WO2013085152A1

WO2013085152A1 - Combined antibiotics comprising cephalosporins and beta-lactamase inhibitors

Info

Publication number: WO2013085152A1
Application number: PCT/KR2012/009391
Authority: WO
Inventors: Byung-Ha BACK; Soon-Ok Kim; Suk-Won SEONG
Original assignee: Union Korea Pharm Co., Ltd.
Priority date: 2011-12-07
Filing date: 2012-11-08
Publication date: 2013-06-13

Abstract

The present invention is related to compositions comprising the combination of cephalosporin selected from the group consisting of generation 1^st generation, 2^nd generation, 4^th generation and 5^th generation cephalosporin, specifically the 1^st generation or the 2^nd generation cephalosporin with beta-lactamase inhibitor, especially, clavulanate or sulbactam and it showed synergistic antibacterial effect on the antibiotic resistant bacteria compared with the sole treatment, therefore, it can be used as the effective and safe therapeutics for treating and preventing bacterial infection caused by multi-drug resistance (MDR).

Description

COMBINED ANTIBIOTICS COMPRISING CEPHALOSPORINS AND BETA-LACTAMASE INHIBITORS

The present invention relates to combined antibiotics comprising cephalosporins and beta-lactamase inhibitors.

The antibiotic resistance has been reported to be very serious problem recently since most of clinical bacteria found in hospital get resistance to most of conventional antibiotic, which makes difficult to treat the bacterial infection (Gold, S. H., N. Engl. J. Med., 335, pp1445, 1996). Moreover, the representative antibiotic resistant bacteria, for example, MRSA (Methicillinresistant Staphylococcus aureus) or MRSE (Methicillinresistant Staphylococcus epidermidis), has been resistant to vancomycin which had been used as an sole antibiotic to treat such antibiotic resistant bacteria infection (Sieradzki K et al., N. Eng. J. Med., 240, p517, 1999).

Antibiotic resistant grampositive bacteria such as Staphylococcus aureus is reported that it is surrounded with cell wall called as peptidoglycan and various surface proteins expressed on the cell surface are adhered thereto. The proteins are known to being as playing essential roles in human infection, for example, it helps contacting bacteria with human cells and deviating from human immune system, which make human body be vulnerable to a bacterial infection and infected disease (Navarre, W.W and Schneewind, O., Microbial. Mol Biol. Rev., 63, p174, 1999).

It has been reported that the bacterial infection caused by resistant bacteria make the patients be longer hospitalization, increase the death rate and higher cost (Cohen, Science, 257, pp.10511055, 1992). The efficacy of novel antibiotics is rapidly reduced by the significantly increased resistance of bacteria. Accordingly, there has been still needs to develop new antibiotics to overcome the antibiotic resistance (Neu, Science, 257, pp10641073, 1992).

However, there has been not reported or disclosed about the therapeutic effect of the combination of cephalosporin with beta-lactamase inhibitors on bacterial infection caused by multi-drug resistance (MDR) in any of above cited literatures, the disclosures of which are incorporated herein by reference.

Accordingly, the present inventors have confirmed that the combination of cephalosporin, specifically the 1^st generation or the 2^nd generation cephalosporin with beta-lactamase inhibitor, especially, clavulanate or sulbactam, showed synergistic antibacterial effect on antibiotic resistant bacteria, therefore, it can be used as the effective and safe therapeutics for treating and preventing bacterial infection caused by multi-drug resistance (MDR).

According to one aspect of the present invention, the present invention provides a combined composition comprising cephalosporin, specifically the 1^st generation or the 2^nd generation cephalosporin with beta-lactamase inhibitor, especially, clavulanate or sulbactam, for the prevention and treatment of the bacterial infection caused by multi-drug resistance (MDR).

Accordingly, it is an object of the present invention to provide a combined composition comprising cephalosporin selected from the group consisting of generation 1^st generation, 2^nd generation, 4^th generation and 5^th generation cephalosporin, with beta-lactamase inhibitor for the prevention and treatment of bacterial infection caused by multi-drug resistance (MDR).

It is another object of the present invention to provide an antibiotic composition comprising cephalosporin selected from the group consisting of generation 1^st generation, 2^nd generation, 4^th generation and 5^th generation cephalosporin and beta-lactamase inhibitor as an active ingredient in amount effective to prevent or treat bacterial infection caused by multi-drug resistance (MDR) together with pharmaceutically acceptable carriers or diluents.

The term “cephalosporin” disclosed herein comprises, but it is not intended to limit therein, conventionally available cephalosporin (cephamycin) such as generation 1^st generation, 2^nd generation, 4^th generation and 5^th generation cephalosporin, preferably, the 1^st generation or 2^nd generation cephalosporin.

The term “the 1^st generation cephalosporin” disclosed herein comprises, but it is not intended to limit thereto, Cefacetrile (Cephacetrile), Cefadroxil (cefacroxyl, Duricef®), Cephalexin (Cefalexin, Keflex®), Cefaloglycin (Cephaloglycin), Cefalonium (Cephalonium), Cefaloridine (Cephaloridine), Cefalotin (Cephalothin, Keflin®), Cefapirin (Cephapirin, Cefadryl®), Cefatriazine, Cefazaflur, Cefazedone, Cefazolin (Cephazolin, Ancef®, Kefzol®), Cefradine (Cephradine, Velosef®), Cefroxadine, or Ceftezole and the like, preferably, Cefadroxil (cefacroxyl, Duricef®), Cefradine (Cephradine, Velosef®) or Ceftezole, more preferably, Ceftezole.

The term “the 2^nd generation cephalosporin” disclosed herein comprises, but it is not intended to limit thereto, Cefaclor (Ceclor, Distaclor, Keflor, Raniclor), Cefonicid (Monicid), Cefprozil (Cefproxil, Cefzil), Cefuroxime (Zefu, Zinnat®, Zinacef®, Ceftin, Biofuroksym), Cefuzonam, Cefmetazole, Cefotetan, Cefoxitin and the like, preferably, Cefaclor (Ceclor, Distaclor, Keflor, Raniclor), Cefuroxime (Zefu, Zinnat®, Zinacef®, Ceftin, Biofuroksym), or Cefmetazole, more preferably, Cefaclor (Ceclor, Distaclor, Keflor, Raniclor), or Cefmetazole.

The term “the 4^th generation cephalosporin” disclosed herein comprises, but it is not intended to limit thereto, Cefclinidine, Cefepime (Maxipime), Cefluprenam, Cefoselis, Cefozopran, Cefpirome (Cefrom), or Cefquinome and the like.

The term “the 5^th generation cephalosporin” disclosed herein comprises, but it is not intended to limit thereto, Ceftobiprole or Ceftaroline and the like.

The term “beta-lactamase inhibitor” disclosed herein comprises, but it is not intended to limit thereto, conventionally available beta-lactamase inhibitor such as clavulanic acid, sulbactam or tazobactam and the like, preferably, clavulanic acid or sulbactam.

The term “combined composition comprising cephalosporin with beta-lactamase inhibitor” disclosed herein includes the combined composition comprising (a) cephalosporin with (b) beta-lactamase inhibitor with the mixed ratio ranging from 1:10∼10:1 by weight(w/w%), preferably, 1:5∼5:1 by weight(w/w%), most preferably 1:2∼2:1 by weight(w/w%).

Accordingly, it is an object of the present invention to provide a combined composition comprising cephalosporin, preferably, the 1^st generation or 2^nd generation cephalosporin, more preferably, a cephalosporin selected from Cefadroxil, Cefradine, Ceftezole, Cefaclor or Cefmetazole; with beta-lactamase inhibitor selected from clavulanic acid, sulbactam or tazobactam, for the prevention and treatment of bacterial infection caused by multi-drug resistance (MDR).

The term “multi-drug resistance (MDR)” disclosed herein comprises, but it is not intended to limit thereto, conventionally known multi-drug resistance in the art, specifically, VRE (Vancomycin Resistant Enterococci), MRSA (Methicillin resistant Staphylococcus aureus), ESBL(Extended spectrum beta-lactamase) producing Gram-negative bacteria, KPC (Klebsiella pneumonia carbapenemase) producing negatives, Imipenem Resistant or Multidrug Resistant Organisms Acinobacter baumanii, Imipenem Resistant or Multidrug Resistant Organisms Klepsiella pneumonia etc, more specifically, MRSA (Methicillin resistant Staphylococcus aureus) or ESBL(Extended spectrum beta-lactamase) producing Gram-negative bacteria, most specifically, the bacteria deposited on ATCC, for example, Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), Escherichia coli (ATCC 35218), Staphylococcus aureus (ATCC 25923), Staphylococcus aureus (ATCC 29213), Staphylococcus aureus (ATCC 43300), Pseudomonas aeruginosa (ATCC 27853), Haemophilus influenzae (ATCC 49247), Streptococcus pneumoniae (ATCC 49619), Neisseria gonorrhoeae (ATCC 49226), Bacteroides fragilis (ATCC 25285), Klebsiella pneumonia (ATCC 700603), or Helicobacter pylori (ATCC 43504).

The term “bacterial infection caused by multi-drug resistance (MDR)” disclosed herein comprises, but it is not intended to limit thereto, conventionally known bacterial infection caused by multi-drug resistance in the art, specifically, otitis, pneumonia, laryngopharyngitis, tonsillitis, bronchitis, pyelonephritis, cystitis, gonococcal urethritis, furuncle, carbuncle, folliculitis, cellulitis, infectious arthrosclerosis, subcutaneous abscess, paronnychia, wound infection, sepsis, bronchitis, infectious bronchiectasis, secondary infection of bronchitis, lung abscess, empyema, cholangitis, cholecystitis, peritonitis, acute pyelonephritis, endometritis, salpingitis, pelveoperitonitis, parametritis, perimaxillary inflammation, maxillary inflammation, postoperative infection or non-gonococcal urethritis.

The inventive compound can be transformed into their pharmaceutically acceptable salt and solvates by the conventional method well known in the art. For the salts, acid-addition salt thereof formed by a pharmaceutically acceptable free acid thereof is useful and can be prepared by the conventional method. For example, after dissolving the compound in the excess amount of acid solution, the salts are precipitated by the water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile to prepare acid addition salt thereof and further the mixture of equivalent amount of compound and diluted acid with water or alcohol such as glycol monomethylether, can be heated and subsequently dried by evaporation or filtrated under reduced pressure to obtain dried salt form thereof.

As a free acid of above-described method, organic acid or inorganic acid can be used. For example, organic acid such as methansulfonic acid, p-toluensulfonic acid, acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonylic acid, vanillic acid, hydroiodic acid and the like, and inorganic acid such as hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used herein.

Further, the pharmaceutically acceptable metal salt form of inventive compound may be prepared by using base. The alkali metal or alkali-earth metal salt thereof can be prepared by the conventional method, for example, after dissolving the compound in the excess amount of alkali metal hydroxide or alkali-earth metal hydroxide solution, the insoluble salts are filtered and remaining filtrate is subjected to evaporation and drying to obtain the metal salt thereof. As a metal salt of the present invention, sodium, potassium or calcium salt are pharmaceutically suitable and the corresponding silver salt can be prepared by reacting alkali metal salt or alkaliearth metal salt with suitable silver salt such as silver nitrate.

The pharmaceutically acceptable salt of the compound comprise all the acidic or basic salt which may be present at the compounds, if it does not indicated specifically herein. For example, the pharmaceutically acceptable salt of the present invention comprise the salt of hydroxyl group such as the sodium, calcium and potassium salt thereof; the salt of amino group such as the hydrogen bromide salt, sulfuric acid salt, hydrogen sulfuric acid salt, phosphate salt, hydrogen phosphate salt, dihydrophosphate salt, acetate salt, succinate salt, citrate salt, tartarate salt, lactate salt, mandelate salt, methanesulfonate(mesylate) salt and p-toluenesulfonate (tosylate) salt etc, which can be prepared by the conventional method well known in the art.

The present inventors have confirmed that the combination of cephalosporin, specifically the 1^st generation or the 2^nd generation cephalosporin with beta-lactamase inhibitor, especially, clavulanate or sulbactam, showed synergistic antibacterial effect on the antibiotic resistant bacteria compared with the sole treatment, therefore, it can be used as the effective and safe therapeutics for treating and preventing bacterial infection caused by multi-drug resistance (MDR).

It is another object of the present invention to provide a pharmaceutical composition comprising cephalosporin selected from the group consisting of generation 1^st generation, 2^nd generation, 4^th generation and 5^th generation cephalosporin and beta-lactamase inhibitor as an active ingredient in amount effective to prevent or treat a bacterial infection caused by multi-drug resistance (MDR) together with pharmaceutically acceptable carriers or diluents.

In accordance with another aspect of the present invention, there is also provided a use of a combined composition comprising cephalosporin selected from the group consisting of generation 1^st generation, 2^nd generation, 4^th generation and 5^th generation cephalosporin with beta-lactamase inhibitor for manufacture of medicines employed for treating or preventing a bacterial infection caused by multi-drug resistance (MDR).

In accordance with another aspect of the present invention, there is also provided a method of treating or preventing a bacterial infection caused by multi-drug resistance (MDR) in mammals, wherein the method comprises administering a therapeutically effective amount of a combined composition comprising cephalosporin selected from the group consisting of generation 1^st generation, 2^nd generation, 4^th generation and 5^th generation cephalosporin with beta-lactamase inhibitor into the mammal suffering with the bacterial infection caused by multi-drug resistance (MDR).

The inventive composition for treating and preventing the bacterial infection caused by multi-drug resistance (MDR) may comprises the above-described extract as 0.1~50% by weight based on the total weight of the composition.

The inventive composition may additionally comprise conventional carrier, adjuvants or diluents in accordance with a using method well known in the art. It is preferable that said carrier is used as appropriate substance according to the usage and application method, but it is not limited. Appropriate diluents are listed in the written text of Remington’s Pharmaceutical Science (Mack Publishing co, Easton PA).

Hereinafter, the following formulation methods and excipients are merely exemplary and in no way limit the invention.

The composition according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, antiagglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient by employing any of the procedures well known in the art.

For example, the compositions of the present invention can be dissolved in oils, propylene glycol or other solvents that are commonly used to produce an injection. Suitable examples of the carriers include physiological saline, polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, etc., but are not limited to them. For topical administration, the extract of the present invention can be formulated in the form of ointments and creams.

Pharmaceutical formulations containing present composition may be prepared in any form, such as oral dosage form (powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like), or injectable preparation (solution, suspension, emulsion).

The composition of the present invention in pharmaceutical dosage forms may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.

The desirable dose of the inventive composition varies depending on the condition and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging 0.0001 to 1000 mg/kg, preferably, 0.001 to 100 mg/kg by weight/day of the inventive compound of the present invention. The dose may be administered in single or divided into several times per day.

The pharmaceutical composition of present invention can be administered to a subject animal such as mammals (rat, mouse, domestic animals or human) via various routes. All modes of administration are contemplated, for example, administration can be made orally, rectally or by intravenous, intramuscular, subcutaneous, intracutaneous, intrathecal, epidural or intra-cerebroventricular injection.

Inventive compounds of the present invention have no toxicity and adverse effect therefore can be used with safe.

It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.

The present invention comprising the combination of cephalosporin selected from the group consisting of generation 1^st generation, 2^nd generation, 4^th generation and 5^th generation cephalosporin, specifically the 1^st generation or the 2^nd generation cephalosporin with beta-lactamase inhibitor, especially, clavulanate or sulbactam, showed synergistic antibacterial effect on the antibiotic resistant bacteria compared with the sole treatment, therefore, it can be used as the effective and safe therapeutics for treating and preventing bacterial infection caused by multi-drug resistance (MDR).

The following Comparative Example, Reference Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

Example 1. Sample Preparation

Cefaclor (C6895, Sigma Co. Ltd.) and Sulbactam (S9701, Sigma Co. Ltd) were used as test samples. Ampicillin:Sulbactam disc (BD 231653, Becton, Dickinson and Company, BD) and Cefaclor disc (BD 231660, Becton, Dickinson and Company, BD) were used in the experiment.

The combination of Cefaclor, one of cephalosporin, and Sulbactam, one of beta-lactamase, was diluted with various dilution ratio to be 30 microgram/disc of total volume, i.e., 1:2 (w/w), 1:1 (w/w) and 2:1 (w/w) (which are designated as CB 12, CB 11 and CB 21, respectively, hereinafter) and inoculated into blank disc (5mm diameter, BD 231039) as a preliminary test prior to following Disc diffusion test.

Experimental Example 1. Disc diffusion test

In order to the synergistic anti-bacterial antibacterial activity of the combination prepared in Example 1, Disc diffusion test was performed by the method according to the Performance Standard for Antimicrobial Susceptibility Testing recommended by CLSI (Clinical and Laboratory Standards Institute, 2007. Performance standards for antimicrobial susceptibility testing; Seventeenth Informational Supplement M100-S17, Wayne, Pa.; Wang, F. D., M. L. Lin, W. S. Lee, and C. Y. Liu. 2004. In vitro activities of β-lactam antibiotics alone and in combination with sulbactam against Gramnegative bacteria. Int. J. Antimicrob. agents 23; 590-595).

1.1. Preparation

All the bacteria used in the experiment were procured from ATCC (American Type Culture Collection, Table 1) and all the preparation such as selection of liquid or culture medium, culture method, germ management etc, was performed by the method according to the recommendation of ATCC Product Information sheet.

Table 1

US Clinical and Laboratory Standards Institute (CLSI)

Organism	Characteristics	ATCC ^® Strain Ref
Enterococcus faecalis		ATCC 29212
Escherichia coli	β-lactamase negative	ATCC 25922
Escherichia coli	TEM1 β-lactamase (NonESBL) producer	ATCC 35218
Staphylococcus aureus	β-lactamase negative, mecA negative	ATCC 25923
Staphylococcus aureus	Weak β-lactamase positive, mecA negative	ATCC 29213
Staphylococcus aureus	Oxacillin resistant, mecA positive	ATCC 43300
Pseudomonas aeruginosa	Contains inducible AmpC β-lactamase	ATCC 27853
Haemophilus influenzae	BLNAR (β-lactamase negative, ampicillin resistant)	ATCC 49247
Streptococcus pneumoniae	Penicillin intermediate (altered penicillin binding protein)	ATCC 49619
Neisseria gonorrhoeae	CMRNG (Chromosome-mediated resistant Neisseria gonorrhoeae)	ATCC 49226
Bacteroides fragilis		ATCC 25285
Klebsiella pneumoniae	SHV18 ESBL producing	ATCC 700603
Helicobacter pylori		ATCC 43504

Recommended performance standards for antimicrobial susceptibility testing (M100-S21)

1.2. Schedule

In order to efficient test, (a) preliminary disc diffusion test to confirm the MIC (Minimal inhibitory concentration) and inhibitory range of bacterial growth for respective antibiotics (Cefaclor and Sulbactam) was performed and then (b) Microbial Viabillity Assay for determining the inhibitory range of bacterial growth and MIC for combined antibiotics. Finally, (c) Disc fusion test using by selected combinations with various ratio of antibiotics was performed to determine the specific inhibitory range of bacterial growth and MIC for combined antibiotics.

1.3. modified MIC test

In order to overcome the limited accuracy of conventional MIC test (turbidity test), modified MIC test using by 96 well-plates to determine their absorbance was performed together with conventional MIC test (turbidity test) as follows;

The equal volume of culture medium (Muller-Hinton broth) inoculated with the testing strains was distributed to a number of test tubes. Turbidity was adjusted to Mcfarland standard (Absorbance: 0.125 at 600nm) and then diluted to 1:200 to the extent that the number of strains reach to about 10⁵-10⁶ CFU/ml.

1ml of culture was inoculated to each test tube to reach 5 x 10⁵ CFU/ml of final inoculated strain number and the concentration of test sample (Cefaclor + subactam) with the ratio of 1:2 (21.3 microgram + 42.6 microgram), 1:1 (32 microgram + 32 microgram), and 2:1 (42.6 microgram + 21.3 microgram), was serially diluted to various concentrations, i.e., 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.12 and 0.06 microgram/disc.

None-treated culture medium with antibiotics was used as a control to compare with test sample group and the concentration of test sample was adjusted to 64 microgram/disc at maximum and diluted to 1 microgram/disc at minimum to determine the MIC of test sample.

The culture medium was cultured for 16 hours at 35℃ and 100 microliter of the medium was transferred to 96 well plates to determine the absorbance of respective turbidity.

After confirming the growth of culture medium using by turbidity test, the MIC concentration of antibiotics in “last” clear tube was confirmed and compared by their absorbance.

1.4. Microbial Viability Assay

In order to determine specific MIC of test sample, conventionally available antimicrobial susceptibility test using by microbial viability assay kit (WST, Dojindo) was performed as follows;

The turbidity of culture medium (Muller-Hinton broth, MHA) inoculated with the testing strains was adjusted to Mcfarland standard (Absorbance: 0.125 at 600nm) and then diluted with saline to 1:10 to the extent that the number of strains reach to about 10⁷ CFU/ml.

The concentration of test sample (Cefaclor + subactam) with the ratio of 1:2 (21.3 microgram + 42.6 microgram), 1:1 (32 microgram + 32 microgram), and 2:1 (42.6 microgram+21.3 microgram), was serially diluted to various concentrations, i.e., 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.12 and 0.06 microgram/disc.

180 microliter of test sample in the culture was inoculated to each well plate and 10 microloter of culture medium was added to each well to reach 10⁴ CFU/ml of final inoculated strain number.

None-treated culture medium with antibiotics was used as a control to compare with test sample group and the culture medium was cultured for 6 hours at 35℃.

After the incubation, 10 microliter of coloring agent (Microbial viability Assay kit) was added to each well to react for 3 hours at 35℃.

At the end of the reaction, the absorbance of each well was determined by microplate reader at 450 nm and analyzed.

1.5. Agar Disc Diffusion Assay

In order to determine specific MIC of test sample, Disc diffusion test was performed by the method according to the Performance Standard for Antimicrobial Susceptibility Testing recommended by CLSI (Clinical and Laboratory Standards Institute) as follows:

The various strains procured from ATCC were cultured in the culture medium (Muller-Hinton broth) according to the recommended instruction of ATCC (Table 2).

Sterilized agar was cooled to 45-50℃ and poured to petri dish at the height of 4 mm (25-30 mm in 100 mm dish) to be harden in clean bench.

Prior to the medium preparation, the agar was cultured for 24 hour at 30-35℃ to check its sterilization. Anti-biotictesting disc was prepared by subjecting blank disc (6mm diameter) to liquid dropping of test samples, drying on clean bench and kept in refrigerator within 1 week for use.

The inoculated amount of strains was determined by using the turbidity of precipitated standard suspension (H₂SO₄+BaCl₂) adjusted to 0.5 according to McFarland turbidity standard and the approximate number of strain was found to 1.5 x 10⁸ CFU/ml.

The determined amount of strains was smeared on the agar plate in three directions and the cap of agar plate was open for 3-5 minutes to let absorb a humid air. The antibiotics disc was put on the surface of the agar using by sterilized pincette and the agar plate was transferred to culture room to incubate for 16 hours at 35℃.

The size of inhibition region was determined by auto caliper.

The culture condition was varied with the sort of strains, for example, the agar plate with Helicobacter pylori had been culture for 30 hours; with the particular strains demanding CO₂gas for growth such as H. influenza, S. pneumoniae, and N. gonorrhoeae; and with those demanding mixed gas such as B. fragilis and H. pylori etc has been cultured in the other culture room.

Table 2

ATCC Product Information Sheet

Organism	ATCC MEDIA/Classification	Culture Condition
Enterococcus faecalis(ATCC 29212)	#44 Broth: Brain Heart infusion (BD 237500),#260 Agar: Trypticase Soy Agar (BD 236950) with 5% defibrinated sheep blood,nonmotile, grampositive, spherical bacterium	37℃, aerobic
Escherichia coli(ATCC 25922)	#18 Broth: Tryptic Soy Broth (BD 211825),#18 Agar: Tryptic Soy Agar (BD 236950), Gramnegative, rodshaped bacterium	37℃, aerobic
Escherichia coli(ATCC 35218)	#3 Broth: Nutrient Broth (BD 234000),#3 Agar: Nutrient Agar (BD 213000), rodshaped bacterium	37℃, aerobic
Staphylococcus aureus(ATCC 25923)	#18 Broth: Tryptic Soy Broth (BD 211825),#18 Agar: Tryptic Soy Agar (BD 236950), facultative anaerobic Grampositive coccal bacterium	37℃, aerobic
Staphylococcus aureus(ATCC 29213)	#18 Broth: Tryptic Soy Broth (BD 211825),#18 Agar: Tryptic Soy Agar (BD 236950),, Grampositive coccal bacterium	37℃, aerobic
Staphylococcus aureus(ATCC 43300)	#18 Broth: Tryptic Soy Broth (BD 211825),#18 Agar: Tryptic Soy Agar (BD 236950),, Grampositive coccal bacterium	37℃, aerobic
Pseudomonas aeruginosa(ATCC 27853)	#18 Broth: Tryptic Soy Broth (BD 211825),#18 Agar: Tryptic Soy Agar (BD 236950),, Gramnegative, aerobic, rodshaped bacterium with unipolar motility	37℃, aerobic
Haemophilus influenzae(ATCC 49247)	#2167 Broth: Haemophilus Test Medium,#814 Agar: GC medium, Gramnegative, rodshaped bacterium	37℃, 5% CO₂
Streptococcus pneumoniae(ATCC 49619)	#44 Broth: Brain Heart infusion (BD 237500),#260 Agar: Trypticase Soy Agar (BD 236950) with 5% defibrinated sheep blood, Grampositive, alphahemolytic, aerotolerant anaerobic member	37℃, 5% CO₂
Neisseria gonorrhoeae(ATCC 49226)	#814 Broth: GC medium,#814 Agar: GC medium, Gramnegative coffee beanshaped diplococci bacteria	37℃, 5% CO₂
Bacteroides fragilis(ATCC 25285)	#1490 Broth: Modified Chopped Meat Medium,Solid medium: Add 2% agar to formula listed in ATCC Producr Information Sheet, Gramnegative bacillus bacterium	37℃, anaerobic gas mixture(80% N₂10% CO₂10% H₂)
Klebsiella pneumoniae(ATCC 700603)	#3 Broth: Nutrient Broth (BD 234000),#3 Agar: Nutrient Agar (BD 213000), Gramnegative, nonmotile, encapsulated, lactose fermenting, facultative anaerobic, rod shaped bacterium	37℃, aerobic
Helicobacter pylori(ATCC 43504)	#18 Broth: Tryptic Soy Broth (BD 211825),#260 Agar: Tryptic Soy Agar (BD 236950)with 5% defibrinated sheep blood, Gramnegative, microaerophilic bacterium	37℃, Microaerophilic (35% O₂, 10% CO₂)

1.6. Result

As can be seen in following Tables 3-16, the inventive combination of cephalosporin with beta-lactamase inhibitor, especially, combination of cefaclor and sulbactam, showed synergistic antibacterial effect on the antibiotic resistant bacteria, especially, Haemophilus influenzae, Neisseria gonorrhoeae, Helicobacter, Bacterioides fragilis, Escherichia coli (ATCC 35218) and Staphylcoccus aureus (ATCC 29213) compared with the sole treatment, i.e., cefaclor or sulbactam.

Therefore, the combination can be used as the effective and safe therapeutics for treating and preventing bacterial infection caused by multi-drug resistance (MDR).

Table 3

Escherichia coli (ATCC 25922)

Antimicrobial Agent		Disc Content	Zone Diameter Break Points, nearest whole mm			MIC Interpretive Standard (μg/mL)
			S	I	R	S	I	R
CLSI Ref.	Cefaclor	30 μg	≥ 18	15-17	≤ 14	≤ 8	16	≥ 32
	Sulbactam	-	-	-	-	-	-	-
Result	Cefaclor	5 μg	10.2			32
		10 μg	12.0
		15 μg	12.2
		20 μg	12.3
		30 μg	13.4
	Sulbactam	5 μg	-			64
		10 μg	-
		15 μg	7.7
		20 μg	8.1
		30 μg	9.3
Ampicillin/Sulbactam		10/10 μg	≥ 15	12-14	≤ 11	≤ 8/4	16/8	≥ 32/16

Table 4

Escherichia coli (ATCC 35218)

Antimicrobial Agent		Disc Content	Zone Diameter Break Points, nearest whole mm			MIC Interpretive Standard (μg/mL)
			S	I	R	S	I	R
CLSI Ref.	Cefaclor	30 μg	≥ 18	15-17	≤ 14	≤ 8	16	≥ 32
	Sulbactam	-	-	-	-	-	-	-
Result	Cefaclor	5 μg	17.7			8
		10 μg	18.1
		15 μg	18.2
		20 μg	18.6
		30 μg	19.4
	Sulbactam	5 μg	-			64
		10 μg	-
		15 μg	-
		20 μg	8.6
		30 μg	8.6
Ampicillin/Sulbactam		10/10 μg	≥ 15	12-14	≤ 11	≤ 8/4	16/8	≥ 32/16

Table 5

Psudomonas aeruginosa (ATCC 27853)

Antimicrobial Agent		Disc Content	Zone Diameter Break Points, nearest whole mm			MIC Interpretive Standard (μg/mL)
			S	I	R	S	I	R
CLSIRef.	Cefaclor	30 μg	-	-	-	-	-	-
	Sulbactam	-	-	-	-	-	-	-
Result	Cefaclor	5 μg	8.4			64
		10 μg	8.5
		15 μg	8.5
		20 μg	8.5
		30 μg	8.6
	Sulbactam	5 μg	-			64
		10 μg	-
		15 μg	-
		20 μg	-
		30 μg	-
Ampicillin/Sulbactam		10/10 μg	-	-	-	-	-	-

Table 6

Staphylococcus aureus (ATCC 25923)

Antimicrobial Agent		Disc Content	Zone Diameter Break Points, nearest whole mm			MIC Interpretive Standard (μg/mL)
			S	I	R	S	I	R
CLSI Ref.	Cefaclor	30 μg	≥ 18	15-17	≤ 14	≤ 8	16	≥ 32
	Sulbactam	-	-	-	-	-	-	-
Result	Cefaclor	5 μg	12.9			32
		10 μg	13.2
		15 μg	13.5
		20 μg	13.9
		30 μg	14.9
	Sulbactam	5 μg	-			64
		10 μg	6.7
		15 μg	8.5
		20 μg	9.6
		30 μg	9.9
Ampicillin/Sulbactam		10/10 μg	≥ 15	12-14	≤ 11	≤ 8/4	16/8	≥ 32/16

Table 7

Staphylococcus aureus (ATCC 29213)

Antimicrobial Agent		Disc Content	Zone Diameter Break Points, nearest whole mm			MIC Interpretive Standard (μg/mL)
			S	I	R	S	I	R
CLSI Ref.	Cefaclor	30 μg	≥ 18	15-17	≤ 14	≤ 8	16	≥ 32
	Sulbactam	-	-	-	-	-	-	-
Result	Cefaclor	5 μg	16.8			32
		10 μg	17.4
		15 μg	17.7
		20 μg	18.7
		30 μg	19.6
	Sulbactam	5 μg	-			64
		10 μg	-
		15 μg	-
		20 μg	-
		30 μg	-
Ampicillin/Sulbactam		10/10 μg	≥ 15	12-14	≤ 11	≤ 8/4	16/8	≥ 32/16

Table 8

Staphylococcus aureus (ATCC 43300)

Antimicrobial Agent		Disc Content	Zone Diameter Break Points, nearest whole mm			MIC Interpretive Standard (μg/mL)
			S	I	R	S	I	R
CLSI Ref.	Cefaclor	30 μg	≥ 18	15-17	≤ 14	≤ 8	16	≥ 32
	Sulbactam	-	-	-	-	-	-	-
Result	Cefaclor	5 μg	9.9			16
		10 μg	12.2
		15 μg	12.5
		20 μg	12.6
		30 μg	13.4
	Sulbactam	5 μg	-			64
		10 μg	-
		15 μg	6.8
		20 μg	8.6
		30 μg	9.2
Ampicillin/Sulbactam		10/10 μg	≥ 15	12-14	≤ 11	≤ 8/4	16/8	≥ 32/16

Table 9

Entrococcus faecalis (ATCC 29212)

Antimicrobial Agent		Disc Content	Zone Diameter Break Points, nearest whole mm			MIC Interpretive Standard (μg/mL)
			S	I	R	S	I	R
CLSI Ref.	Cefaclor	30 μg	-	-	-	-	-	-
	Sulbactam	-	-	-	-	-	-	-
Result	Cefaclor	5 μg	12.8			32
		10 μg	13.3
		15 μg	14.4
		20 μg	14.8
		30 μg	15.0
	Sulbactam	5 μg	-			64
		10 μg	-
		15 μg	8.4
		20 μg	10.3
		30 μg	11.4
Ampicillin/Sulbactam		10/10 μg	-	-	-	-	-	-

Table 10

Haemophilus influenza (ATCC 49247)

Antimicrobial Agent		Disc Content	Zone Diameter Break Points, nearest whole mm			MIC Interpretive Standard (μg/mL)
			S	I	R	S	I	R
CLSI Ref.	Cefaclor	30 μg	≥ 20	17-19	≤ 16	≤ 8	16	≥ 32
	Sulbactam	-	-	-	-	-	-	-
Result	Cefaclor	5 μg	10.4			32
		10 μg	10.9
		15 μg	12.9
		20 μg	15.3
		30 μg	17.2
	Sulbactam	5 μg	-			64
		10 μg	6.7
		15 μg	8.5
		20 μg	9.6
		30 μg	9.9
Ampicillin/Sulbactam		10/10 μg	≥ 20	-	≤ 19	≤ 2/1	-	≥ 4/2

Table 11

Neisseria gonorrhoeae (ATCC 49226)

Antimicrobial Agent		Disc Content	Zone Diameter Break Points, nearest whole mm			MIC Interpretive Standard (μg/mL)
			S	I	R	S	I	R
CLSI Ref.	Cefaclor	30 μg	-	-	-	-	-	-
	Sulbactam	-	-	-	-	-	-	-
Result	Cefaclor	5 μg	12.0			4
		10 μg	20.5
		15 μg	25.0
		20 μg	29.1
		30 μg	33.0
	Sulbactam	5 μg	11.0			16
		10 μg	15.5
		15 μg	21.4
		20 μg	22.6
		30 μg	24.1
Ampicillin/Sulbactam		10/10 μg	-	-	-	-	-	-

Table 12

Streptococcus pneumoniae (ATCC 49619)

Antimicrobial Agent		Disc Content	Zone Diameter Break Points, nearest whole mm			MIC Interpretive Standard (μg/mL)
			S	I	R	S	I	R
CLSI Ref.	Cefaclor	30 μg	-	-	-	≤ 1	2	≥ 4
	Sulbactam	-	-	-	-	-	-	-
Result	Cefaclor	5 μg	9.4			64
		10 μg	11.4
		15 μg	13.3
		20 μg	13.5
		30 μg	14.4
	Sulbactam	5 μg	-			64
		10 μg	-
		15 μg	7.3
		20 μg	8.7
		30 μg	9.7
Ampicillin/Sulbactam		10/10 μg	-	-	-	-	-	-

Table 13

Klebsiella pneumoniae (ATCC 700603)

Antimicrobial Agent		Disc Content	Zone Diameter Break Points, nearest whole mm			MIC Interpretive Standard (μg/mL)
			S	I	R	S	I	R
CLSI Ref.	Cefaclor	30 μg	-	-	-	-	-	-
	Sulbactam	-	-	-	-	-	-	-
Result	Cefaclor	5 μg	15.1			32
		10 μg	16.3
		15 μg	16.4
		20 μg	16.4
		30 μg	17.8
	Sulbactam	5 μg	-			64
		10 μg	-
		15 μg	-
		20 μg	-
		30 μg	9.4
Ampicillin/Sulbactam		10/10 μg	-	-	-	-	-	-

Table 14

Helicobacter pylori (ATCC 43504)

Antimicrobial Agent		Disc Content	Zone Diameter Break Points, nearest whole mm			MIC Interpretive Standard (μg/mL)
			S	I	R	S	I	R
CLSI Ref.	Cefaclor	30 μg	-	-	-	-	-	-
	Sulbactam	-	-	-	-	-	-	-
Result	Cefaclor	5 μg	23.6			1
		10 μg	24.7
		15 μg	25.9
		20 μg	26.4
		30 μg	29.6
	Sulbactam	5 μg	-			64
		10 μg	-
		15 μg	-
		20 μg	-
		30 μg	-
Ampicillin/Sulbactam		10/10 μg	-	-	-	-	-	-

Table 15

Bacterioides fragilis (ATCC 25285)

Antimicrobial Agent		Disc Content	Zone Diameter Break Points, nearest whole mm			MIC Interpretive Standard (μg/mL)
			S	I	R	S	I	R
CLSI report	Cefaclor	30 μg	-	-	-	-	-	-
	Sulbactam	-	-	-	-	-	-	-
Result	Cefaclor	5 μg	12.9			4
		10 μg	13.0
		15 μg	13.0
		20 μg	15.8
		30 μg	21.8
	Sulbactam	5 μg	7.3			64
		10 μg	10.6
		15 μg	15.3
		20 μg	16.4
		30 μg	20.0
Ampicillin/Sulbactam		10/10 μg	-	-	-	≤ 8/4	16/8	≥ 32/16

Table 16

Viability assay and zone diameter breakpoint of various ratio of test sample

Organisms	Viability assay (μg)			Zone Diameter Breakpoints (mm)
Organisms	1:2	1:1	2:1	1:2	1:1	2:1
Escherichia coli(ATCC 25922)	≥ 16	＞ 8	≥ 8	15.2	15.5	16.0
Escherichia coli(ATCC 35218)	≥ 32	≥ 16	≥ 8	20.8	21.7	22.7
Psudomonas aeruginosa(ATCC 27853)	-	-	-	13.8	15.2	14.8
Staphylcoccus aureus(ATCC 25923)	≥ 8	≥ 1	≥ 1	14.7	15.0	14.9
Staphylcoccus aureus(ATCC 29213)	≥ 8	≥ 1	≥ 1	20.9	21.7	22.2
Staphylcoccus aureus(ATCC 43300)	≥ 16	≥ 32	≥ 8	14.7	14.8	15.1
Enterococcus faecalis(ATCC 29212)	≥ 32	≥ 16	≥ 4	15.4	15.9	16.7
Haemophilus influenza(ATCC 49247)	＞ 32	≥ 32	≥ 16	13.4	13.0	19.1
Neisseria gonorrhoeae(ATCC 49226)	≥ 64	≥ 32	≥ 32	23.6	24.4	25.9
Streptococcus pneumoniae(ATCC 49619)	≥ 64	≥ 32	≥ 32	16.2	15.7	11.7
Klebsilella pneumoniae(ATCC 700603)	-	-	-	17.4	17.4	18.6
Helicobacter pylori(ATCC 43504)	≥ 16	≥ 8	≥ 8	35.2	37.7	40.7
Bacterioides fragilis(ATCC 25285)	-	-	-	30.1	32	32.9

Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.

Preparation of injection

CB 12 100mg

Sodim methabifulfite 3.0mg

Methyl paraben 0.8mg

Propyl paraben 0.1mg

Distilled water for injection optimum amount

Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2 ml ample and sterilizing by conventional injection preparation method.

Preparation of powder

CB 11 500mg

Corn Starch 100mg

Lactose 100mg

Talc 10mg

Powder preparation was prepared by mixing above components and filling sealed package.

Preparation of tablet

CB 21 200mg

Corn Starch 100mg

Lactose 100mg

Magnesium stearate optimum amount

Tablet preparation was prepared by mixing above components and entabletting.

Preparation of capsule

CB 12 100mg

Lactose 50mg

Corn starch 50mg

Talc 2mg

Magnesium stearate optimum amount

Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.

Preparation of liquid

CB 11 1000mg

Sugar 20g

Polysaccharide 20g

Lemon flavor 20g

Liquid preparation was prepared by dissolving active component, and then filling all the components in 1000㎖ ample and sterilizing by conventional liquid preparation method.

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

As described in the present invention, the inventive compositions comprising the combination of cephalosporin selected from the group consisting of generation 1^st generation, 2^nd generation, 4^th generation and 5^th generation cephalosporin, specifically the 1^st generation or the 2^nd generation cephalosporin with beta-lactamase inhibitor, especially, clavulanate or sulbactam, showed synergistic antibacterial effect on the antibiotic resistant bacteria compared with the sole treatment, therefore, it can be used as the effective and safe therapeutics for treating and preventing bacterial infection caused by multi-drug resistance (MDR).

Claims

An antibiotic composition comprising cephalosporin selected from the group consisting of generation 1^st generation, 2^nd generation, 4^th generation and 5^th generation cephalosporin and beta-lactamase inhibitor as an active ingredient in amount effective to prevent or treat bacterial infection caused by multi-drug resistance (MDR) together with pharmaceutically acceptable carriers or diluents.
The antibiotic composition of claim 1, wherein said cephalosporin is the 1^st generation or 2^nd generation cephalosporin.
The antibiotic composition of claim 1, wherein said 1^st generation cephalosporin is selected from Cefacetrile (Cephacetrile), Cefadroxil (cefacroxyl, Duricef®), Cephalexin (Cefalexin, Keflex®), Cefaloglycin (Cephaloglycin), Cefalonium (Cephalonium), Cefaloridine (Cephaloridine), Cefalotin (Cephalothin, Keflin®), Cefapirin (Cephapirin, Cefadryl®), Cefatriazine, Cefazaflur, Cefazedone, Cefazolin (Cephazolin, Ancef®, Kefzol®), Cefradine (Cephradine, Velosef®), Cefroxadine or Ceftezole .
The antibiotic composition of claim 1, wherein said 2^nd generation cephalosporin is selected from Cefaclor (Ceclor, Distaclor, Keflor, Raniclor), Cefonicid (Monicid), Cefprozil (Cefproxil, Cefzil), Cefuroxime (Zefu, Zinnat®, Zinacef®, Ceftin, Biofuroksym), Cefuzonam, Cefmetazole, Cefotetan, Cefoxitin and the like, preferably, Cefaclor (Ceclor, Distaclor, Keflor, Raniclor), Cefuroxime (Zefu, Zinnat®, Zinacef®, Ceftin, Biofuroksym), or Cefmetazole.
The antibiotic composition of claim 1, wherein said 4^th generation cephalosporin is selected from Cefclinidine, Cefepime (Maxipime), Cefluprenam, Cefoselis, Cefozopran, Cefpirome (Cefrom), or Cefquinome.
The antibiotic composition of claim 1, wherein said 5^th generation cephalosporin is selected from Ceftobiprole or Ceftaroline.
The antibiotic composition of claim 1, wherein said beta-lactamase inhibitor is selected from clavulanic acid, sulbactam or tazobactam.
The antibiotic composition of claim 1, wherein said combined composition comprises (a) cephalosporin and (b) beta-lactamase inhibitor with the mixed ratio ranging from 1:10∼10:1 by weight (w/w).
The antibiotic composition of claim 1, wherein said multi-drug resistance (MDR) is selected from VRE (Vancomycin Resistant Enterococci), MRSA (Methicillin resistant Staphylococcus aureus), ESBL (Extended spectrum beta-lactamase) producing Gram-negative bacteria, KPC (Klebsiella pneumonia carbapenemase) producing negatives, Imipenem Resistant or Multidrug Resistant Organisms Acinobacter baumanii, Imipenem Resistant or Multidrug Resistant Organisms Klepsiella pneumonia.
The antibiotic composition of claim 1, wherein said bacterial infection caused by multi-drug resistance (MDR) is selected from otitis, pneumonia, laryngopharyngitis, tonsillitis, bronchitis, pyelonephritis, cystitis, gonococcal urethritis, furuncle, carbuncle, folliculitis, cellulitis, infectious arthrosclerosis, subcutaneous abscess, paronnychia, wound infection, sepsis, bronchitis, infectious bronchiectasis, secondary infection of bronchitis, lung abscess, empyema, cholangitis, cholecystitis, peritonitis, acute pyelonephritis, endometritis, salpingitis, pelveoperitonitis, parametritis, perimaxillary inflammation, maxillary inflammation, postoperative infection or non-gonococcal urethritis.
A use of a combined composition comprising cephalosporin selected from the group consisting of generation 1^st generation, 2^nd generation, 4^th generation and 5^th generation cephalosporin with beta-lactamase inhibitor for manufacture of medicines employed for treating or preventing a bacterial infection caused by multi-drug resistance (MDR).
A method of treating or preventing a bacterial infection caused by multi-drug resistance (MDR) in mammals, wherein the method comprises administering a therapeutically effective amount of a combined composition comprising cephalosporin selected from the group consisting of generation 1^st generation, 2^nd generation, 4^th generation and 5^th generation cephalosporin with beta-lactamase inhibitor into the mammal suffering with the bacterial infection caused by multi-drug resistance (MDR).