JP2952767B2 - Β-lactam agent sensitivity-inducing effect on methicillin-resistant Staphylococcus aureus (MRSA) by flavone derivative - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an anti-MRSA active pharmaceutical composition containing a flavone derivative as an active ingredient, comprising an anti-MRSA active pharmaceutical composition comprising a β-lactam antibiotic and a flavone derivative in combination. A method for enhancing antibacterial activity and a method for preventing or treating bacterial infection.

[0002]

BACKGROUND OF THE INVENTION Penicillin, the first antibiotic, has a β-lactam ring,
Demonstrates excellent efficacy against staphylococci. However, penicillinase (β-lactamase) production is relatively easily induced in staphylococci, β-lactam ring is cleaved,
It will be inactivated. It is called a penicillin-resistant bacterium. For this penicillin-resistant bacterium, for example,
Although research and development of penicillinase-resistant penicillins such as methicillin and cephem antibiotics seemed to have solved most clinically, MRSA in which all β-lactam drugs were ineffective appeared. That is, MRSA
It is a multidrug-resistant Staphylococcus aureus that is widely resistant not only to penicillins but also to cephem antibiotics and aminoglycoside antibiotics. In recent years, the abuse of third-generation cephem antibiotics, which have weak antibacterial activity against staphylococci, has resulted in the selective growth of these resistant bacteria and their spread in hospitals. It is becoming a serious social problem. Vancomycin (VCM) is currently used as an antibacterial agent for MRSA infections.
The short-term bactericidal action of M is by no means strong and has serious side effects such as ototoxicity and nephrotoxicity. In addition, a combination of a plurality of antibacterial agents has been studied for the purpose of enhancing the antibacterial activity against MRSA. For example, attempts have been made to use aminoglycoside agents and β-lactam agents or fosfomycin and β-lactam agents in combination, but the combined effect is not satisfactory. There is an urgent need to develop new antibacterial agents effective against such resistant bacteria.

[0003] The present inventors have searched for compounds having anti-MRSA activity from weak Chinese herbal medicines even if they have no side effects or not, and found that apigenin, a component contained in Hanren, Luteolin (lute
olin) and flavone derivatives with similar structures were found to have anti-MRSA activity. They have also found the interesting fact that flavone derivatives suppress β-lactam drug resistance and induce sensitivity. The present invention has been completed based on such findings.

[0004]

SUMMARY OF THE INVENTION Apigenin and luteolin are used in Scutellaria barbata.
D. Don) is a component contained in the whole plant, half-branch lotus is also other alkaloids, flavonoid glycosides, phenols,
Contains fullerol bodies. Hanjian Lotus is a perennial herb in the Labiatae family. Dried whole grasses, many of which have already fallen off, have stems and branches with spikes and are 15-25
cm, square, yellow-green or purple-brown, glossy and smooth. Pharmacological actions include fever, detoxification, hemostasis, and painful action. However, there has never been a report that Hanjingren has anti-MRSA activity, and there is no report that flavone derivatives such as apigenin and luteolin have anti-MRSA activity.

[0005] The present invention relates to an anti-MRSA active pharmaceutical composition comprising a combination of a flavone derivative and a series of compounds selected from the group consisting of β-lactam antibiotics and their pharmaceutically acceptable salts. The anti-MRSA activity of a combination of a flavone derivative and a β-lactam antibiotic is not known, and this finding is novel.

In the present invention, the β-lactam antibiotic used is methicillin.
n, DMPPC), oxacillin (oxacilli)
n, MCIPC), penicillin G (penicilli)
nG, PCG), ampicillin (ampicilli)
n, ABPC), cephalothin (cephalothi)
n, CET), cefoxitin,
CFX), cefuroxime, CX
M) and cefotaxime, C
TX), and the like. As the flavone derivative, flavone, apigenin (apigen)
in), luteolin, kaempferol, quercetin (que
rcetin), sakuranetin (sakranetti)
n), eriodictyol
1), (+)-taxifolin ((+)-taxifo
lin) and phloretin.

The generic names and abbreviations of the antibiotics used in the present invention encompass all of the free acids and their pharmaceutically acceptable salts. Pharmaceutically acceptable salts are usually used as salts of β-lactam antibiotics, such as salts of sodium, potassium, calcium and the like, and acid addition salts of amine salts such as procaine and dibenzylamine and hydrochlorides. Pharmaceutically acceptable salts.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Purification of Apigenin and Luteolin A half-bran lotus (1.5 kg) was refluxed with 50% ethanol for 2 hours, suction-filtered at a warm temperature, and the filtrate was concentrated under reduced pressure. Ether was added to the concentrated ethanol extract, and liquid-liquid extraction was performed. The ether layer was concentrated at 70 ° C. under reduced pressure to dryness to obtain a brown viscous substance. This was dissolved in anhydrous ethanol to obtain an ether-extracted fraction. The ether extract was layered on a glass column (70 × 200 mm) packed with silica gel (Kieselgel 60) and fractionated with n-hexane-ethyl acetate to obtain two fractions having antibacterial activity. Further, these fractions were layered on a glass column (40 × 75 mm) packed with ODS-Q3 (Wako) and fractionated with 50% methanol. One fraction having antibacterial activity is LiCh
Purification with chloroform-methanol (9: 1) using rosorb Si 60 (25 × 250 mm) resulted in the isolation of apigenin in a yield of 920 mg. Still another fraction was purified by n-hexane-ethyl acetate (1: 9), and as a result, luteolin was isolated in a yield of 25 mg.

[0009]

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[0010]

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Antibacterial activity against MRSA As test bacteria, 20 strains of MRSA (methicillin-resistant Staphylococcus aureus) and 7 strains of MSSA (methicillin-sensitive Staphylococcus aureus) were used. The measurement of the antibacterial activity against MRSA, which was used as an index when isolating and extracting the active ingredient, was carried out by a disk diffusion method. MRSA bacterial solution about 10 ⁶ CFU / ml
(Colony forming unit: colony fo
rming unit) was spread on an agar plate, and a disk (diameter 1) having 100 μl of various concentrations of the test sample absorbed thereon was absorbed.
3 mm), and cultured at 37 ° C. for 20 hours. The antibacterial activity of the purified preparation was determined by the agar plate dilution method (Chemotherap,
y 29 (1), 76-79 (1981)). The plating medium for sensitivity measurement was a semi-synthetic medium based on Mueller-Hinton Agar, and the inoculum was Mueller-HintonBrot.
In h), the test bacteria were cultured at 37 ° C. for 20 hours, and then diluted with 8.5% physiological saline to adjust to 10 ⁶ CFU / ml. Test samples were prepared in a two-fold serial dilution series. The medium for inoculation was inoculated into the medium for measuring a sensitive plate using a microplanter (Sakuma Seisakusho), cultured at 37 ° C. for 20 hours, and then subjected to a minimum inhibitory concentration (MIC).
(nhibition concentration) was determined. The MIC value was defined as the minimum concentration at which growth was completely inhibited.

An ether-extracted fraction from Hanjian lotus, SB-
Table 1 shows the results of antimicrobial activities of 1, apigenin and luteolin against MRSA and MSSA.

[0013]

[Table 1]

Result: MRSA20 strain, MSSA7 of ether extract of Haneda lotus
The MIC values for the strains were 125-> 250 ftg / ml. The isolated SB-1 and apigenin show almost the same antibacterial activity and have MIC values of 3.9 to> 250 μg / ml.
And showed strong activity. SB-1 and apigenin are M
It was shown that there was considerable variation in activity intensity regardless of RSA or MSSA. Luteolin did not differ between strains, and the MIC value showed an almost constant activity of 62.5 to 125 μg / ml (Table 1). The MIC could not be measured because SB-2 had a poor yield.

Next, the relationship between the chemical structure of flavone and the antibacterial activity was examined. Flavones, flavones (fl
avone), chrysin, acacetin, baicalein (baical)
ein), the flavonol kaempferol (kae)
mpferol), quercetin (querceti)
n), rhamnetin, myricetin, quercetagetin (quer)
cetqgetin), the flavanones liquiritigenin, sakuranetin, naringenin (narin)
genin), hesperetin (hespereti)
n), eriodictyol
1), (+)-taxifolin (+)-taxifolin, which is a dihydroflavonol, formononetin (formonenetin), which is an isoflavone, daidzein, genistein (gen)
The antibacterial activity of istein, catechins (+)-catechin ((+)-catechin), and chalcones phloretin was measured. The flavone is methanol and the others are 0.1N Na
Dissolved in OH. As described above, the antimicrobial activity is measured by the MIC according to the agar plate dilution method determined by the Japanese Society of Chemotherapy.
The MIC is determined as the concentration that suppresses the growth of 80% of the strain.
The concentration that suppresses the growth of 80, 50% of the strain is MIC ₅₀
And

[0016]

[Table 2]

Results The antibacterial activities against MRSA and MSSA were kaempferol, quercetin, sacranetin, eriodictyol and phloretin in addition to apigenin and luteolin. However, none of apigenin and luteolin were as active as MIC, and the MIC ₅₀ value was 250 μg / ml (Table 2).

The expression of the activity and the structural correlation are summarized as follows. Among the flavones, the only active compounds were apigenin and luteolin, and also the flavonols showed activity only in kaempferol and quercetin. , 5,7,4 'hydroxyl groups were considered to be important for the expression of activity. In addition, dihydroflavonols, isoflavones, and catechins did not show any activity, and chalcones, phloretin, showed activity.Thus, the three-dimensional structure of the flavone and the position of the hydroxyl group were involved in the expression of the activity. Was thought to be.

Since MRSA is resistant to many chemotherapeutic agents as well as methicillin, combination therapy with various chemotherapeutic agents has recently been attempted. We are the MIC
When apigenin at a concentration less than
Methicillin was found to exhibit an antibacterial effect on That is, by adding 4 μg / ml of apigenin, the MIC value of methicillin alone was 1000 μg / ml.
MIC ₅₀ was 15.6 μg / ml.
And the activity was increased about 100 times (Table 3).

Therefore, a combination of 4 μg / ml of apigenin and other antibiotics (oxacillin, penicillin G, ampicillin, cephalotin, cefoxitin, cefuroxime, cefotaxime, vancomycin, streptomycin, chloramphenicol, kanamycin, erythromycin, nalidic acid, tetracycline) Tried.

Results Only methicillin and cefoxitin were found to have the effect of suppressing β-lactam drug resistance in the presence of 4 μg / ml apigenin (Table 3).

[0022]

[Table 3]

Next, another apigenin structure analogous compound (21
Seed) An experiment using 50 μg / ml and methicillin in combination was attempted.

Results Both luteolin and kaempferol, which exhibited activity alone, exhibited MIC values of 3.9 to 31.3 μg / ml, exhibiting the effect of suppressing tolerance. Even more surprisingly, the flavone and taxifolin, which did not show activity alone, also suppressed the resistance, of which flavone had an MIC ₅₀ of 3.9 μg / m 2.
1 and very strong activity. Further, the effect of suppressing the β-lactam agent resistance was remarkably observed only in MRSA (Table 4).

[0025]

[Table 4]

Next, MRSAN with a strong combined effect
o. The effect of flavones on the antimicrobial activity of methicillin on No. 5 was examined with broth at different concentrations. The experimental method was a microfluid dilution method (Chemot) specified by the Japanese Society of Chemotherapy.
herpy 38 (1), 102-105 (199)
0)). The culture medium for sensitivity measurement was Mueller-Hinton broth.
Broth) and calcium (Ca) ion 50 m
g / ml, magnesium (Mg) ion 25 mg / m1
And 2% sodium chloride (NaCl) was used. The inoculum was inoculated into Mueller Hinton broth at 37 ° C. for 20 hours, and then diluted with Mueller Hinton broth to a final inoculum of 10 ⁵ CFU / ml. The non-test sample was prepared in a two-fold serial dilution series, and after culturing at 37 ° C. for 20 hours using a U-shaped well microplate, the minimum inhibitory concentration (MIC) was determined. In the determination, the MIC was defined as the minimum drug concentration of the well in which the growth of bacteria was not visually observed.

Results Although the effect of suppressing the resistance was not observed at 0.11 mM of flavone, it was remarkably observed at 0.2 mM, and it was found that the antibacterial activity of methicillin increased as the amount of flavone added increased (Table 5). ).

[0028]

[Table 5]

MRS in the presence of 0.5 mM flavone
ANo. The antibacterial activities of cephalotin, cefuroxime, cefotaxime, cefoxitin, streptomycin, and vancomycin against 5 were measured with bouillon.

Results Unlike the results of apigenin in Table 3, in addition to methicillin and cefoxitin, all of the tested β-lactam agents (cephalotin, cefuroxime, cefotaxime) exhibited an effect of suppressing the resistance, and in particular, showed an effect on cefoxitin. It showed the strongest effect. In addition, it was not observed in the other antibiotics tested, and was considered to suppress the resistance only to the β-lactam agent (Table 6).

[0031]

[Table 6]

Effective Dosage and Administration Method The flavone derivative of the present invention can be prepared as an external preparation having an MRSA antibacterial activity. The antibacterial agent or the bactericide for the flavone derivative of the present invention is prepared using a usual method, but it is used by adding to a commercially available disinfectant, for example, cresol water, cresol soap solution, phenol water for disinfection and the like. Can be. These antibacterial agents or bactericides are used at a concentration of about 0.1 to 10 (weight or dose)% for disinfecting instruments, patient excrement, and cleaning skin, mucus, and wounds.

The administration form of the flavone derivative of the present invention may be parenteral administration, oral administration or external administration as in the case of ordinary β-lactam antibiotic preparations. Generally, administration by injection is preferred. In this case, the injection is prepared by a conventional method, and the injection form includes the case where the injection is dissolved with a suitable vehicle, for example, sterilized distilled water, physiological saline or the like. The flavone derivative can be administered orally by combining it with a β-lactam in various dosage forms. For example, tablets, capsules, tablets coated with sugar and the like, liquid solutions or suspensions.

The flavone derivative of the present invention can be used in combination with a β-lactam antibiotic to treat human bacterial infections caused by MRSA or the like as causative bacteria. The total dose of both the flavone derivative and the β-lactam antibiotic used in prophylaxis and treatment can be varied depending on the type of drug to be combined, the combination ratio, age, weight, patient symptoms and administration route. For example, when administered to an adult (body weight of about 70 kg), 10 mg to 100 mg of the combined drug is orally administered once to three times a day per administration. By changing these dosages and administration routes, the best therapeutic effect can be obtained.

In the present invention, it is possible to apply a very wide range of weight ratio when both drugs are used or mixed. The combination ratio varies depending on the type and severity of the infection and the type of the β-lactam antibiotic used in combination.Therefore, the combination ratio is not particularly limited. A combination of concentrations that can be expected to be effective can be realized.

The pharmaceutical composition of the present invention is usually prepared according to a conventional method and administered in a pharmaceutically suitable form. For example, solid oral forms include lactose,
Diluents such as dextrose, saccharose, cellulose, corn starch and potato starch, silica,
Lubricants such as talc, stearic acid, magnesium stearate or calcium stearate and / or polyethylene glycol, binders such as starch, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, polyvinylpyrrolidine, starch, alginic acid, alginate, glycolic acid Disintegrating agents such as sodium starch, effervescent agents, dyes, sweeteners such as wetting agents such as lecithin, polysorbate, lauryl sulfate;
And may contain non-toxic and pharmaceutically inactive substances commonly used in pharmaceutical formulations.

The above pharmaceutical preparations are produced by known methods, for example, mixing, granulating, tableting, sugar-coating or coating methods.

In the case of parenteral administration, a suppository intended for rectal application is possible, but a general-purpose dosage form is an injection. Injectables are available in different dosage forms, such as liquid preparations, dissolution-type preparations, and suspension preparations.However, basically, the active ingredient is sterilized by an appropriate method, then placed directly in a container and sealed. Are considered the same.

The simplest method of formulation is to sterilize the active ingredient by an appropriate method, then separately or physically mix the active ingredients, and then formulate a fixed amount of the active ingredient into divided formulations. When a liquid form is selected, a method in which the active ingredient is dissolved in an appropriate medium, sterilized and filtered, and then filled in an appropriate ampoule or vial and sealed can be adopted. In this case, the commonly used medium is distilled water for injection, but this is not a promise in the present discovery. In addition, if present, soothing agents having local anesthetic effects such as procaine hydrochloride, xylocaine hydrochloride, benzyl alcohol and phenol, preservatives such as benzyl alcohol, phenol, methyl, or propyl bacben, and chlorobutanol, citric acid, Acetic acid, buffering agents such as sodium salt of phosphoric acid, as well as tonicity agents, stabilizers,
It is also possible to add additives such as solubilizers.

[0040]

EXAMPLE 1 (Disinfectant) 0.5 g of apigenin is dissolved in 1000 ml of ordinary water and used as a disinfectant.

Examples 2 to 6 (Disinfectant) Disinfectants are prepared in the same manner as in Example 1 by using luteolin, quercetin, sakuranetin, eriodictyol and phloretin in place of apigenin.

Example 7 (Tablets) Apigenin 50 mg, methicillin 50 m
g, lactose 1g, starch 300mg, methylcellulose 50mg, talc 30mg are prepared into 10 tablets and sugar-coated with white sugar.

Examples 8 to 11 (Tablets) Tablets are prepared in the same manner as in Example 7 using luteolin, kaempferol, flavone, and (+)-taxifolin instead of apigenin.

Example 12 (Tablets) According to a conventional method, 50 mg of flavone, 50 mg of cefoxitin sodium, 1 g of lactose, 300 mg of starch, 50 mg of methylcellulose, and 30 mg of talc are prepared into 10 tablets and sugar-coated with sucrose.

Examples 13 to 15 (Tablets) Tablets are prepared in the same manner as in Example 12 except that cephalothin sodium, cefuroxime sodium and cefotaxime sodium are used instead of cefoxitin sodium.

Example 16 (injection) 500 mg of flavone, 500 sodium cefoxitin
The sterile mixture of mg is placed in a sterile vial and sealed. At the time of use, this mixture is dissolved in physiological saline to prepare an injection.

Examples 17 to 19 (Injection) Injections are prepared in the same manner as in Example 16 except that cephalothin sodium, cefuroxime sodium and cefotaxime sodium are used instead of cefoxitin sodium.

Example 20 (injection) Flavone 400 mg, cefoxitin sodium 400
The sterile mixture of mg was dissolved in 20 ml of physiological saline, filtered through a 0.22 μm Millipore filter, filled into a sterilized glass bottle and sealed to prepare an injection.

Examples 21 to 22 (Injections) Injections are prepared in the same manner as in Example 20, except that cephalothin sodium, cefuroxime sodium and cefotaxime sodium are used instead of cefoxitin sodium.

Continuation of the front page (51) Int.Cl. ⁶ Identification symbol FI C07D 311/32 C07D 311/32 (56) References Dokkyo J. Med. Sc i. , 22 [4] (1995) p. 253-261 "Antibacterial activity of flavonoids against methicillin in-resistant Staphylococcus aureus (MRSA)" Ethnopharmacl o. , 50 [1] (1996) p. 27-34 "Comparative study on the antibacteri al activity of phy tochemical flavono es against methici llin-resistant Sta phyloccoccus aureu s" (58) investigated the field (Int.Cl. ^6, DB name) A61K 31/35 C07D 311/26 -311/38 CA (STN) (54) [Title of the Invention] Induction of β-lactam drug susceptibility to methicillin-resistant Staphylococcus aureus (MRSA) by flavone derivatives

Claims (9)

(57) [Claims] 1. An anti-MRSA active pharmaceutical composition comprising apigenin as an active ingredient. 2. An anti-MRSA active pharmaceutical composition comprising quercetin as an active ingredient. 3. An anti-MRSA active pharmaceutical composition comprising Sakuranetin as an active ingredient. 4. An anti-MRSA active pharmaceutical composition comprising eriodictyol as an active ingredient. 5. An anti-MRSA active pharmaceutical composition obtained by mixing a flavone with a β-lactam antibiotic. 6. An anti-MRSA active pharmaceutical composition obtained by mixing apigenin with a β-lactam antibiotic. 7. An anti-MRSA active pharmaceutical composition obtained by mixing luteolin with a β-lactam antibiotic. 8. An anti-MRSA active pharmaceutical composition obtained by mixing kaempferol with a β-lactam antibiotic. 9. An anti-MRSA active pharmaceutical composition obtained by mixing (+)-taxifolin with a β-lactam antibiotic.