to any one of Items 1 to 3, which is one or more compounds selected from the group, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.
Item 5. NLRP3 Inflammasome NLRP3 Inflammasome in vitro in non-human animal subjects or in mammalian-derived isolated or cultured cells, consisting of administration to the subject in an amount effective to inhibit the formation and / or expression of the NLRP3 inflammasome. A method for inhibiting the formation and / or expression of a mammal.
Item 6. From the group consisting of gout, pseudo-gout, rheumatoid arthritis, hyperuricemia, type II diabetes, urticaria, criopyrinopathy, and cryopyrin-associated periodic fever syndrome (CAPS) containing effective amounts of cephem compounds. A pharmaceutical composition for the prevention or treatment of selected inflammatory diseases.
Item 7. The cephem compound has the following formula (I).

(In the formula, Z is sulfur or oxygen, R ¹ is hydrogen or methoxy, R ² is an acyl, CO ₂ R ³ is a carboxyl group or a carboxylate anion, R ⁴ is hydrogen, halogen, -CH ₂ -R ⁵ or -CH ₂ OCONH ₂ , where R ⁵ is hydroxy, optionally substituted C1-6 alkoxy, optionally substituted aminocarbonyloxy, optionally substituted C1- 6 Alkoxycarbonyloxy, —S— (optionally substituted heteroaryl), or nitrogen-containing cyclic group)
Item 6. The pharmaceutical composition according to Item 6, which is a compound represented by, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.
Item 8. The cephem compound has the following formula (I).

(In the formula, R ¹ is hydrogen or methoxy, R ² is an acyl, CO ₂ R ³ is a carboxyl group or carboxylate anion, and R ⁴ is hydrogen, halogen, or -CH _2- R ⁵ . Yes, R ⁵ is hydroxy, optionally substituted C1-6 alkoxy, optionally substituted aminocarbonyloxy, optionally substituted C1-6 alkylcarbonyloxy, -S- (even substituted) Good heteroaryl), or nitrogen-containing cyclic group)
Item 6. The pharmaceutical composition according to Item 6, which is a compound represented by, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.
Item 9. The cephem compounds consist of cefoxytin, cefamandole, cephalotin, cefacrol, cefoniside, cefloxim, cefotaxime, ceftazidime, cefmenoxime, cefazolin, cefpyrom, cefepime, ceftoriazone, ceftizoxime, cefoperazone, ceftizoxime, cefoperazone, cefotetan, cefotetan, cefoperazone, cefotetan. Item 6. The pharmaceutical composition according to any one of Items 6 to 8, which is one or more compounds selected from the group, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.
Item 10. Item 6. The pharmaceutical composition according to any one of Items 6 to 9, further containing a quinolone antibacterial agent.
Item 11. A therapeutic agent for inflammatory diseases containing an effective amount of a cephem compound.
Item 12. NLRP3 inflammasome inhibitor or selected from the group consisting of gout, pseudogout, rheumatoid arthritis, hyperuricemia, type II diabetes, renal lung, criopyrinopathy, and cryopyrin-associated periodic fever syndrome (CAPS) Use of cephem compounds to produce pharmaceutical compositions for the prevention or treatment of inflammatory diseases.
Item 13. A combination drug of a cephem compound and a quinolone antibacterial agent for inhibiting the expression and / or release of IL-1β from immune cells.
Item 14. A product containing a cephem compound and a quinolone antibacterial agent as a combination drug for use simultaneously, sequentially or at intervals in preventing or treating an inflammatory disease.
Item 15. A combination drug of a cephem compound and a quinolone antibacterial agent for use in the prevention or treatment of inflammatory diseases.

According to the NLRP3 inflammasome inhibitor of the present invention, the formation of the NLRP3 inflammasome complex can be inhibited with little or no side effects. In addition, according to the pharmaceutical composition of the present invention, it is possible to prevent or treat an inflammatory disease in a subject with little or no side effects. Since cephem compounds are inexpensive drugs that are widely used as antibacterial drugs, they are also advantageous in terms of manufacturing cost as compared with antibody drugs.

Formation of NLRP3 inflammasome complex using ASC (apoptosis-associated speck-like protein containing caspase recruitment domain; apoptosis-related spec-like protein) by activation stimulus (nigericin), NLRP3 inflammasome by cephem compound (cefotaxim) Schematic diagram showing inhibition of formation and suppression of IL-1β release. Schematic diagram showing the effects of NLRP3 inflammasome and quinolone antibacterial agents on inflammation suppression. (A) Schedule of Example 1, (B) Western blot showing the effects of various cephem compounds on IL-1β release. Western blot showing the effect of cefoxitin on IL-1β release compared to cefotaxime. Western blot showing the relationship between the presence of nigericin and cefotaxime and the formation of NLRP3 infrasomes. Western blot showing the effects of the cephem compounds (A) cefcapene and (B) flomoxef on IL-1β release. Sup: Culture supernatant, Lys: Cytolytic extract (A) Schematic diagram showing the schedule of cefotaxime administration and LPS administration to C57BL / 6N mice. (B) A graph showing the inhibitory effect of cefotaxime administration on IL-1β release. (A) Schematic diagram showing the schedule of gefitinib administration, cefotaxime administration, and LPS administration to C57BL / 6N mice. (B) A graph showing the inhibitory effect of cefotaxime administration on IL-1β release. Inhibitory effect on IL-1β release in cells by the combination of cefotaxime and levofloxacin. (A) A graph showing the inhibitory effect of a drug on IL-1β release in THP1 cells. (B) A graph showing the inhibitory effect of the drug on IL-1β release in BMBD cells, and (C) a graph showing the inhibitory effect of various drugs on IL-1β release. Sup: culture supernatant, Lys: cytolytic extract, non: cells and no reagent added, Ctrl: control without reagent added, C, CTX: cefotaxime, L, Lev: levofloxacin. *** <0.0001. (A) A schematic diagram showing a schedule of administration of a drug and LPS to C57BL / 6N mice. (B) The graph which shows the inhibitory effect on IL-1β release by administration of cefotaxime and levofloxacin. *** <0.001, N.S. No statistically significant difference.

According to the first aspect of the present invention, there is provided an NLRP3 inflammasome inhibitor containing a cephem compound as an active ingredient. As used herein, the cephem-based compound refers to a compound having a structure in which a hetero-six-membered ring is linked to a β-lactam ring, and includes cephalosporin-based compounds, cephamycin-based compounds, and oxacephem-based compounds. The cephalosporin-based compound has a heterocycle in which a hetero6-membered ring containing a sulfur atom S is linked to a β-lactam ring. It may have an oxyimino group (> C = NOR) on the side chain extending from the 7th position of the β-lactam ring. Cephamycin compounds do not have an oxyimino group and have a methoxy group (-O-CH ₃ ) at the 7-position of the β-lactam ring. The oxacephem-based compound is a compound in which the sulfur S of the six-membered ring is replaced with the oxygen atom O.

In some embodiments, the cephem compound is a compound represented by the following formula (I), a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.

In the formula, Z is sulfur or oxygen, R ¹ is hydrogen or methoxy, R ² is an acyl, CO ₂ R ³ is a carboxyl group or carboxylate anion, and R ⁴ is hydrogen, halogen,-. CH _2- R ⁵ or -CH ₂ OCONH ₂ , where R ⁵ is hydroxy, optionally substituted C1-6 alkoxy, optionally substituted aminocarbonyloxy, optionally substituted C1-6. Alkoxycarbonyloxy, —S— (optionally substituted heteroaryl), or nitrogen-containing cyclic group.

In some embodiments, the cephem compound is a compound represented by the following formula (Ia), a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.

In the formula, R ¹ is hydrogen or methoxy, R ² is an acyl, CO ₂ R ³ is a carboxyl group or carboxylate anion, and R ⁴ is hydrogen, halogen, -CH _2- R ⁵ . R ⁵ is hydroxy, optionally substituted C1-6 alkoxy, optionally substituted aminocarbonyloxy, optionally substituted C1-6 alkylcarbonyloxy, —S— (optionally substituted hetero). Aryl), or a nitrogen-containing cyclic group. The compound represented by the formula (Ia) is a compound represented by the formula (I) in which Z is sulfur and R ⁴ is hydrogen, halogen, or -CH _2- R ⁵ .

In the compounds represented by the formulas (I) and (Ia), R ² is any of the following formulas (a) to (e).

In the formula, p is 0, 1 or 2.
m is 0, 1 or 2
A ₁ is a substituted or unsubstituted (C1-6) alkyl, (C3-6) cycloalkyl, cyclohexenyl, cyclohexadienyl, aromatic (including heterocyclic aromatic) group (eg, substituted or unsubstituted phenyl). , Thienyl, pyridyl, or substituted or unsubstituted thiazolyl group), (C1-6) alkylthio group or (C1-6) alkyloxy.
X ₁ is a hydrogen, halogen atom, carboxylic acid, carboxylic acid ester, sulfonic acid, azide, tetrazolyl, hydroxy, acyloxy, amino, ureido, acylamino, heterocyclylamino, guanidino or acylureido group.
A ₂ is an aromatic group (eg, a substituted or unsubstituted phenyl, 2-alkoxy-1-naphthyl, 3-arylisooxazolyl, or 3-aryl-5-methylisooxazolyl group), or a substituted or substituted group. It is an unsubstituted alkyl group and
A ₃ is an aromatic group (eg, substituted or unsubstituted phenyl, 2-alkoxy-1-naphthyl, 3-arylisooxazolyl, or 3-aryl-5-methylisooxazolyl group), or substituted or substituted. It is an unsubstituted alkyl group and
X ₂ is an oxygen or sulfur atom
q is 0, 1 or 2, and A ₄ is a substituted or unsubstituted aryl or heteroaryl group (eg, phenyl, frill, aminothiazolyl, or aminothiazolyl).
A ₅ is hydrogen, (C1-6) alkyl, (C3-8) cycloalkyl, (C3-8) cycloalkyl (C1-6) alkyl, (C1-6) alkoxycarbonyl (C1-6) alkyl, ( C2-6) Alkoxy, carboxy (C1-6) alkyl, (C2-6) alkynyl, aryl, or (C1-6) alkyl substituted with 1, 2, or 3 aryl groups.
A ₆ is a substituted or unsubstituted aryl or heteroaryl group (eg, phenyl, frill, aminothiazolyl, or aminothiazolyl).
A ₇ is hydrogen, (C1-6) alkyl, (C3-8) cycloalkyl, (C3-8) cycloalkyl (C1-6) alkyl, (C1-6) alkoxycarbonyl (C1-6) alkyl, ( C2-6) alkenyl, carboxy (C1-6) alkyl, (C2-6) alkynyl, aryl, or (C1-6) alkyl substituted with 1, 2, or 3 aryl groups.

C1-6 has 1 to 6 carbon atoms, C2-6 has 2 to 6 carbon atoms, and C3-8 has 3 to 8 carbon atoms.

Substituent when A ₁ is a substituted (C1-6) alkyl, substituent when A ₁ is a substituted phenyl, substituent when A ₁ is a substituted thiazolyl group, A ₃ Substituent when is a substituted phenyl, substituent when A ₃ is a substituted alkyl group, substituent when A ₄ is a substituted aryl (eg phenyl) or heteroarylphenyl, A _{When 6} is a substituted aryl (eg, phenyl) or heteroarylphenyl, the substituents are halogen (F, Cl, Br, or I), mercapto, C1-6 alkyl, phenyl, C1-6 alkoxy, respectively. Hydroxy (C1-6) alkyl, mercapto (C1-6) alkyl, halo (C1-6) alkyl, hydroxy, amino, nitro, carboxy, (C1-6) alkylcarbonyloxy, alkoxycarbonyl, formyl, or (C1-C1- 6) It may be an alkylcarbonyl.

In one embodiment, in the compound represented by the formula (I) or the formula (Ia), when R ² is a (a) group, A ₁ is (C1-6) alkyl, (C3-6) cycloalkyl. , Cyclohexenyl, cyclohexadienyl, phenyl, hydroxyphenyl, thienyl or pyridyl, where X ₁ is a hydrogen or halogen atom, or carboxy, carboxylic acid ester, azide, tetrazolyl, hydroxy, acyloxy, amino, ureido, guanidino or acylureide. It is a group, p is 0 or 1, and m is 0 or 1.

In one embodiment, in a compound of formula (I) or formula (Ia), where R ² is the group of formula (c), A ₃ is phenyl, X ₂ is oxygen and q is It is 0.

In one embodiment, in the compound represented by the formula (I) or the formula (Ia), when R ² is a group of the formula (d), the A ₄ group is phenyl, thien-2-yl, thien-3. -Il, Flu-2-yl, Flu-3-yl, Pyrido-2-yl, Pyrido-3-yl, Pyrido-4-yl, 5-Amino-1,2,4-Thiasiazol-3-yl or 2 - an-4-yl, a ₅ groups are hydrogen, methyl, ethyl, cyclopropylmethyl, triphenylmethyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, phenyl, carboxymethyl, carboxypropyl and t -Contains butoxycarbonylmethyl.

In one embodiment, in the compound represented by the formula (I) or the formula (Ia), when R ² is a group of the formula (e), the A ₆ group is phenyl, thien-2-yl, thien-3. -Il, Flu-2-yl, Flu-3-yl, Pyrido-2-yl, Pyrido-3-yl, Pyrido-4-yl, 5-Amino-1,2,4-Thiasiazol-3-yl or 2 - an-4-yl, a ₇ group selected from the group consisting of hydrogen, methyl, ethyl, cyclopropylmethyl, triphenylmethyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, phenyl, carboxymethyl, carboxypropyl and t -Contains butoxycarbonylmethyl.

When CO ₂ R ³ is a carboxyl group, in the compound represented by the formula (I) or the formula (Ia), R ³ is hydrogen, and when CO ₂ R ³ is a carboxylate anion, CO ₂ R ³ is COO ^- represented by. ^{Cations such as K +} and Na ⁺ can be ionic bonded to the carboxylate anion.

In the compound represented by the formula (I) or the formula (Ia), the substituents of C1-6 alkoxy, aminocarbonyloxy, C1-6 alkylcarbonyloxy and —S-heteroaryl of ^{R 4 are each halogen (F).} , Cl, Br, or I), C1-6 alkyl, hydroxy, amino, nitro, or carboxy.

In the compound represented by the formula (I) or the formula (Ia), when R ⁵ is —S— (a heteroaryl which may be substituted), it may be monocyclic or condensed polycyclic. , Preferably thienyl, furanyl, thiazolyl, tetrazolyl, pyridyl, or thiadiazolyl.

In the compound represented by the formula (I) or the formula (Ia), when R ⁵ is a nitrogen-containing cyclic group, it may be monocyclic or condensed polycyclic, preferably aminothiazolyl, thienyl,. Furanyl, pyridyl, or cyclopentapyridyl.

In a preferred embodiment, the cephem compound comprises cefotetan, cefcapene, cefotetan, cefcapene, cefcapene, cefcapene, cefcapene, cefotaxime, cefmenoxime, ceftazidime, cefazolin, cefpirom, cefepim A pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.
In another preferred embodiment, the cephem compound comprises cefoxitin, cephalotin, cefacrol, cephonicid, cefroxime, cefotaxime, ceftazidime, cefazolin, cefpyrom, cefepime, ceftoriazone, ceftizoxime, cefoperazone, cefotezol, cephalexin, and cefotetan. One or more compounds to be made, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.

In a more preferred embodiment, the cephem compound is one or more compounds selected from the group consisting of cefoxitin, cephalotin, cefacrol, cephonicid, cefuroxime, cefotaxime, ceftazidime, cefazolin, cefpyrom, and cefepime, pharmaceutically thereof. An acceptable salt, or a pharmaceutically acceptable ester thereof.
In a more preferred embodiment, the cephem compound is one or more compounds selected from the group consisting of cefoxitin, cephalotin, cefacrol, cephonicid, cefuroxime, cefotaxime, ceftazidime, cefazolin, cefpyrom, and cefepime, pharmaceutically thereof. An acceptable salt, or a pharmaceutically acceptable ester thereof.
In terms of the strength of suppressing IL-1β release from immune cells, the above cephem compounds are cefoxitin, cefamandole, cefarotin, cefachlor, cefoniside, cefuroxime, cefotaxime, cefmenoxime, ceftazidime, cefazolin, cefpyrom, cefepim, cefcapene, And one or more compounds selected from the group consisting of Flomoxef, pharmaceutically acceptable salts thereof, or pharmaceutically acceptable esters thereof.
These compounds have a stronger IL-1β release inhibitory effect when compared with ceftizoxime, cefoperazone, ceftezol and cephalexin.

The structural formulas of these compounds are shown below. These compounds have excellent NLRP3 inflammasome inhibitory activity and are highly safe as they have few or no side effects.

In one embodiment, cefadrin, cephalotin, cefotetan, cefprozil, cefixime, cefprozil, cefuroxime, cephalexin, cephalosporins, cefuroxime, cefotetan, cefmenoxime are excluded from the compounds represented by the above formula (I) or formula (Ia). NS.

A pharmaceutically acceptable salt of a cephem compound is a salt having the desired pharmacological activity of the cephem compound, which is a pharmaceutically acceptable base or acid containing an inorganic base or an organic base and an inorganic acid or an organic acid. Means a salt prepared from.

Examples of the inorganic base include alkali metals (eg, Na, K) and alkaline earth metals (eg, Ca, Mg). Examples of the organic base include triethylamine and pyridine. Examples of the salt with the inorganic acid include salts with hydrochloric acid, hydrofluoric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, perchloric acid, hydroiodic acid and the like. Examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, mandelic acid, ascorbic acid, lactic acid and the like. ..

The pharmaceutically acceptable salt of the compound represented by the formula (I) can be a solvate with an organic solvent and / or water. When forming a hydrate, it may be coordinated with any number of water molecules. For example, monohydrochloride monohydrate and the like can be mentioned.

The pharmaceutically acceptable ester of the compound represented by the formula (I) or the formula (Ia) is preferably of the following partial formulas (i), (ii), (iii), (iv) and (v). Any in vivo hydrolyzable ester.

In the formula, ^Ra is hydrogen, (C1-6) alkyl, (C3-7) cycloalkyl, methyl, or phenyl.
R ^b is (C1-6) alkyl, (C1-6) alkoxy, phenyl, benzyl, (C3-7) cycloalkyl, (C3-7) cycloalkyloxy, (C1-6) alkyl (C3-7). Cycloalkyl, 1-amino (C1-6) alkyl, or 1-((C1-6) alkyl) amino (C1-6) alkyl,
Alternatively, R ^a and R ^b together form a 1,2-phenylene group optionally substituted with one or two methoxy groups;
R ^c represents a (C1-6) alkylene optionally substituted with a methyl or ethyl group, and R ^d and R ^e independently represent (C1-6) alkyl.
R ^f indicates (C1-6) alkyl,
R ^g represents a phenyl optionally substituted with up to 3 groups selected from hydrogen or halogen, (C1-6) alkyl, or (C1-6) alkoxy.
Q is oxygen or NH,
R ^h is hydrogen or (C1-6) alkyl,
R ⁱ is hydrogen, a (C1-6) alkyl, optionally substituted with halogen (C2-6) alkenyl, (C1-6) alkoxycarbonyl, aryl or heteroaryl,
Alternatively, R ^h and R ⁱ together form (C1-6) alkylene,
R ^j represents hydrogen, (C1-6) alkyl or (C1-6) alkoxycarbonyl, R ^k is (C1-8) alkyl, (C1-8) alkoxy, (C1-6) alkoxy (C1-) 6) Indicates alkoxy or aryl.

Preferred ester groups include, for example, acyloxyalkyl groups such as acetoxymethyl, pivaloyloxymethyl, α-acetoxyethyl, α-pivaloyloxyethyl, 1- (cyclohexylcarbonyloxy) prop-1-yl, and ( 1-Aminoethyl) carbonyloxymethyl; alkoxycarbonyloxyalkyl groups such as ethoxycarbonyloxymethyl, α-ethoxycarbonyloxyethyl and propoxycarbonyloxyethyl; dialkylaminoalkyls in particular di-lower alkylaminoalkyl groups such as dimethylaminomethyl, Dimethylaminoethyl, diethylaminomethyl or diethylaminoethyl; 2- (alkoxycarbonyl) -2-alkyl group, eg 2- (isobutoxycarbonyl) pent-2-enyl and 2- (ethoxycarbonyl) but-2-enyl; lactone group And so on.

The above ester is produced, for example, by reacting an acidic group of a compound represented by the formula (I) or the formula (Ia) with an appropriate alcohol or amine. Esters that are hydrolyzed in vivo and pharmaceutically active in vivo are called "prodrugs" and such esters are known to those of skill in the art. A C1-C6 alkyl ester having an acidic group (for example, a methyl ester or an ethyl ester) is preferable.

The compound represented by the formula (I) or the formula (Ia) of the present invention can be produced by a known method or a commercially available product can be obtained.

The compound represented by the formula (I) or the formula (Ia) of the present invention exists as a trans isomer (E isomer) and a cis isomer (Z isomer) or a mixture thereof, or a diastereo isomer. And any isomer is included in the scope of the present invention.

The inhibitor of the first aspect of the present invention can be used for subjects including mammals including humans (for example, humans, cows, horses, pigs, monkeys, dogs, cats, mice, rats, rabbits, goats, sheep, etc.). It is possible and preferably used for humans.

Examples of the administration form of the inhibitor include oral preparations, injection preparations, suppositories and the like, and oral preparations or injection preparations are preferable. Each of these dosage forms can be produced by a conventional formulation method known to those skilled in the art.

In addition, the administration form can be selected according to the drug, and cefoxitin, cephalotin, cefotaxime, ceftazidime, cefazolin, cefpyrom, cefepime, ceftoriazone, cefoperazone, ceftezol, and cefotetan are injections, cefoperazone, cefoniside, cefuroxime, ceftizoxime, and cefotetan. Ceftizoxime is preferably an oral preparation.

The amount of the cephem compound to be blended in each administration unit form is not constant depending on the symptom of the subject to which the compound is applied or the dosage form thereof, but generally, about 250 to about 250 to oral preparations per administration unit form. 2000 mg, about 1000 to 6000 mg for injections, and about 250 mg for suppositories.

The daily dose of the drug having the above-mentioned administration form varies depending on the subject's symptoms, body weight, age, gender, etc. and cannot be unconditionally determined, but is usually about 1000 to 6000 mg per day for an adult (body weight 50 kg). The dose is preferably 1000 to 2000 mg, and this is preferably administered once a day or in 2 to 3 divided doses.

As schematically shown in FIG. 1, the NLRP3 inflammasome is a protein complex consisting of the NLRP3 protein, the adapter protein ASC, and caspase-1, and is caspase-1 by a specific activation stimulus (for example, nigericin). When activated, it is thought to induce the inflammatory cytokines IL-1β and IL-18 from the precursor to the mature body, causing inflammation.
Since the inhibitor of the first aspect of the present invention inhibits the formation of the NLRP3 inflammasome, it causes the expression and / or release of interleukin-1β (hereinafter referred to as IL-1β), which is a cytokine involved in the inflammatory reaction. Suppress. When the expression and / or release of IL-1β in a subject is suppressed, the inflammatory reaction is suppressed, which is considered to be effective for the prevention or treatment of inflammatory diseases.

At least selected from the group consisting of gout, pseudo-gout, rheumatoid arthritis, hyperuricemia, type II diabetes, pneumoconiosis, criopyrinopathy, and cryopyrin-associated periodic fever syndrome (CAPS) as inflammatory diseases. There is one kind. These inflammatory diseases are autoinflammatory diseases, and it is thought that the NLRP3 inflammasome is involved in the development of the disease.

Therefore, according to the second aspect of the present invention, an IL-1β inhibitor containing a cephem compound as an active ingredient is provided.

In the above second aspect, the details of the cephem compound, the administration target, the dose, and the administration form are as described above with respect to the inhibitor of the first aspect of the present invention.

According to the third aspect of the present invention, a pharmaceutical composition for preventing or treating an inflammatory disease containing an effective amount of a cephem compound is provided.

The inflammatory disease can preferably be an inflammatory disease with increased release of IL-1β or IL-18 from immune cells due to the formation of the NLRP3 inflammasome, where the immune cells are preferably monocytes, macrophages, trees. Macrophages or combinations thereof.
Inflammatory diseases are selected from the group consisting of, for example, gout, pseudogout, rheumatoid arthritis, hyperuricemia, type II diabetes, pneumoconiosis, criopyrinopathy, and cryopyrin-associated periodic fever syndrome (CAPS). At least one of them can be mentioned.

In some embodiments, the cephem compound includes a compound represented by the above formula (I), a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.

(In the formula, Z, R ¹ , R ² , CO ₂ R ³ , R ⁴ are as described above)
In some embodiments, the cephem compound includes a compound represented by the above formula (I) or formula (Ia), a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof. Be done.

(In the formula, R ¹ , R ² , CO ₂ R ³ , R ⁴ are as described above)
A compound represented by the above formula (I) or formula (Ia), a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof inhibits the expression of the NLRP3 inflammasome. A pharmaceutical composition containing a compound represented by I) or formula (Ia), a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester compound thereof prevents or excessive inflammation in a subject. It acts to suppress.

In a preferred embodiment, the cephem compounds are cefoxitin, cefamandole, cephalotin, cefaclor, cefoniside, cefuroxime, cefotaxime, ceftazidime, cefmenoxime, cefazolin, cefpyrom, cefepime, ceftoriazone, ceftizoxime, cefoperazone, ceftizoxime, cefoperazone. , And one or more compounds selected from the group consisting of Flomoxef, pharmaceutically acceptable salts thereof, or pharmaceutically acceptable esters thereof.
In another preferred embodiment, the cephem compound comprises cefoxitin, cephalotin, cefacrol, cephonicid, cefroxime, cefotaxime, ceftazidime, cefazolin, cefpyrom, cefepime, ceftoriazone, ceftizoxime, cefoperazone, cefotezol, cephalexin, and cefotetan. One or more compounds to be made, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.

In a more preferred embodiment, the cephem compound is one or more compounds selected from the group consisting of cefoxitin, cephalotin, cefacrol, cefoniside, cefuroxime, cefotaxime, ceftazidime, cefazolin, cefpyrom, and cefepime, pharmaceutically thereof. An acceptable salt, or a pharmaceutically acceptable ester thereof.
The pharmaceutical composition may further contain a drug for preventing or suppressing inflammation other than the above-mentioned cephem compound. Examples of such a drug include a quinolone antibacterial drug. Quinolone antibacterial agents are agents having a 4-quinolone skeleton, and are old quinolone antibacterial agents in which hydrogen at the 6-position of the quinolone ring is not substituted, and new quinolone-based antibacterial agents in which a fluorine atom is introduced at the 6-position of the quinolone ring. Is included. Preferably, the quinolone antibacterial agent is a new quinolone antibacterial agent. Preferred examples of new quinolone antibacterial agents are levofloxacin, norfloxacin, ofloxacin, ciprofloxacin, ciprofloxacin, lomefloxacin, tosfloxacin, pazufloxacin, purlifloxacin, moxifloxacin, garenoxacin, sitafloxacin, trobuff. Examples thereof include loxacin, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof, and levofloxacin, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof is particularly preferable. .. The pharmaceutically acceptable salt of the quinolone antibacterial agent can be a solvate with an organic solvent and / or water. When forming a hydrate, it may be coordinated with any number of water molecules.
As shown in FIG. 2, at least two signalings are thought to be involved in the activation of the inflammatory response, one (Signal 1) is TLR, TNF receptor, IL on immune cells. When the pattern recognition receptor (PRR) such as -1 receptor binds to the common molecular structure (PAMP), TNF, IL-1, etc. of pathogenic microorganisms, the intracellular transcription factor NF-kB is activated. Activated NF-kB migrates into the nucleus and induces transcription and translation of pro-IL-1β3, a precursor of pro-inflammatory cytokines. The other (Signal 2) is the activation of caspase 1 by the activation of NLRP3 inflammasome by ATP, Alum, etc., and the induction of inflammatory cytokines IL-1β and IL-18 from the precursor to the mature body. This is as described in FIG.
Some new quinolone antibiotics are known to suppress the inflammatory response by inhibiting Signal 1. For example, levofloxacin and ciprofloxacin have been reported to bind directly to TLR4 and inhibit TLR4 dimerization (Zusso et al., Journal of Neuroinflammation (2019) 16: 148). Trovafloxacin has been reported to inhibit TNF, LPS-induced p65 nuclear translocation (G Giustarini et al., Texicology and Applied Pharmacology 391 (2020) 114915).
The combination of a cephem compound and a quinolone antibacterial agent brings about an effect (additive effect or synergistic effect) that exceeds the additive effect of suppressing the inflammatory reaction. In particular, the combination of a cephem compound with a particular quinolone antibacterial agent produces additive or synergistic effects.
The amount of the quinolone antibacterial agent to be blended in each administration unit form is not constant depending on the symptom of the subject to which it is applied, its dosage form, etc., but generally, about 50 oral preparations per administration unit form. It is about 2000 mg, about 100 to 6000 mg for injections, and about 50 to 1000 mg for suppositories.
The daily dose of the quinolone antibacterial agent having the above-mentioned administration form varies depending on the subject's symptoms, body weight, age, gender, etc. and cannot be unconditionally determined, but is usually about 50 per day for an adult (body weight 50 kg). It is about 6000 mg, preferably 100 to 2000 mg, and it is preferable to administer this once a day or in 2 to 3 divided doses.
In a preferred embodiment, the half-life of the cephem compound is around 2 hours and the half-life of the quinolone antibacterial agent is around 6 to 10 hours, so that the number of administrations of cefotaxime is larger than the number of administrations of levofloxacin (3: 1 to 5: 1). ) Set.

When other optional agents such as cephem compounds and quinolone antibacterial agents are included in the pharmaceutical composition, they may be blended with a pharmaceutical carrier as necessary, and various administration forms may be used depending on the prophylactic or therapeutic purpose. It can be adopted.

As the pharmaceutical carrier, various conventional organic or inorganic carrier substances are used as the preparation material, and excipients, binders, disintegrants, lubricants, colorants in solid preparations; solvents, dissolution aids, suspensions in liquid preparations. It is blended as a turbidizing agent, an isotonic agent, a buffering agent, a pain-relieving agent, and the like. Further, if necessary, pharmaceutical additives such as preservatives, antioxidants, colorants, sweeteners and stabilizers can be used.

Examples of the administration form of the pharmaceutical composition include oral preparations, injection preparations, suppositories and the like, and oral preparations or injection preparations are preferable. Each of these dosage forms can be produced by a conventional formulation method known to those skilled in the art.

When preparing an oral solid preparation, an excipient, if necessary, a binder, a disintegrant, a lubricant, a colorant, a flavoring or a odorant, etc. are added to the compound of the present invention, and then tablets are prepared by a conventional method. , Coated tablets, granules, powders, capsules and the like can be produced.

Examples of excipients include calcium carbonate, kaolin, sodium hydrogen carbonate, lactose, D-mannitol, starches, crystalline cellulose, talc, granulated sugar, porous substances and the like.

Binders include water, ethanol, 1-propanol, 2-propanol, glucose, dextrin, α-starch, gelatin, D-mannitol, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shelac, calcium phosphate, Examples thereof include polyvinylpyrrolidone.

Disintegrants include dried starch, sodium alginate, powdered canten, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, lactose, carboxymethyl cellulose calcium, croscarmellose sodium, crospovidone, low-substituted hydroxypropyl cellulose, Examples include partially pregelatinized starch.

Examples of the lubricant include magnesium stearate, calcium stearate, talc, starch, sodium benzoate and the like.

Examples of the colorant include tar pigment, caramel, iron sesquioxide, titanium oxide, riboflavins and the like.

Examples of the flavoring or deodorizing agent include sucrose, orange peel, citric acid, tartaric acid and the like.

When preparing an oral liquid preparation, an oral liquid preparation, a syrup agent, an elixir agent, etc. can be produced by adding a flavoring agent, a buffering agent, a stabilizer, a odorant, etc. to a cephem compound. In this case, the flavoring or odorant may be those listed above, the buffering agent may be sodium citrate or the like, and the stabilizer may be tragant, gum arabic, gelatin or the like.

When preparing an injection, add a pH regulator, buffer, stabilizer, isotonic agent, local anesthetic, etc. to the cephem compound, and use a conventional method to make subcutaneous, intramuscular, and intravenous injections. Can be manufactured. Examples of the pH adjuster and buffer in this case include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid and the like. Examples of the local anesthetic include procaine hydrochloride, lidocaine hydrochloride and the like. Examples of the tonicity agent include sodium chloride, glucose, D-mannitol, glycerin and the like.

When preparing a suppository, the compound of the present invention is added with a pharmaceutical carrier known in the art, for example, polyethylene glycol, lanolin, cacao butter, fatty acid triglyceride, etc., and if necessary, an interface such as Tween80 (registered trademark). After adding an activator or the like, it can be produced by a conventional method.

The pharmaceutical composition of the third aspect of the present invention is used for a subject which is a mammal including a human (for example, human, cow, horse, pig, monkey, dog, cat, mouse, rat, rabbit, goat, sheep, etc.). Is possible and is preferably used for humans.

The amount of the cephem compound to be blended in each of the above-mentioned administration unit forms is not constant depending on the symptom of the patient to which this is applied, the dosage form thereof, etc. It is about 250 to 2000 mg, about 1000 to 6000 mg for injections, and about 250 mg for suppositories.

The daily dose of the drug cephem compound having the above-mentioned administration form varies depending on the subject's symptoms, body weight, age, gender, etc. and cannot be unconditionally determined, but is usually about 1000 per day for an adult (body weight 50 kg). It is about 6000 mg, preferably 1000 to 2000 mg, and it is preferable to administer this once a day or in 2 to 3 divided doses.

According to the fourth aspect of the present invention, a therapeutic agent for an inflammatory disease containing an effective amount of a cephem compound is provided. Details of the cephem compound, administration target, dose, and administration form are as described above with respect to the pharmaceutical composition of the third aspect of the present invention.

The inflammatory disease can preferably be an inflammatory disease with increased IL-1β or IL-18 release from immune cells due to the formation of the NLRP3 inflammasome, where the immune cells are preferably monocytes, macrophages, dendritic cells. Cells, or a combination thereof.
Inflammatory diseases are selected from the group consisting of, for example, gout, pseudogout, rheumatoid arthritis, hyperuricemia, type II diabetes, urticaria, criopyrinopathy, and cryopyrin-associated periodic fever syndrome (CAPS). At least one of them can be mentioned.
In order to enhance the therapeutic effect of inflammatory diseases, the above-mentioned quinolone antibacterial agents may be further administered to a subject or patient in combination with a cephem compound. In the seventh aspect, the above-mentioned quinolone antibacterial agent can be further used in combination with a cephem compound to produce an IL-1β inhibitor. Preferably, the quinolone antibacterial agent is a new quinolone antibacterial agent. Preferred examples of new quinolone antibacterial agents are levofloxacin, norfloxacin, ofloxacin, ciprofloxacin, ciprofloxacin, lomefloxacin, tosfloxacin, pazufloxacin, purlifloxacin, moxifloxacin, garenoxacin, sitafloxacin, trobuff. Examples thereof include loxacin, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof, and levofloxacin, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof is particularly preferable. .. The pharmaceutically acceptable salt of the quinolone antibacterial agent can be a solvate with an organic solvent and / or water. When forming a hydrate, it may be coordinated with any number of water molecules.
Details of the quinolone antibacterial agent, administration target, dose, and administration form are as described above with respect to the pharmaceutical composition of the third aspect of the present invention.
The cephem compound and the quinolone antibacterial agent can be used as a combination preparation at the same time, sequentially or at intervals. The administration forms of the cephem compound and the quinolone antibacterial agent may be the same, for example, an oral preparation or an injection preparation, or may be different such that one is an oral preparation and the other is an injection preparation. ..

According to the fifth aspect of the present invention, the use of a cephem compound for producing an NLRP3 inflammasome inhibitor is provided.

According to a sixth aspect of the present invention, the formation and / or formation of the NLRP3 inflammasome in the subject comprises administering the cephem compound to the subject in an amount effective for inhibiting the formation and / or expression of the NLRP3 inflammasome. Or a method for inhibiting expression.

Such a method can be an in vivo method of administering a cephem compound to a mammal including humans, or an in vitro method using isolated cells or cultured cells derived from mammals including humans. ..

In the fifth and sixth aspects described above, the details of the cephem compound, the administration target, the dose, and the administration form are as described above with respect to the inhibitor of the first aspect of the present invention.

According to a seventh aspect of the present invention, the use of a cephem compound for producing an IL-1β inhibitor is provided.
In the seventh aspect, the above-mentioned quinolone antibacterial agent can be further used in combination with a cephem compound to produce an IL-1β inhibitor. Details of the quinolone antibacterial agent, administration target, dose, and administration form are as described above with respect to the pharmaceutical composition of the third aspect of the present invention.

According to the eighth aspect of the present invention, the expression and / or release of IL-1β in a subject comprises administering the cephem compound to the subject in an amount effective for inhibiting the expression and / or release of IL-1β. Regarding the method of inhibiting the above.

Such a method can be an in vivo method of administering a cephem compound to a mammal including humans, or an in vitro method using isolated cells or cultured cells derived from mammals including humans. ..
In the eighth aspect, the above-mentioned quinolone antibacterial agent can also be administered in combination with a cephem compound in order to enhance the inhibitory effect on the expression and / or release of IL-1β.

In the seventh aspect and the eighth aspect, the details of the cephem compound, the administration target, the dose, and the administration form are as described above with respect to the inhibitor of the second aspect of the present invention.
In the seventh aspect and the eighth aspect, the details of the quinolone antibacterial agent, the administration target, the dose, and the administration form are as described above with respect to the pharmaceutical composition of the third aspect of the present invention.
The cephem compound and the quinolone antibacterial agent can be used as a combination preparation at the same time, sequentially or at intervals. The administration forms of the cephem compound and the quinolone antibacterial agent may be the same, for example, an oral preparation or an injection preparation, or may be different such that one is an oral preparation and the other is an injection preparation. ..

According to a ninth aspect of the present invention, the use of a cephem compound for producing a pharmaceutical composition for the prevention or treatment of an inflammatory disease is provided.
In the ninth aspect, the above-mentioned quinolone antibacterial agent can be further used in combination with a cephem compound to produce a pharmaceutical composition for the prevention or treatment of an inflammatory disease.

According to a tenth aspect of the present invention, there is provided a method for preventing or treating an inflammatory disease, which comprises administering a cephem compound to a subject in an amount effective for treatment and / or prevention.
In the tenth aspect, the above-mentioned quinolone antibacterial agent may be administered in combination with a cephem compound in order to enhance the preventive or therapeutic effect of an inflammatory disease.

According to the eleventh aspect of the present invention, a combination drug of a cephem compound and a quinolone antibacterial agent for inhibiting the expression and / or release of IL-1β from immune cells is provided.
According to the twelfth aspect of the present invention, a cephem compound and a quinolone antibacterial agent as a combination drug for use simultaneously, sequentially or at intervals when preventing or treating an inflammatory disease. Products including and are provided.
According to the thirteenth aspect of the present invention, a combination drug of a cephem compound and a quinolone antibacterial agent for use in the prevention or treatment of an inflammatory disease is provided.
In each of the ninth to thirteenth aspects, the details of each of the cephem compound and the quinolone antibacterial agent, the subject to be administered, the dose, the dosage form, and the inflammatory disease are described in the pharmaceutical composition of the third aspect of the present invention. As described above with respect to.

All patent applications and literature disclosures cited herein are incorporated herein by reference in their entirety.

The present invention will be described in more detail with reference to examples below, but the present invention is not limited thereto.

Example 1 Suppression of IL-1β release using cephem compounds In this example, the present inventors added various cephem compounds to a medium containing human monocytes in the presence or absence of nigericin, and used them from cells. The effect on IL-1β release was investigated.

(Method)
Experiments were performed according to Cell Death and Differentiation (2007) 14, 1583-1589. THP1 cells were cultured in RPMI medium supplemented with 10% FCS and 50 mM 2-mercaptoethanol and differentiated in 12-O-tetradecanoylformol 13-acetate (PMA) for 3 hours for this experiment. Human monocytes are purified, seeded on a plate, cultured overnight (O / N) and wait for the differentiated cells to stick to the plate. Then, 200 μg / ml of various cephem compounds was added (premedication), 5 μM of nigercin was added 24 hours later, and cells and culture supernatant were collected after 3 hours. The schedule is shown in FIG. 3 (A).

As cephem compounds, cefotaxime, cefotaxime, ceftriazone, cefazolin, cefoxitin, ceftizoxime, cefoperazone, cephalexin, ceftezol, cefoperol, cephalexin, cefpyrom, cefepime, cefotetan, cefoniside, and cefotetan were used.

Protein was extracted from the cell culture supernatant by methanol / chloroform precipitation. Briefly, the cell-free supernatant was mixed with methanol: chloroform at a ratio of 5: 5: 1 (cell culture supernatant / methanol / chloroform), the mixture was vortexed and centrifuged at 15,000 rpm for 10 minutes. .. The clear upper layer was discarded and 1000 μl methanol was added to the intermediate layer. The mixture was centrifuged at 15,000 rpm for 10 minutes to remove the liquid layer. The protein pellets were dried and resuspended in 8M urea. The cells were lysed in an ice-cooled lysis buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1% protease and phosphatase inhibitor mixture (Nacalai Tesque). The cell culture supernatant and the protein extracted from the cells were sampled, separated by SDS-PAGE, and analyzed by Western blotting (WB).

(result)
As shown in FIG. 3 (B), IL-1β was not released when nigericin was not added. It was shown that the addition of nigericin releases IL-1β extracellularly, but the addition of various cephem compounds reduces the amount of IL-1β released and suppresses the release of IL-1β.

When any cephem compound was added, the amount of IL-1β released was lower than that of the control without the addition of the cephem compound (second lane from the left of each membrane).

In this experiment, based on cefotaxime, cefotaxime, cephalotin, cefacrol, cefoniside, and cefuroxime have a stronger inhibitory effect on IL-1β release than cefotaxime, and cefotaxime, cefazolin, cefpyrom, and cefepime have a stronger inhibitory effect on cefotaxime and IL-1β release. Ceftriazone, ceftizoxime, cefoperazone, ceftezol, cephalexin, and cefotetan were less effective in suppressing IL-1β release than cefotaxime.

Example 2 Relationship between concentration of cephem compound and IL-1β release inhibitory effect In this experiment, the IL-1β release inhibitory effect of cefoxitin compared with cefotaxime was compared, and its concentration dependence was investigated.
(Method)
The IL-1β release inhibitory effect of cefoxitin and cefotaxime was investigated using the same schedule as in Example 1. The concentration of cefoxitin added was 0, 50, 100, 200, 300 μg / ml.
(result)
As shown in FIG. 4, IL-1β was not released without the addition of nigericin. When nigericin was added, IL-1β was released, but as the concentration of cefoxitin added increased, the amount of IL-1β released became more suppressed. Cefoxitin suppressed IL-1β release more strongly than cefotaxime at the same concentration (200 μg / ml).

Example 3 Inhibition of NLRP3 inflammasome formation using cefotaxime In this experiment, cefotaxime was added in the presence or absence of nigericin, and the effect on the formation of NLRP3 inflammasome was investigated.
(Method)
The ASC oligomerization (inflammasome formation) assay was performed with a few minor modifications to the method described in Bio Protoc .; 7 (10): .doi: 10.21769 / BioProtoc.2292. Briefly, cells are washed with PBS to buffer A (20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl ₂ , 1 mM EDTA, 1 mM EGTA, 320 mM sucrose, 1% protease inhibitor. Collected in agent cocktail). Cells were lysed by shearing 15 times through a 27 gauge needle and the cytolysate was centrifuged at 600 xg to remove bulk nuclei and unbroken cells. The resulting supernatant was centrifuged at 17,700 xg to pelletize the ASC oligomer. The pellet was resuspended in CHAPS buffer (20 mM HEPES-KOH (pH 7.5), 5 mM MgCl ₂ , 0.5 mM EGTA, 0.1% CHAPS, 1% protease inhibitor cocktail) with 1.5 mM dysuccinimidyl sorbate (DSS). The reaction was allowed for 30 minutes and the mixture was quenched with SDS sample buffer.
(result)
As shown in FIG. 5, the NLRP3 inflammasome complex was not formed without the addition of nigericin. The addition of nigericin formed the NLRP3 inflammasome, but the addition of cefotaxime (200 μg / ml) reduced the expression level of the NLRP3 inflammasome.

Example 4 Suppression of IL-1β release using a cephem compound Under the same experimental conditions as in Example 1, the effect of a further cephem compound on IL-1β release was investigated. Specifically, cefcapene and floodoxef were tested.
(result)
As shown in FIGS. 6 (A) and 6 (B), IL-1β was not released when nigericin was not added. It was shown that the addition of nigericin releases IL-1β extracellularly, but the addition of cefcapene and floodoxef reduces the amount of IL-1β released and suppresses the release of IL-1β.

Example 5 Effect of Cefotaxime on IL-1β release in an in vivo mouse sepsis model (method)
C57BL / 6N mice were divided into two groups and cefotaxime (CTX) or PBS was pre-administered intraperitoneally according to the schedule shown in FIG. 7 (A), and then LPS (InvivoGen) was intraperitoneally administered to both groups. To induce sepsis. After that, blood is collected from the inferior vena cava, and the blood IL-1β concentration is measured by ELISA. Dosage: 1 point of 20 mg / kg Dosing time: 1 point of 8 hours after the first administration of cefotaxime (CTX) or PBS
2 groups including Control (each group n = 6)
Examination using 12 8-week-old mice (results)
As shown in FIG. 7 (B), the blood IL-1β concentration increased in the group to which PBS was administered without pre-administration of cefotaxime, whereas the blood IL-1β concentration was increased in the group to which cefotaxime was pre-administered. Significantly decreased (Student's t-test, * <0.05). This result suggests that premedication with cefotaxime is effective in suppressing IL-1β release in septic mice.

Example 6 Effect of Cefotaxime on IL-1β Release in In vivo Mouse Peritonitis Model (Method)
After intraperitoneal administration of cefotaxime (CTX) and LPS (InvivoGen) to C57BL / 6N mice according to the schedule shown in FIG. 8 (A), the mice were divided into 4 groups, and cefotaxime was administered to 1 group at 2-hour intervals. A total of 5 doses were administered, and gefitinib was co-administered at the time of the last cefotaxime administration to induce peritonitis. Another group received cefotaxime alone, another group received only gefitinib, and another group received neither cefotaxime nor gefitinib. One hour after the last drug administration, a peritoneal lavage fluid was collected from the mice, and the IL-1β concentration in the fluid was measured by ELISA.
Gefitinib dosing time: 1 point 8 hours after the first cefotaxime and LPS dosing
4 groups including Control (each group n = 6)
Examination using 24 8-week-old mice (results)
As shown in FIG. 8 (B), administration of gefitinib induces peritonitis. Therefore, in the group to which cefotaxime was not administered and gefitinib was administered (second column from the left), a control to which cefotaxime was not administered and gefitinib was not administered was used. The IL-1β concentration was significantly increased compared to the group (leftmost column) (** <0.01). Furthermore, the IL-1β concentration in the sample solution was highest in the group receiving gefitinib but not cefotaxime, but the group receiving gefitinib and receiving cefotaxime (second column from the right) did not receive photaxime. The IL-1β concentration was significantly reduced compared to (* <0.05). The group receiving cefotaxime without gefitinib (rightmost column) had almost the same level of IL-1β concentration as the control group without cefotaxime and no gefitinib (one-way ANOVA (One-way ANOVA)). way ANOVA)). This result suggests that premedication with cefotaxime is effective in suppressing IL-1β release in mice with gefitinib-induced peritonitis.

Example 7 Suppression of IL-1β release using a combination of a cephem compound and a quinolone antibacterial agent Under the same experimental conditions as in Example 1, levofloxacin, a quinolone antibacterial agent, was used as a medium in addition to the cephem compound cefotaxime. The effects of addition of cephem compounds, levofloxacin, and addition of both agents on IL-1β release were investigated.
(result)
As shown in FIGS. 9A and 9B, IL-1β was not released in either THP1 cells or bone marrow-derived macrophages (BMBD) without the addition of nigericin. The addition of nigericin releases IL-1β extracellularly, but the addition of cefotaxime and levofloxacin alone reduces the amount of IL-1β released compared to controls, and both cefotaxime and levofloxacin administration of the drug The amount released was even lower than that of single agent administration. The combination of cefotaxime and levofloxacin has been shown to more effectively suppress the release of IL-1β.
As shown in FIG. 9C, the effects of the drug concentration were investigated by further changing the concentration of cefotaxim to 100 μg / ml and 200 μg / ml and the concentration of levofloxacin to 100 μg / ml and 200 μg / ml. In the drug-administered group, the amount of IL-1β released was significantly lower than that in the control (*** <0.0001, one-way ANOVA). When 100 μg / ml cefotaxime and 100 μg / ml levofloxacin were added, the release of IL-1β in THP1 cells was suppressed as compared with the addition of 100 μg / ml cefotaxime alone and 100 μg / ml levofloxacin alone. .. When 100 μg / ml cefotaxime and 200 μg / ml levofloxacin are added in combination, the release of IL-1β is further suppressed as compared with the case where 100 μg / ml cefotaxime and 100 μg / ml levofloxacin are added. It was suppressed.

Example 8 Effect of combined administration of NLRP3 infrasome inhibitor and quinolone antibacterial agent on IL-1β release in gefitinib-induced peritonitis mice Cefotaxime (CTX) in C57BL / 6N mice on a schedule as shown in FIG. 10 (A). , Levofloxacin (LVLX) was intraperitoneally administered, and LPS (InvivoGen) and gefitinib were further intraperitoneally administered to induce peritonitis. Mice were divided into four groups: a group that received neither cefotaxime nor levofloxacin (control), a group that received cefotaxime alone, a group that received levofloxacin alone, and a group that received both. One hour after the last drug administration, a peritoneal lavage fluid was collected from the mice, and the IL-1β concentration in the fluid was measured by ELISA.
4 groups including Control (each group n = 3)
(result)
As shown in FIG. 10 (B), since peritonitis was induced by administration of gefitinib, the IL-1β concentration increased in the control group, and both the group to which cefotaxime was administered and the group to which levofloxacin was administered were compared with the control group. The IL-1β concentration was significantly reduced (*** <0.001). In the group receiving both cefotaxime and levofloxacin, the IL-1β concentration was significantly reduced compared to the group receiving levofloxacin alone (*** <0.001). (All are one-way ANOVA). This result suggests that administration of the combination of cefotaxime and levofloxacin further suppresses IL-1β release in mice with gefitinib-induced peritonitis as compared with administration of each drug of cefotaxime and levofloxacin alone.

Claims (15)

1. An NLRP3 inflammasome inhibitor containing a cephem compound as an active ingredient.
2. The cephem compound has the following formula (I).
   

   

   (In the formula, Z is sulfur or oxygen, R ¹ is hydrogen or methoxy, R ² is an acyl, CO ₂ R ³ is a carboxyl group or a carboxylate anion, R ⁴ is hydrogen, halogen, -CH ₂ -R ⁵ or -CH ₂ OCONH ₂ , where R ⁵ is hydroxy, optionally substituted C1-6 alkoxy, optionally substituted aminocarbonyloxy, optionally substituted C1- 6 Alkoxycarbonyloxy, —S— (optionally substituted heteroaryl), or nitrogen-containing cyclic group)
   The NLRP3 inflammasome inhibitor according to claim 1, which is a compound represented by, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.
3. The cephem compound has the following formula (Ia).
   

   

   (In the formula, R ¹ is hydrogen or methoxy, R ² is an acyl, CO ₂ R ³ is a carboxyl group or carboxylate anion, and R ⁴ is hydrogen, halogen, or -CH _2- R ⁵ . Yes, R ⁵ is hydroxy, optionally substituted C1-6 alkoxy, optionally substituted aminocarbonyloxy, optionally substituted C1-6 alkylcarbonyloxy, -S- (even substituted) Good heteroaryl), or nitrogen-containing cyclic group)
   The NLRP3 inflammasome inhibitor according to claim 1, which is a compound represented by, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.
4. The cephem compounds consist of cefoxitin, cefamandole, cefarotin, cefaclor, cefoniside, cefloxim, cefotaxime, ceftazidime, cefmenoxime, cefazoline, cefpyrom, cefepim, ceftoriazone, ceftizoxime, cefoperazone, ceftizoxime, cefoperazone, ceffoperazone, ceftizoxime, cefoperazone. The NLRP3 inframasome inhibitor according to any one of claims 1 to 3, which is one or more compounds selected from the group, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.
5. NLRP3 Inflammasome NLRP3 Inflammasome in vitro in non-human animal subjects or in mammalian-derived isolated or cultured cells, consisting of administration to the subject in an amount effective to inhibit the formation and / or expression of the NLRP3 inflammasome. A method for inhibiting the formation and / or expression of a mammal.
6. From the group consisting of gout, pseudo-gout, rheumatoid arthritis, hyperuricemia, type II diabetes, urticaria, criopyrinopathy, and cryopyrin-associated periodic fever syndrome (CAPS) containing effective amounts of cephem compounds. A pharmaceutical composition for the prevention or treatment of selected inflammatory diseases.
7. The cephem compound has the following formula (I).
   

   

   (In the formula, Z is sulfur or oxygen, R ¹ is hydrogen or methoxy, R ² is an acyl, CO ₂ R ³ is a carboxyl group or carboxylate anion, R ⁴ is hydrogen, halogen, -CH ₂ -R ⁵ or -CH ₂ OCONH ₂ , where R ⁵ is hydroxy, optionally substituted C1-6 alkoxy, optionally substituted aminocarbonyloxy, optionally substituted C1- 6 Alkoxycarbonyloxy, —S— (optionally substituted heteroaryl), or nitrogen-containing cyclic group)
   The pharmaceutical composition according to claim 6, which is a compound represented by the above, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.
8. The cephem compound has the following formula (Ia).
   

   

   (In the formula, R ¹ is hydrogen or methoxy, R ² is an acyl, CO ₂ R ³ is a carboxyl group or carboxylate anion, and R ⁴ is hydrogen, halogen, or -CH _2- R ⁵ . Yes, R ⁵ is hydroxy, optionally substituted C1-6 alkoxy, optionally substituted aminocarbonyloxy, optionally substituted C1-6 alkylcarbonyloxy, -S- (even substituted) Good heteroaryl), or nitrogen-containing cyclic group)
   The pharmaceutical composition according to claim 6, which is a compound represented by the above, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.
9. The cephem compounds consist of cefoxytin, cefamandole, cephalotin, cefacrol, cefoniside, cefloxim, cefotaxime, ceftazidime, cefmenoxime, cefazolin, cefpyrom, cefepime, ceftoriazone, ceftizoxime, cefoperazone, ceftizoxime, cefoperazone, cefotetan, cefotetan, cefoperazone, cefotetan. The pharmaceutical composition according to any one of claims 6 to 8, which is one or more compounds selected from the group, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.
10. The pharmaceutical composition according to any one of claims 6 to 9, further comprising a quinolone antibacterial agent.
11. A therapeutic agent for inflammatory diseases containing an effective amount of a cephem compound.
12. NLRP3 inflammasome inhibitor or selected from the group consisting of gout, pseudogout, rheumatoid arthritis, hyperuricemia, type II diabetes, renal lung, criopyrinopathy, and cryopyrin-associated periodic fever syndrome (CAPS) Use of cephem compounds to produce pharmaceutical compositions for the prevention or treatment of inflammatory diseases.
13. A combination drug of a cephem compound and a quinolone antibacterial agent for inhibiting the expression and / or release of IL-1β from immune cells.
14. A product containing a cephem compound and a quinolone antibacterial agent as a combination drug for use simultaneously, sequentially or at intervals in preventing or treating an inflammatory disease.
15. A combination drug of a cephem compound and a quinolone antibacterial agent for use in the prevention or treatment of inflammatory diseases.

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