POTENTIATED ANTIBIOTIC COMPOSITION

Abstract: Disclosed are antibiotic compositions comprising an antibiotic and a potentiator of antimicrobial activity, including an antibiotic composition comprising an antibiotic selected from the group consisting of a beta -lactam antibiotic (e.g. methicillin) and a C11-C15 saturated or unsaturated, linear or branched aliphatic alcohol antibiotic and a potentiator of antimicrobial activity having the structure 2-hydroxy-6-R-benzoic acid, where R is a C10-C15 saturated or unsaturated aliphatic hydrocarbon group, (e.g. an anacardic acid), and an antibiotic composition comprising a polymyxin and a potentiator of antimicrobial activity selected from the group consisting of indole, a 3-substituted indole and an alkenyl aldehyde. The composition of the present invention is able to kill or inhibit the growth of microorganisms using much lower concentrations of the antibiotic. The specific potentiator used in the composition of the present invention also enhances the efficacy of antibiotics against both Gram-negative and Gram-positive bacteria, and also against fungi, such as yeasts.

Description: TITLE OF THE INVENTION POTENTIATED ANTIBIOTIC COMPOSITION BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a potentiated antibiotic composition, in particular to an antibiotic composition comprising an antibiotic and a specific potentiator of antimicrobial activity. Discussion of Related Art J_Q The development of antibiotic resistance has become a serious problem in medical microbiology. Newer generation β-lactam antibiotics, such as methi¬ cillin, were developed largely to overcome the resist¬ ance to earlier developed types of penicillins; such Tc resistance has spread rapidly among pathogenic bacte¬ ria. However, methicillin resistant bacterial strains are now frequently encountered, notably among the Staphylococci . Non-β-lactam antibiotics are often used for 2o treating particular types of infection. Polymyxins are often used for treating skin infections due to Gram- negative bacteria, such as Pseudomonas . However, the toxicity of these compounds restricts their parenteral use, and these compounds are usually administered 25 topically in ointments. Polymyxins are not known to be effective against microorganisms other than Gram- negative bacteria. Fungal infections generally are treated with quite different types of antibiotics. Notably, topical Candida infections are usually treated with imidazole derivatives (e.g., clotrimazole) or nystatin. SUMMARY OF THE INVENTION The present invention provides a potentiated antibiotic composition, especially an antibiotic compo- sition comprising an antibiotic which has an antimicro¬ bial (microbiocidal or microbiostatic) activity and a specific potentiator of antimicrobial activity (which is frequently referred to simply as "potentiator", hereinafter) . The antibiotic composition of the present invention combining an antibiotic and a poten¬ tiator is able to kill or inhibit the growth of mi¬ croorganisms using much lower concentrations of the antibiotic. The potentiator also enhances the efficacy of antibiotics against antibiotic-resistant strains of microorganisms . Accordingly, it is an object of the present inven¬ tion to provide an antibiotic composition which is capable of killing or inhibiting the growth of microor¬ ganisms using much lower concentrations of the antibi- otic. It is another object of the present invention to provide an antibiotic composition which exhibits im¬ proved antimicrobial activity. It is a further object of the present invention to provide an antibiotic composition of the character described above, which can be easily manufactured at a reasonable cost. DETAILED DESCRIPTION OF THE INVENTION The present invention provides an antibiotic composition comprising an antibiotic and a potentiator of antimicrobial activity. More particularly, in one aspect of the present invention, there is provided an antibiotic composition comprising: an antibiotic selected from the group consisting of a β-lactam antibiotic and a C^η . ~ ^15 saturated or unsaturated, linear or branched aliphatic alcohol antibiotic; and a potentiator of antimicrobial activity having the structure 2-hydroxy-6-R-benzoic acid, where R is a CJ_Q - Cic saturated or unsaturated aliphatic hydrocarbon group. In another aspect of the present invention, there is provided an antibiotic composition comprising a polymyxin and a potentiator of antimicrobial activity selected from the group consisting of indole, a 3- substituted indole and an alkenyl aldehyde. The compositions of the present invention are useful in suppressing the growth or infectivity of microorganisms which are resistant to the antibiotic in the absence of potentiators . With respect to the antibiotic to be used in the present invention, a β-lactam antibiotic, such as methicillin, a C^ - C-^ saturated or unsaturated, linear or branched aliphatic alcohol antibiotic, or a polymyxin can be used. Examples of C^ - C-^ saturated or unsaturated, linear or branched aliphatic alcohol antibiotics in¬ clude n-alcohols and 2-alcohols . With re- spect to the C^ - C-^ saturated or unsaturated ali¬ phatic 2-alcohols, those which have 11 to 13 carbon atoms in the longest chain from the hydroxyl group of the 2-alcohols are preferred. Specific preferred examples of aliphatic alcohols include n-undecanol, n-dodecanol, n-tridecanol, farne- sol, 2-dodecanol, 2-tridecanol and 2-tetradecanol . The aliphatic alcohols mentioned above have moder¬ ate to weak antibiotic activity by themselves, and may be combined with a potentiator in the compositions of the present invention. The polymyxins, including polymyxin B and polymyx¬ in E, are polypeptide antibiotics. On the other hand, with respect to the potentiator to be used in the present invention, an anacardic acid or a derivative thereof, indole or a 3-substituted indole, or an alkenyl aldehyde can be used. As used herein, the term "anacardic acid" refers to a 2-hydroxy-6-R-benzoic acid, wherein R is a C-^ Q - saturated or unsaturated aliphatic hydrocarbon group. Anacardic acids of particular interest include those wherein R is a 8( Z) ,-11(Z) ,-14-pentadecatrienyl chain; a 8(Z ) ,-11 ( Z )-pentadecyldienyl chain; a 8(Z)- pentadecylenyl chain; a dodecyl chain; and a decyl chain. These compounds may be, respectively, denoted the (15:3), (15:2), (15:1), (12:0), and (10:0) anacar¬ dic acids . The so-called "anacardic acid", which naturally occurs, particularly in the nut shell oil of the cash¬ ew, Anacardlum occldentale L. (Anacardiaceae) , is a mixture of (15:3), (15:2), (15:1) and (15:0) anacardic acids . The natural anacardic acids (15:3), (15:2) and (15:1) to be used in the present invention may be obtained conveniently by isolation from the nut shell oil of the cashew. Purification may be effected by medium pressure liquid chromatography, as previously described [Kubo et al . , J. Agric. Food Chem. (1986) .34.: 970-973; Bagchi et al . , Planta Med. (1985) 467]; fur¬ ther purification may be performed by high performance liquid chromatography (HPLC) [Kubo et al., Chem. Let¬ ters (1987) 1101-1104; Kubo and Nakatsu, LC-GC (1991) 8 . : 933-939]. The non-natural anacardic acid derivatives (12:0) and (10:0) may be synthesized as described in litera- ture [Kubo et al . , J. Agric. Food Chem. (1993) 4J.: 1016-1019] . Anacardic acid is an effective potentiator in combination with a β-lactam antibiotic, such as methi¬ cillin, or in combination with a C^ - C-^ saturated or unsaturated, linear or branched aliphatic alcohol antibiotic, to provide an antibiotic composition inhib¬ iting methicillin-resistant Staphylococcus aureus . Indole and 3-substituted indoles are effective potentiators in combination with a polymyxin antibiot- ic . Preferred examples of 3-substituted indoles in¬ clude 3-alkyl indoles, especially 3-methylindole (skatole) and 3-ethylindole, and also include 3-carbox- ylic indole. The N-H ring group preferably is left intact. An alkenyl aldehyde is also an effective potentiator in combination with a polymyxin antibiotic . Especially preferred is trans-2-hexenal . The potentiators by themselves usually exhibit weak antibiotic activity. However, the compositions comprised of a combination of antibiotic and potentia¬ tor have synergistic antibiotic activities which enable use of reduced amounts of the antibiotic component. In the present invention, it is preferred to use an antibiotic and a potentiator individually having a purity of pharmaceutically acceptable grade. In the antibiotic composition of the present invention, with respect to the ratio of the antibiotic to the potentiator, there is no specific limitation as long as the synergistic effect between the antibiotic and the potentiator can be exerted to allow use of a reduced amount of the antibiotic. However, the weight ratio of the antibiotic to the potentiator is usually 1 : 10000 to 100 : 1, preferably 1 : 5000 to 10 : 1, more preferably 1 : 3000 to 1 : 1. The compositions of the present invention are effective against both Gram-negative and Gram-positive bacteria, and also against fungi, such as yeasts. In this connection, it is noted that a composition com¬ prising a combination of a β-lactam antibiotic such as methicillin, or a C^ - C-^ saturated or unsaturated, linear or branched aliphatic alcohol antibiotic, with an anacardic acid potentiator, is especially effective against Gram-positive bacteria, such as methicillin resistant Staphylococcus aureus (MRSA) . Particularly striking is the ability of the present compositions, comprising an antibiotic and a potentiator, to extend the range of antimicrobial effectiveness against mi¬ croorganisms previously considered unreactive towards the antimicrobial compound. For example, antibiotic activities of polymyxins have been considered to be restricted to Gram-negative bacteria, such as E. coli and Pseudomonas aeruginosa . However, compositions of the present invention extend the antibiotic effect of polymyxins to Gram-positive bacteria such as Staphylo- coccus aureas, and to fungi, including yeasts such as Candida albicans . A particular advantage of these compositions is their reduced toxicity. Because of the synergy between the antibiotic and potentiator components, the amount of antibiotic required is reduced. Since the antibiot¬ ics are toxic both to humans and the environment, the reduced use of antibiotics is highly desirable. Formulations The antibiotic composition of the present inven- tion comprising an antibiotic and a potentiator may be used to treat in vi vo infections, or to disinfect topical surfaces, such as intact skin, and nonliving surfaces, such as bandages, bodily appliances, cathe¬ ters, surgical instruments, patient examination tables, etc. For treatment of in vi vo infections, the composi¬ tions may be administered either internally or exter¬ nally. For external administration, the compositions may be used to treat infections of the skin or mucosal surfaces, infected cuts, burns, or abrasions, bacterial skin infections, or fungal infections (e.g., athlete's foot). For internal administration, the compositions are useful for treating systemic bacterial infections, especially Staphylococcus infections . The compositions may also be administered internally by topical adminis¬ tration to mucosal surfaces, such as vaginal mucosa, for treatment of infections, particularly yeast infec¬ tion. That is, examples of applications of the composi- tions of the present invention include: topical disinfectants for sterilization of sur¬ faces, such as countertops, surgical instruments, bandages, and intact skin; pharmaceutical compositions for external applica- tion to skin and mucosal surfaces, including the cor- nea, dermal cuts and abrasions, burns, and sites of bacterial or fungal infection; pharmaceutical compositions for administration to internal mucosal surfaces, such as the oral cavity or vagina to inhibit the growth of bacteria or fungi, including yeasts; pharmaceutical compositions for coating indwelling catheters and similar implants which are susceptible to harboring bacterial or fungal infection; and pharmaceutical compositions for treatment of systemic bacterial infections, especially Staphylococ- cus infections . The topical disinfectant and pharmaceutical compo¬ sition of the present invention can be in various forms, such as a tablet, powder, granule, syrup, sus¬ pension, capsule, liposome dispersion and reagent for injection. Depending on the form of the disinfectant or medicine, a suitable type of a carrier or an addi¬ tive to be used for the composition of the present invention is selected. Examples of carriers to be used for a medicine for oral administration include excipi¬ ents, such as lactose, refined sugar, glucose, starch and crystalline cellulose; binders, such as hydroxypro- pyl cellulose, carboxymethyl cellulose, starch, gum arabic, gelatin, glucose, refined sugar, tragacanth and sodium alginate; disintegrators, such as carboxymethyl cellulose, starch and calcium carbonate; lubricants, such as stearic acid, refined tare, sucrose fatty acid ester, hydrated vegetable oil, magnesium stearate and calcium stearate; surfactants, such as sodium lauryl sulfate, soybean lecithine, sucrose fatty acid ester and polysorbate 80; additives such as lecithine, soy¬ bean oil and glycerin; a flowability agent; and a coloring agent. When the composition of the present invention is formulated into an inhalant, polychloromo- nofluoromethane or the like may be used as a solvent. If desired, other additives, such as a germicide, an antiseptics, a stabilizer, an isotonizing agent and a soothing agent, can be used in combination with the diluent. When the composition of the present invention is formulated into a medicine for parenteral adminis¬ tration (e.g., a reagent for injection), distilled water for injection, physiological saline, an aqueous solution of glucose, a vegetable oil for injection, propylene glycol and polyethylene glycol and the like can be used as a diluent. For example, compositions for in vivo administra¬ tion may be provided as solutions, especially aqueous solutions, but optionally may include alcoholic solu- tions to increase the solubility of hydrophobic compo- nents . Such solutions are convenient for oral adminis¬ tration but may be formulated for parenteral adminis¬ tration. For oral administration, ethanol is preferred because of its low toxicity. Usually ethanol is used in the minimum concentration needed to keep the compo¬ nents in solution. For external topical application, isopropanol may be used also. Other formulations for oral administration may include solid dosage forms such as tablets or capsules . Compositions preferred for topical administration may be provided as emulsions, creams, or liposome dispersions, or as an ointment in a hydrophobic carrier such as petrolatum. For topical disinfection of nonliving surfaces, compositions may be provided as solutions, either aqueous or organic. Where direct human contact with the disinfectant is to be limited, such as in disinfec¬ tion of work surfaces or restrooms, mixed organic solutions may be appropriate, e.g., ethanol or isoprop¬ anol in water. Preferred alcohols for solvent purposes include ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and t-butyl alcohols. Concentration of the alcohol in a mixed solvent system may range from 5 % to essential¬ ly 100 %. Usually the cosolvent is water or an aqueous buffer. In most cases, the alcohol component is used in an amount necessary to keep the antibiotic and potentiator in solution. Anacardic acid, C^ - C-^ aliphatic alcohols and polymyxins have limited solubilities in aqueous solu¬ tion. Compositions comprising any of these compounds may optionally be formulated with a lipophilic phase, as in emulsions and liposome dispersions . For external application to intact skin or for disinfection of nonliving surfaces, an organic solvent or cosolvent, such as ethanol or propanol may be employed. Evaporation of the solvent leaves a residue of the antibiotic and potentiator on the treated surface to inhibit reinfection. Particular formulations may be manufactured ac¬ cording to methods well known in the art. Formulations are given in, for example, Remington ' s Pharmaceutical Sciences and similar reference works . The dose of the pharmaceutical composition of the present invention is varied depending on the age, weight, condition, etc. of the patient. However, the dose may generally be in the range of from 1 μg to 100 g per day for an adult. The daily dose may be administered at one time, or it may also be divided into 2 or 3 portions and these portions are administered at intervals. The administration is generally continued for a period of from several days to 2 months. The daily dose and the administration period are varied to some extent, depending on the condition of the patient. The antimicrobial activity of the composition of the present invention is demonstrated by test data showing the minimum inhibitory concentrations (MIC) and minimum biocidal concentrations (MBC) of compositions against various pathogenic organisms cultured in vi tro under standard conditions. These in vi tro tests strongly correlate with in vi vo activity, as is evi¬ denced by the widespread use of the MIC and MBC deter¬ minations to predict utility of antibiotic compositions in treatment of infection in animals, including humans. BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail with reference to the following Examples . In Examples, the synergistic effect of an antibi¬ otic with a potentiator in antimicrobial activity was evaluated by determining the ratio of the MIC and MBC exhibited by the use of the individual antibiotic alone to the MIC and MBC exhibited by the use of the antibi¬ otic combined with 1/2 MIC of the potentiator. The test microorganisms to be used and the methods for determination of MIC and MBC are as follows. Experimental Procedures: (1) Test Microorganisms The below-mentioned four types of microorganisms to be tested were strains supplied by the ATCC. Staphylococcuε aureus is a clinically significant member of the Gram-positive group of bacterial patho¬ gens. It gives rise to serious infections, and may produce bacteremia, endocarditis, and meningitis. Methicillin-resistant strains of Staphylococcus aureus (MRSA) were chosen for evaluation because they are a significant medical problem, in view of the fact that methicillin is the drug of choice for treatment of S. aureus infection in the common penicillin-resistant strains . Pseudomonas aeruginosa is a Gram-negative patho- genic bacteium which may cause urinary tract infections or pneumonia. Escherichia coli is also a Gram-negative pathogenic bacterium which may cause infections of urinary or genital organs, or infantile diarrhea. Candida albicanε is a eukaryotic fungus rather than a bacterium, and consequently would be expected to exhibit very different antibiotic susceptibilities from bacteria. In particular, C. albicans is a yeast, and is responsible for vaginal and oral thrush, and for skin and lung infections. (2) Methods of Testing With respect to each of an antibiotic alone, a potentiator alone and the antibiotic combined with the potentiator, MIC and MBC were determined according to the following procedures. (i) MIC Determination MIC was determined by the broth dilution method as described in J. Agric . Food Chem., 41, 1780-1783 (1993) . That is, a test compound was dissolved in N,N- dimethylformamide (DMF) (except methicillin, which was dissolved in sterile distilled water) to make stock solution at concentrations of 80, 40, 20, or 10 mg/ml depending upon the potency and solubility. An aliquot of 0.01 ml of the stock solution was added to a tube containing 0.99 ml of a liquid medium [Nutrient broth (GIBCO, U.S.A.) for MRSA, E. coli and P. aeruginosa ; Reinforced clostridial medium (Oxoid, England) for C. albicans] to get the initial concentration of testing solution. Serial two-fold dilutions were made by mixing 0.5 ml of the testing solution with 0.5 ml of blank medium (which was same as the above-mentioned liquid medium). A 0.5 ml aliquot of diluted inoculum containing an appropriate concentration of the test microorganism (1:100 dilution of the initial inoculum described below; which had been diluted with the liquid medium) was then added into each tube containing 0.5 ml serial dilutions of the test compound. After incubat¬ ing for 2 days at 37 °C, the tube was evaluated for visible growth. The MIC was determined as the lowest concentration of a compound which prevented visible microorganism growth. A culture growth control without compound (solvent only) and several culture sensitive reference agents [gentamycin for MRSA and P. aerugino- sa ; ampicillin for E. coli ; and amphotericin B for C. albicans] were used as positive controls. The assays were performed in duplicate. In the MIC determination mentioned above, the initial population was 1 x 10 colony forming units (CFU) per ml for E. coli ; 1 x 10 6 CFU per ml for P. aeruginosa ; 2 x 10° CFU per ml for methicillin-resist- ant Staphylococcuε aureus (MRSA) ; and 5 x 10 CFU per ml for Candida alubicanε . (ii) MBC Determination MBC was also determined according to the method as described in J. Agric. Food Chem., 41, 1780-1783 (1993) . That is, after determining the MIC, 0.1 ml of a ten-fold dilution from each of the tubes showing no turbidity (these tubes were those which had been ob- tained and confirmed with respect to the prevention of visible microorganism growth in the above MIC determi¬ nation test), wherein the ten-fold dilution was made using the liquid medium described above, was plated onto a chemical free nutrient agar plate [Bacto-Agar (Difco Laboratories, U.S.A.) for MBC determination]. After 24 hours of incubation at 37 °C, the colonies were counted. The MBC was the lowest concentration of the test compound that decreased the initial inoculum population of the test microorganism by >99.9 percent. (iii) Synergistic Effect Studies Synergistic effects of a putative potentiating substance in combination with an antibiotic reference compound were evaluated by a broth checkerboard method. Serial two-fold dilutions of the antibiotic reference compound in combination with a concentration of 1/2 MIC of the putative potentiating substance were made by the above described tube dilution method for determination of MICs and MBCs in each testing strain. Since the potentiator is present at 1/2 of its MIC, the MIC determined for the antibiotic should be 1/2 of its usual value, if the effects of the two compounds are merely additive; greater than 1/2, if the compounds are antagonistic; and less than 1/2, if the compounds are synergistic. The "synergistic effect" shown in the following Tables is the ratio of the MIC exhibited by the use of the antibiotic alone to the MIC exhibited by the use of the antibiotic in the presence of 1/2 MIC of the potentiator. A 2x synergistic effect means that the activities of the antibiotic and poten¬ tiator are merely additive, whereas an effect greater than 2x indicates the occurrence of true synergy. Example 1: Synergistic Effect of Methicillin Combined with Anacardic Acid on Methicillin- resistant Staphylococcus aureus Tests were performed to determine the synergistic effect of methicillin combined with anacardic acid (15:3) on methicillin-resistant Staphylococcus aureus (MRSA; ATCC 33591). That is, MICs of methicillin alone, anacardic acid (15:3) alone, and methicillin in combination with 1/2 MIC of anacardic acid (15:3) against MRSA (ATCC 33591) were determined according to the procedures described in items (i) and (iii) above. Results are given in Table 1. In Table 1, the expression "Anacardic Acid (1/2 MIC) + Methicillin" is defined to mean methicillin in combination with 1/2 MIC of anacardic acid (15:3). This definition also applies to Tables 2 to 7 below. Table 1 Synergistic Testing Strain MIC (μ-g/ml) Effect Methicillin Anacardic Anacardic Acid Alone Acid (15:3) (1/2 MIC) (15:3) Alone +Methicillin Methicillin- 800 6.25 25 32x resistant Staphyl ococcus aureus tATCC 33591) Example 2 : Synergistic Effect of Polymyxin Antibiotic Combined with Indole or 2-Hexenal (1) Synergistic Effect of Polymyxin B Combined with Indole or 2-Hexenal on Gram-negative Bacteria Tests were performed to determine the synergistic effect of Polymyxin B combined with indole or 2-hexenal on Gram-negative bacteria of the strains Escherichia coli (ATCC 10536) and Pseudo onas aeruginosa (ATCC 10145) . That is, MICs and MBCs of polymyxin B alone, indole alone and polymyxin B in combination with 1/2 MIC of indole against E. coli (ATCC 10536) and P. aeruginosa (ATCC 10145) were determined according to the procedures described in items (i) to (iii) above. Results are given in Table 2. Table Synergistic Testing Strain MIC and MBC (μg/ml) Effect Indole Polymyxin B Indole (1/2 MIC) Alone Alone +Polymyxin B MIC MBC MIC MBC MIC MBC MIC MBC Escherichia coli 3.13 3.13 800 800 0.20 0.39 16x 8x (ATCC 10536) Synergistic Testing Strain MIC and MBC (μg/ml) Effect 2-Hexenal Polymyxin B 2-Hexenal (1/2 MIC) Alone Alone +Polymyxin B MIC MBC MIC MBC MIC MBC MIC MBC Escheri chia coli 3.13 3.13 400 400 0.39 0.39 8x 8x (ATCC 10536) Synergistic Testing Strain MIC and MBC (μg/ml) Effect Indole Polymyxin B Indole (1/2 MIC) Alone Alone +Polymyxin MIC MBC MIC MBC MIC MBC MIC MBC Pseudomonas 6.25 6.25 400 800 0.39 0.78 16x 8x aeruginosa (ATCC 10145) Synergistic Testing Strain MIC and MBC (μg/ml) Effect 2-Hexenal Polymyxin B 2-Hexenal (1/2 MIC) Alone Alone +Polymyxin MIC MBC MIC MBC MIC MBC MIC MBC Pseudomonas 6.25 6.25 200 400 0.78 0.78 8x 8x aeruginosa (ATCC 10145) (2) Synergistic Effect of Polymyxin B Combined with Indole or 2-Hexenal on Gram-Positive Bacteria Tests were performed to determine the synergistic effect of Polymyxin B combined with indole or 2-hexenal on Gram-positive bacteria of a methicillin and polymyx¬ in B-resistant strain of Staphylococcus a ureus (ATCC 33591) . That is, MICs and MBCs of polymyxin B alone, indole alone or 2-hexenal alone and polymyxin B in combination with 1/2 MIC of indole or 2-hexenal against MRSA (ATCC 33591) were determined according to the procedures described in items (i) to (iii) above. Results are given in Table 3. Table 3 Synergistic Testing Strain MIC and MBC (μg/ml) Effect Indole Polymyxin B Indole (1/2 MIC) Alone Alone +Polymyxin B MIC MBC MIC MBC MIC MBC MIC MBC Methicillin- 800 800 800 800 100 100 8x 8x resistant Staphylococcus aureus (ATCC 33591) Synergistic Testing Strain MIC and MBC (μg/ml) Effect 2-Hexenal Polymyxin B 2-Hexenal (1/2 MIC) Alone Alone +Polymyxin B MIC MBC MIC MBC MIC MBC MIC MBC Methicillin- 800 800 400 800 25 50 32x 16x resistant Staphylococcus aureus (ATCC 33591) (3) Synergistic Effect of Polymyxin B Combined with Indole on Candida albicans Tests were performed to determine the synergistic effect of polymyxin B combined with indole on yeast fungi of particular of Candida albicans (ATCC 10231). That is, MICs and MBCs of polymyxin B alone, indole alone and polymyxin B in combination with 1/2 MIC of indole against C. albicans (ATCC 10231) were determined according to the procedures described in items (i) to (iii) above. Results are given in Table 4. Table 4 Synergistic Testing Strain MIC and MBC (μg/ml) Effect Indole Polymyxin B Indole (1/2 MIC) Alone Alone +Polymyxin B MIC MBC MIC MBC MIC MBC MIC MBC Candida albicans 800 1600 400 800 200 400 4x 4x (ATCC 10231) (4) Synergistic Effect of Polymyxin E Combined with Indole on Gram-negative Bacteria Tests were performed to determine the synergistic effect of polymyxin E combined with indole on Gram- negative bacteria of the strains Escherichia coli (ATCC 10536) and Pseudomonas aeruginosa (ATCC 10145). That is, MICs and MBCs of polymyxin E alone, indole alone and polymyxin E in combination with 1/2 MIC of indole against E. coli (ATCC 10536) and P. aeruginosa (ATCC 10145) were determined according to the procedures described in items (i) to (iii) above. Results are given in Table 5. Table 5 Synergistic Testing Strain MIC and MBC (μg/ml) Effect Indole Polymyxin E Indole (1/2 MIC) Alone Alone +Polymyxin E MIC MBC MIC MBC MIC MBC MIC MBC Escheri chia coli 3.13 6.25 800 800 0.78 0.78 4x 8x (ATCC 10536) Synergistic Testing Strain MIC and MBC (μg/ml) Effect Indole Polymyxin E Indole (1/2 MIC) Alone Alone +Polymyxin E MIC MBC MIC MBC MIC MBC MIC MBC Pseudomonas 6.25 12.5 400 800 0.39 0.78 16x 16x aeruginosa (ATCC 10145) (5) Synergistic Effect of Polymyxin E Combined with Indole on Yeast Tests were performed to determine the synergistic effect of polymyxin E combined with indole on yeast Candida albicans (ATCC 10231). That is, MICs and MBCs of polymyxin E alone, indole alone and polymyxin E in combination with 1/2 MIC of indole against C. albicans (ATCC 10231) were determined according to the procedures described in items (i) to (iii) above. Results are given in Table 6. Table 6 Synergistic Testing Strain MIC and MBC (μg/ml) Effect Indole Polymyxin E Indole (1/2 MIC) Alone Alone +Polymyxin E MIC MBC MIC MBC MIC MBC MIC MBC Candida albicans 1600 1600 400 800 100 200 >16x >8x (ATCC 10231) Polymyxin antibiotics have been known to be re¬ stricted in activity to Gram-negative bacteria, such as E. coli and Pseudomonas aeruginosa . However, as a result of incorporation of indole or 2-hexenal potenti¬ ator into compositions of the invention, it was found that the range of activity of polymyxins was remarkably extended to include not only Gram-positive bacteria such as Staphylococcus aureus, but even fungi as well. Notably, the composition comprising a polymyxin and indole was effective against yeast, including Candida . Example 3: Synergistic Effect of C-^ - C- j r Alcohols Combined with Anacardic Acid on Methi- cillin-resistant Staphylococcus aureus Tests were performed to determine a synergistic effect of n-alcohols (undecanol, dodecanol, tridecanol) combined with anacardic acid (15:3) against methicillin-resistant Staphylococcus aureus (ATCC 33591) . That is, MICs of undecanol, dodecanol or trideca¬ nol alone, anacardic acid (15:3), and undecanol, dodec¬ anol or tridecanol in combination with 1/2 MIC of anacardic acid (15:3) against MRSA (ATCC 33591) were determined according to the procedures described in items (i) and (iii) above. Results are given in Table 7. Table 7 Synergistic Testing Strain MIC (μg/ml) Effect Undecanol Anacardic Anacardic Alone Acid (15:3) Acid (15:3) Alone (1/2 MIC) +Undecanol Methicillin- 50 6.25 12.5 4x resistant Staphylococcus aureus (ATCC 33591) Synergistic MIC (μg/ml) Effect Dodecanol Anacardic Anacardic Alone Acid (15:3) Acid (15:3) Alone (1/2 MIC) +Dodecanol Methicillin- 80 6.25 10 8x resistant Staphylococcus aureus (ATCC 33591) Synergistic MIC (μg/ml) Effect Tridecanol Anacardic Anacardic Alone Acid (15:3) Acid (15:3) Alone (1/2 MIC) +Tridecanol Methicillin- 50 6.25 25 2x resistant Staphylococcus aureus (ATCC 33591) Industrial applicability As described above, the composition of the present invention combining an antibiotic and a specific poten¬ tiator is able to kill or inhibit the growth of mi- croorganisms using much lower concentrations of the antibiotic. In addition, by virtue of the specific potentiator contained in the composition, the efficacy of antibiotics against antibiotic-resistant strains of microorganisms is also enhanced. The compositions of the present invention are effective against both Gram-negative and Gram-positive bacteria, and also against fungi such as yeasts. It is noted that a composition comprising a combination of a β-lactam antibiotic such as methicillin, or a C-^ - C^ j saturated or unsaturated, linear or branched aliphatic alcohol antibiotic, with an anacardic acid potentiator, is especially effective against Gram-positive bacteria such as methicillin resistant Staphylococcus aureus (MRSA) . Particularly striking is the ability of the present compositions, comprising an antibiotic and a potentiator, to extend the range of antimicrobial effectiveness against microorganisms previously consid¬ ered unreactive towards the antimicrobial compound. A particular advantage of these compositions is their reduced toxicity. Because of the synergy between the antibiotic and potentiator components, the amount of antibiotic required is reduced. Since the antibiot¬ ics are toxic both to humans and the environment, the reduced use of antibiotics is highly desirable. Therefore, the compositions of the present inven¬ tion are useful as: topical disinfectants for sterilization of sur¬ faces such as countertops, surgical instruments, band¬ ages, and intact skin; pharmaceutical compositions for external applica¬ tion to skin and mucosal surfaces, including the cor¬ nea, dermal cuts and abrasions, burns, and sites of bacterial or fungal infection; pharmaceutical compositions for administration to internal mucosal surfaces such as the oral cavity or vagina to inhibit the growth of bacteria or fungi, including yeasts; pharmaceutical compositions for coating indwelling catheters and similar implants which are susceptible to harboring bacterial or fungal infection; and pharmaceutical compositions for treatment of systemic bacterial infections, especially Staphylococ¬ cus infections .

Claims: CLAIMS 1. An antibiotic composition comprising: an antibiotic selected from the group consisting of a β-lactam antibiotic and a C^ - C j c saturated or unsaturated, linear or branched aliphatic alcohol antibiotic; and a potentiator of antimicrobial activity having the structure 2-hydroxy-6-R-benzoic acid, where R is a C^Q - C- ] ,^ saturated or unsaturated aliphatic hydrocarbon group. 2. The antibiotic composition of claim 1, wherein said antibiotic is a β-lactam antibiotic. 3. The antibiotic composition of claim 2, wherein said β-lactam antibiotic is methicillin. 4. The antibiotic composition of claim 1, wherein said antibiotic is a C-^ - C 15 saturated or unsaturat¬ ed, linear or branched aliphatic alcohol antibiotic. 5. The antibiotic composition of claim 4, wherein said C11 - Cic saturated or unsaturated, linear or branched aliphatic alcohol antibiotic is a - C-^ j n-alcohol antibiotic. 6. The antibiotic composition of claim 4, wherein said C- ] ^ - C-^ saturated or unsaturated, linear or branched aliphatic alcohol antibiotic is a C- - -^ saturated or unsaturated aliphatic 2-alcohol antibiotic having 11 to 13 carbon atoms in the longest chain from the hydroxyl group of the 2-alcohol antibiotic. 7 . The antibiotic composition of any of claims 1 to 6, wherein R is an 8(Z), 11 (Z), 14-pentadecatrienyl group. 8. The antibiotic composition of any of claims 1 to 6, wherein R is an 8(Z), 11(Z )-pentadecadienyl group. 9. The antibiotic composition of any of claims 1 to 6, wherein R is an 8( Z)-pentadecenyl group. 10. The antibiotic composition of any of claims 1 to 6, wherein R is a dodecyl group. 11. The antibiotic composition of any of claims 1 to 6, wherein R is a decyl group. 12. An antibiotic composition comprising: a polymyxin; and a potentiator of antimicrobial activity selected from the group consisting of indole, a 3-substituted indole and an alkenyl aldehyde. 13. The antibiotic composition of claim 12, wherein said 3-substituted indole is a 3-alkyl indole. 14. The antibiotic composition of claim 13, wherein said alkyl indole is 3-methylindole. 15. The antibiotic composition of claim 12, wherein said 3-substituted indole is 3-carboxylic indole. 16. The antibiotic composition of claim 12, wherein said alkenyl aldehyde is trans-2-hexenal .

Inventors: KUBO ISAO, SUKIGARA MASAYUKI, KUBO, ISAO, SUKIGARA, MASAYUKI
Applicants: ASAHI CHEMICAL IND, ASAHI KASEI KOGYO KABUSHIKI KAISHA
Application year: 1995
Main classification: A61K31
IPC codes: A61K31, A61K38, A61P31, A61P43