Composition and method of potentiating antibiotics with polyphenols

Abstract: A method of increasing the activity of an antibiotic against methicillin resistant Staphylococcus aureus in a patient by administering to the patient the antibiotic in combination with a catechin of the formula (I) and/or a theaflavin of the formula (II).

Description: FIELD OF THE INVENTION The present invention relates to compositions displaying a strengthening of the antibacterial action of antibiotics. More specifically, the present invention relates to compositions with stronger antibacterial action of antibiotics against methicillin resistant Staphylococcus aureus (abbreviated as MRSA from hereon) by a combined use of the antibiotics with tea catechin and/or theaflavin. BACKGROUND INFORMATION With the popularization and rapid development of medical treatments, various kinds of antibiotics are being used in the medical world. However, bacteria resistant to these antibiotics have emerged, and MRSA is representative of such. MRSA shows resistancy to not only methicillin but to most medications and since up until now there has been no conclusively effective treatment, arbekacin or vancomycin is at present being used in combination with other medications. However, this kind of combination treatment requires the intake of many kinds of medications in large amounts, and thus presents problems such as the increased possibility of detrimental side-effects. So with the increase in "compromised hosts" a method of controlling MRSA is urgently required. The present inventors have shown and reported that tea extractions and tea catechins in clinical use show an antibacterial action against MRSA isolate (Jpn. J. Bacteriol., 46, 839-845, 1991). For example, administration of 250mg/ml of epigallocatechin gallate showed an antibacterial action against MRSA, and within 24 hours 10 4 cells/ml of MRSA were destroyed. We have also reported that tea catechins combined with exotoxin, for example α-toxin of Staphyloccus aureus , and rendered them inactive (Jpn. J. Bacteriol.,45, 561-566, 1990; Jpn. J. Bacteriol.,45, 913-919, 1990.). Further, a method of preventing the transmission of infection caused by MRSA with the use of tea polyphenols is disclosed in USPS, 358, 713. Other investigations have shown, that the combination of an extract of tea or an active fraction isolated thereof, with a β-lactam antibiotic leads to a synergistic activity against MRSA (WO95/23607). The active fraction in tea could be shown to be the fraction containing the tea polyphenol compounds which prevented the infection with an antibiotic-resistant staphylococcus (EP-A-0 443 090). SUMMARY OF THE INVENTION Compositions displaying an increased activity of an antibiotic against methicillin resistant Staphylococcus aureus compositions comparising an effective antibiotic amount of said antibiotic in combination with a synergistic amount of at least one polyphenol selected from the group consisting of a catechin of the formula (I) and/or a theaflavin of the formula (II). BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the synergistic effect of tea catechins (Polyphenon 100) with amikacin against MRSA F-51. Figure 2A shows the synergistic effect of tea catechins (Polyphenon 100) with tetracycline against MRSA F-51. Figure 2B shows the synergistic effect of tea catechins (Polyphenon 100) with chloramphenicol against MRSA F-51. Figure 2C shows the synergistic effect of tea catechins (Polyphenon 100) with gentamicin against MRSA F-51. Figure 3A shows the ineffectiveness of tea catechins ((-)-epigallocatechin gallate) with kanamycin against MRSA F-51. Figure 3B shows the ineffectiveness of tea catechins ((-)((-)-epigallocatechin gallate) with erythromycin against MRSA F-51. Figure 3C shows the ineffectiveness of tea catechins ((-)-epigallocatechin gallate) with climdamycin against MRSA F-51. Figure 3D shows the ineffectiveness of tea catechins ((-)-epigallocatechin gallate) with colistin against MRSA F-51. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The influence of tea catechins on the antibacterial action of each kind of antibiotic to which MRSA showed resistancy was measured using the cup method. Results showed that when 100 µ g/ml of tea catechin were combined with specific antibiotics, i. e. tetracycline (2.5 µ g/ml) or chloramphenicol (12.5 µ g/ml), these antibiotics had an antibacterial action on MRSA, and thus the effectiveness of tea catechins when used in combination with antibiotics was confirmed. According to these investigations it was found that when antibiotics which showed no antibacterial action on MRSA when used independently, were used in conjunction with tea catechins and/or theaflavins in a concentration less than the MIC, there was an apparent antibacterial action on MRSA. On the basis of these discoveries the present inventors developed and completed compositions which displayed an increased antibacterial action of antibiotics against MRSA. Thus the present invention relates to compositions with increased antibacterial action of antibiotic against MRSA in a patient in need thereof, comprising providing as a medicament an effective antibiotic amount of said antibiotic in combination with a synergistic amount of at least one polyphenol selected from the group consisting of a catechin of the following formula (I) and/or a theaflavin of the following formula (II). The antibiotics used in the present invention showed no antibacterial action against MRSA when used independently. Antibiotics such as these included the aminoglycoside type etc., and more specifically, amikacin, tetracycline, chloramphenicol, gentamicin and the like are illustrated as such antibiotics. In the present invention, the antibiotics are used in such a small amount that they would not be effective against MRSA if used independently. Usually, 2 to 35 µ g/ml, preferably 2 to 12 µ g/ml of the antibiotics is used in the method of the present invention. The tea catechin used in the present invention is represented by structural formula (I) as shown below. (wherein R 1 represents H or OH and R 2 represents H or Tea catechins may be for example, (-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin, (-)-epigallocatechin gallate, (-)-gallocatechin etc. (inclusive of isomers thereof) and some of these catechins can be used singly or a combination of two or more compounds may be used. Among these catechins, compounds containing (-)-epicatechin gallate and/or (-)-epigallocatechin gallate are preferable. In particular, tea catechin which contains (-)-epigallocatechin gallate as its main component is desirable; for example, "Polyphenon 100"™ (product of Mitsui Norin Co. Ltd., composition: (+)-gallocatechin 1.44%, (-)-epicatechin 5.81%, (-)-epigallocatechin 17.57%, (-)-epicatechin gallate 12.51%, (-)-epigallocatechin gallate 53.9%) or "Polyphenon E"™ (product of Mitsui Norin Co. Ltd., composition: (-)-epicatechin 10.8%, (-)-epigallocatechin 9.2%, (-)-epicatechin gallate 6.5%, (-)-epigallocatechin gallate 54.8%, (-)-gallocatechin gallate 4.0%) are preferable. The MIC of tea catechin against MRSA is 125-250 µ g/ml, and in the process of the present invention, tea catechin is used in a concentration less than the MIC. Theaflavin used in the present invention is represented by structural formula (II) as shown below. (Wherein R 3 and R 4 represent H or and R 3 and R 4 may be the same or different from each other). Theaflavins may be for example, free theaflavins, theaflavin monogallate A, theaflavin monogallate B, theaflavin digallate etc. (inclusive of isomers thereof) and these theaflavins may be used singly or a combination of two or more compounds may be used. Among these theaflavins, compounds containing theaflavin digallate are preferable. In particular, the compound containing theaflavin digallate as its main component is desirable; for example "Polyphenon TF"™ (product of Mitsui Norin Co. Ltd., composition: theaflavin 16.8%, theaflavin monogallate A 19.5%, theaflavin monogallate B 16.1%, theaflavin digallate 31.4%). The MIC of theaflavin against MRSA is 200 µ g/ml. and in the process of the present invention, theaflavin is used in a concentration less than the MIC. In the process of the present invention a combination of more than two of the above mentioned tea catechins and/or theaflavins may be used. In order to determine the proportional concentrations of tea catechins and/or theaflavins to the antibiotics, the antibacterial action of each antibiotic in the presence of 100 µ g of tea catechins was measured using the cup method, and it was found that in case of 2.5 µ g/ml of tetracycline and 12.5 µ g/ml of chloramphenicol, there was an apparent antibacterial action against MRSA. Thus by using these amounts of antibiotics as a guide, it can be determined in what proportions to mix these two kinds of compounds. It was the result of these investigations, that compositions comprising an antibiotic with a catechin contain the antibiotic in an amount of 2-35 µg/ml and the catechin in an amount of 20-120 µg/ml. When the inventive compositions comprise an antibiotic with a theaflavin, such compositions comprise the antibiotic in an amount of 1,6-25 µg/ml and the theaflavin in an amount of 6,3-100 µg/ml. In order to make use of the anti-bacterial action against MRSA according to the method of the present invention, antibiotics, and tea catechins and/or theaflavins are mixed in the proportions stated above and administered to patients. The mixture may be administered in various forms. For example it may be mixed with an excipient and used as an oral administration and/or non-oral administration. It may be mixed in suitable proportions with supplementary substances such as lubricating agents, emulsifying agents, dispersing agents or the like. As an oral administration, it may be in the form of a liquid, powder, tablet, capsule, granules etc. and in such cases, the excipients used are apart from water, sugar, starch, dextran, calcium phosphate, calcium carbonate, magnesium oxide, magnesium stearate, aluminium silicate, aluminium hydroxide, sodium bicarbonate, glycerin etc. As a non-oral administration, it may be in the form of an injection, a drip, an ointment etc. and it may be mixed with a common substance such as distilled water, physiological saline, vegetable oils such as olive oil etc., alcohol such as ethanol etc., polyethylene glycol and so on. The present invention will be explained in detail with reference to the following examples. Example 1 The influence of tea catechins on the antibacterial action of different antibiotics against MRSA was determined using the cup method. The tea catechin used was "Polyphenon 100"™ (product of Mitsui Norin Co. Ltd.). Tea catechin in a concentration less than the MIC was dissolved in sterilized water and added in final concentrations of 25, 50, 100 µ g/ml to a melting Mueller-Hinton agar by keeping at a temperature of 60°C. 2ml of the Mueller-Hinton broth (Difco Lab. U.S.A.) in which test bacteria had been cultivated for 18 hours was added to 98ml of the agar plate containing the tea catechin, and mixed well. Immediately thereafter, 10ml samples were poured into petri dishes. After cooling, a stainless cup (  8mm) for the examination of the antibiotics was put on the agar plate in the dish, and into the cup was poured 0.1ml of each of the various dilutions of the antibiotics , and after refrigerating for an hour, they were cultured overnight in a 36°C incubator and the following day the differences in inhibition (size of growth inhibition zone) were measured by slide calipers. Results are shown in Figure 1 and Figure 2A-2C. F-51. a strain of bacteria with a high resistancy against oxacillin was used and the tea polyophenols used were in the form of Polyphenon 100 (Mitsui Norin Co. Ltd.). The antibiotics used were amikacin (Ban-yu Seiyaku) (Fig.1), tetracycline (Wako Pure Chemical Industries )(Fig. 28A). chloramphenicol (Wako Pure Chemical Industries )(Fig. 2B). gentamicin (Wako Pure Chemical Industries )(Fig. 2C). In the figure, ○ indicates Polyphenon 100 at a concentration of 100 µ g/ml,  indicates Polyphenon 100 at a concentration of 50 µ g/ml. □ indicates Polyphenon 100 at a concentration of 25 µ g/ml. ▪ indicates the control containing no Polyphenon 100. As is evident from this figure, under the cup method F-51 strain amikacin, tetracycline, chloramphenicol showed a synergistic effect but the antibacterial effect of gentamicin with the addition of tea catechins was low. F-51 was susceptible to vancomycin but the antibacterial activity was not influenced by the addition of tea catechins. Comparative Example This experiment was carried out in the same way as in Example 1 except that the following antibiotics were used: kanamycin (Wako Pure Chemicals), erythromycin (Wako Pure Chemicals), climdamycin (Sigma Chemnical Company), and colistin (Wako Pure Chemicals) and the effect of each catechin on the antibacterial action of each antibiotic was investigated by the cup method as in Example 1. Results obtained are shown in Figure 3A-D. F-51 strain which showed a high resistancy to oxacillin was used as the test bacteria and (-)-epigallocatechin gallate was used as the tea catechin. In the figure, ○indicates (-)-epigallocatechin gallate at a concentration of 50 µ g/ml,  indicates (-)-epigallocatechin gallate at a concentration of 25 µ g/ml, □ indicates (-)-epigallocatechin gallate at a concentration of 12.5 µ g/ml, ▪ indicates (-)-epigallocatechin gallate at a concentration of 6.3 µ g/ml, and Δ indicates no addition of (-)-epigallocatechin gallate. As is evident from the figure, under the cup method F-51 strain showed its resistancy with up to 400 µ g/ml of each of the antibiotics, and no improved synergistic antibacterial effects could be observed even though (-)-epigallocatechin was added at any concentration and combined with the antibiotics, and there was no change in the size of the inhibition zone.

Claims: Composition comprising an antibiotic in combination with at least one polyphenol selected from the group consisting of a catechin of the formula (I) and/or a theaflavin of the formula (II), (wherein R 1 represents H or OH and R 2 represents H or said composition comprising 2-35 µg/ml of the antibiotic and 20-120 µg/ml of the catechin, or (wherein R 3 and R 4 represent H or said composition comprising 1,6-25µg/ml of the antibiotic and 6,3-100µg/ml of the theaflavin, provided that the antibiotic is neither a β-lactam antibiotic nor vancomycin. Composition according to claim 1, wherein the antibiotic is one antibiotic selected from the group consisting of amikacin, tetracycline, chloramphenicol and gentamycin. Composition according to claim 1 or 2, wherein the catechin is epichatechin gallate and/or epigallocatechin gallate. Composition according to claim 1 or 2, wherein the catechin has as its main component epigallocatechin gallate. Composition according to claim 1 or 2, wherein the theaflavin has as its main component theaflavin digallate. Composition according to one or more of claims 1 to 5 for use as a medicament. A method of increasing the activity of an antibiotic composition against methicillin resistant Staphylococcus aureus comprising adding to said antibiotic, at least one polyphenol selected from the group consisting of a catechin of formula (I) as defined in claim 1 and a theaflavin of formula (II) as defined in claim 1, provided that said antibiotic is neither a β-lactam antibiotic nor vancomycin and the method is not a method of treatment of the animal or human body. Use of a polyphenol selected from the group consisting of a catechin of formula (I) as defined in claim 1 and a theaflavin of formula (II) as defined in claim 1 and an antibiotic which is neither a β-lactam antibiotic nor vancomycin for the manufacture of a medicament for treatment of infections caused by methicillin resistant Staphylococcus aureus.

Inventors: HARA YUKIHIKO, SHIMAMURA TADAKATSU, HARA, YUKIHIKO, SHIMAMURA, TADAKATSU
Applicants: MITSUI NORIN KK, MITSUI NORIN CO., LTD.
Application year: 1996
Main classification: A61K31
IPC codes: A61K31, A61P31, A61K36, A61P43