NL8104627A - Antimicrobial compsns. contg. chlorhexidine salts - synergised with p-hydroxybenzoate ester(s) - Google Patents

Abstract

Antimicrobial compsns. contain (a) 0.0032-0.05wt.% chlorhexidine dihydrochloride, diacetate and/or gluconate, and (b) 0.0063-0.2wt.% of one or more lower alkyl p-hydroxybenzoates. (b) is pref. methyl or propyl p-hydroxybenzoate or a eutectic mixt. of isopropyl, isobutyl and n-butyl p-hydroxybenzoate in a ratio of 4:2.95:2.96. The concn. is pref. 0.05wt.% for the methyl ester, 0.0125 wt.% for the propyl ester or 0.0063 wt.% for the eutectic mixt. The concn. of (a) is pref. 0.0032 wt.%. The compsns. can be used as disinfectants and preservatives. Combinations of (a) and (b) have synergistically increased activity against bacteria, fungi and yeasts at very low concns.

Description

- 1- 4 '*

Antimicrobial agent / method of preparation thereof / and "method of controlling microorganisms with said agent.

Certain alkyl esters of p-hydroxybenzoic acid such as isopropyl / isobutyl and n-butyl-p-hydroxybenzoate are known to be suitable antimicrobial or preservative agents. It is also known that chlorhexidine diacetate, chlorohexidine dihydrochloride and chlorhexidine gluconate are suitable disinfectants or preservatives.

British Patent 1,514,469 discloses a mouthwash having the following composition:

Wt% 10 Chlorhexidine Gluconate 0.2

Trichlorobutanol 0.1

Methyl p-hydroxybenzoate 0.2

Propyl p-hydroxybenzoate 0.2

Chloroform 0.5 15 Sodium dioctyl sulfosuccinate 0.1

Peppermint oil DAB 7 1.5

Menthol 0.15

Ethanol (94%) 45.00

Glycerol (86%) 35.00 Water (distilled) 17.05

According to the aforementioned British Patent Specification 1,514,469, tests with aqueous preparations containing 5% by weight of the above composition have shown that it took 30 minutes to kill Streptococcus haemolyticus bacteria and more than 30 minutes.

25 to kill Candida albicans microorganisms. It is further stated that the composition of Example I which does not contain a chlorhexidine compound and no lower alkyl ester of p-hydroxybenzoic acid is better than the above composition or preparation.

US Pat. No. 3,097,131 discloses that eutectic mixtures of alkyl esters of p-hydroxy-8104627% -2 - benzoic acid can be emulsified in an aqueous solution containing colloldal protectants such as gelatin or carboxymethyl cellulose.

U.S. Pat. No. 3,960,745 discloses cleaning compositions (suitable for preoperative cleaning in surgery) containing 0.5-10.0 w / v% of a soluble chlorhexidine salt (e.g., chlorhexidine acetate or chlorhexidine gluconate) , a block copolymer of polyoxyethylene and polyoxypropylene, and an inert diluent or inert carrier, and optionally further contain a perfume, colorant and preservative, for example, ethanol, isopropanol, methyl p-hydroxybenzoate or propyl p-hydroxybenzoate.

It has been found that the combination of a lower alkyl ester of p-hydroxybenzoic acid and a chlorhexidine salt at very low weight concentrations shows a synergistic effect when used as an antimicrobial agent.

The invention has resulted in an antimicrobial agent consisting of a liquid mixture of about 0.0032-0.05 wt% chlorhexidine dihydrochloride, chlorhexidine-20 diacetate and / or chlorhexidine gluconate, and about 0.0063-0.2 wt% of one or more lower alkyl esters of p-hydroxybenzoic acid.

The chlorhexidine dihydrochloride, chlorhexidine diacetate and chlorhexidine gluconate (or chlorhexidine digluconate) are commercially available and can be used individually or as a mixture.

Suitable lower alkyl esters of p-hydroxybenzoic acid are alkyl esters with 1-6 carbon atoms, for example methyl, propyl, n-butyl, sec.-butyl, arayl, and hexyl p-hydroxybenzoate. The alkyl esters of p-hydroxybenzoic acid can also be used individually or as a mixture of two or more esters, such as mixtures of methyl and propyl p-hydroxybenzoate.

Very suitable are eutectic mixtures of three or more alkyl esters of p-hydroxybenzoic acid. Such mixtures can be in the form of oils. A preferred mixture is t 35 that of isopropyl, isobutyl and n-buyl p-hydroxybenzoate with an 8104627 at-3 ratio between about 1: 1: 1 and 4: 2: 2 and in particular between 4: 2.5: 2.5 and 4: 3: 3 (e.g. 4: 2.95: 2.96). Examples of other eutectic mixtures are a mixture of isopropyl, sec.butyl and amyl p-hydroxybenzoate, and a mixture of sec-5 butyl, isobutyl and n-butyl p-hydroxybenzoate.

The antimicrobial agents of the invention are suitable for combating bacteria, yeasts and fungi.

The antimicrobial agents of the invention can be easily prepared by dissolving the chlorhexidine salt in, for example, water, propylene glycol, polyethylene glycol or glycerol. The chlorhexidine salt solution is then mixed with a solution of one or more alkyl esters of p-hydroxybenzoic acid in, for example, propylene glycol. The concentration of components is chosen within the above stated limits in view of the microorganism to be controlled. In addition to solutions, emulsions can also be used.

The synergistic effect of the antimicrobial agents of the invention is evident from the experiments described below. For example, the minimum concentration of a chlorhexidine diacetate solution to completely inhibit the growth of Pseudomonas aeruginosa ATCC 9027, 15442, 13388, and 14502 is 0.05 wt%, while a solution of 0.1 wt% methyl p-hydroxy benzoate and 0.025 wt% propyl p-hydroxybenzoate is ineffective against said microorganisms. However, when only 0.0063% by weight of chlorhexidine diacetate is dissolved in the latter solution, the growth of the aforementioned microorganisms with the exception of Pseudomonas aeruginosa ATCC 14502 is completely inhibited.

Another example: a 0.1 wt% solution of a eutectic mixture of isopropyl, isobutyl and n-butyl p-hydroxybenzoate in a weight ratio of about 4: 2.95: 2.96 is ineffective against Pseudomonas aeruginosa, but a solution of the same eutectic mixture at a concentration of 0.05 wt% to which added 0.025 wt% chlorhexidine gluconate is able to completely inhibit the growth of this highly resistant microorganism.

8104627 ¢ - - 4 -

It is clear that the low concentrations of components in the antimicrobial agent according to the invention are cost effective. But it also reduces toxicological risks. In addition, it increases the spectrum of action against microorganisms while preventing a microorganism from developing resistance to a single component alone. And as far as the preparation of aqueous solutions is concerned, there are no problems with the solubility of alkyl esters of p-hydroxybenzoic acid for 10 such as with higher concentrations (eg 1 wt%) of such esters.

Example I

A 5 wt% stock solution of chlorohexidine diacetate in propylene glycol was prepared by placing 5 g of chloro-hexidine diacetate in a 100 ml flask and adding propylene glycol to a volume of 100 ml. The formed solution was mixed well with a Sybron magnetic stirring plate.

A stock solution of 10 wt% methyl p-hydroxybenzoate and 2.5 wt% propyl p-hydroxybenzoate in propylene-20 glycol was prepared by adding 10 g methyl p-hydroxybenzoate and 2.5 g propyl p-hydroxybenzoate in bring a 100 ml flask and add propylene glycol to a volume of 100 ml. This solution was also mixed well on a magnetic stir plate.

The minimum growth inhibitory concentration of each solution and of mixtures thereof against different microorganisms was determined as follows: A) Agar plate preparation 1) Tryptin soy agar (TSA) was prepared according to the manufacturer's instructions in 100 ml aliquots. The 30 media were autoclaved at 121 ° C for 15 min in a Castle Thermatic GO autoclave.

2) After autoclaving, the media were cooled to 45 ° C in a water bath.

3) The aforementioned stock solutions were now added to the media in varying amounts with stirring 8104627-5 - to achieve the desired test concentrations. Each agar medium and solution mixture was then placed in 100 x 15 mm Petri dishes.

B) Inoculum 5 1} The bacterial inoculum consisted of 0.1 ml of 1 in 100 diluted saline from a TSB culture of 6 7 each

Pseudomonas aeruginosa strain (about 10-10 CFU / ml where CFU is short for colony forming units) that had survived overnight.

2> The inoculum was spread over the entire agar surface with a sterile Dacron plug.

C) Incubation.

The bacterial plates were incubated at 35 ° C for 48 hours.

15 D) The results were expressed in figures with the following meaning: 0: no growth 1+: 10 CFU per Petri scale 1+: sparse growth 20 2 + i medium growth 3+: heavy growth 4+: liquefaction (as with the blank) blank: 1.125 ml propylene glycol 25 8104627 - 6 -

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The results show that the combination of chlorhexidine diacetate with methyl and propyl p-hydroxybenzoate exhibits a synergistic effect in very low concentrations.

Example II

Example I was repeated with chlorhexidine dihydrochloride and a mixture of methyl and propyl p-hydroxybenzoate, and of chlorhexidine dihydrochloride and an architectural oil containing a mixture of isopropyl, isobutyl and n-butyl p-hydroxybenzoate in a weight ratio of about 4: 2.25: 10 2.26. The results are shown in Table B below.

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ü O HO

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15 5 555 P fl -0) O O O H (1) O U Ο H

dPOS (I «# E dP« Q * O

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Note: The eutectic oil per se does not show growth inhibiting activity against Pseudomonas aeruginosa at a concentration of 0.05%.

Example ΙΙΙ-Λ 5 Example I was repeated with chlorhexidine gluconate and a mixture of methyl and propyl p-hydroxybenzoates.

The results are shown in Table C-1 below.

Example III-B

Example I was repeated with chlorhexidine gluconinate and an eutectic oil of a mixture of isopropyl, iso-butyl and n-butyl p-hydroxybenzoates in a weight ratio of about 4: 2.25: 2.26. The results are shown in Table C2 below.

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% - 11 -

Example IV

An eutectic oil (100 g) containing a mixture of isopropyl, isobutyl and n-butyl p-hydroxybenzoates in a weight ratio of about 4: 2.25: 2.26 was mixed with 80 g of a 20 wt.% chlorhexidine gluconate solution in water, 9 g of a 70 wt.% sorbitol solution, 3 g of disodium hydrogen phosphate (NajHPO4) and 8 g of gelatin ("Crotein SPC") at 60-70 ° C, after which the mixture was stirred on a magnetic stir plate until cooled and homogenized.

The antimicrobial activity of the emulsions against bacteria, yeasts and fungi was determined in the same manner as in Example I. The results are shown in Table D below.

TABLE D

15 Microorganism Test emulsion Blank 0.05%

Bacillus subtills ATCC 6633 0 4+

Staphylococcus aureus ATCC 6538 0 4+

Staphylococcus epidermidis ATCC 1228 0 4+ 20

Streptococcus faecalis 0 4+

Escherichia coli ATCC 8739 0 4+

Klebsiella pneumoniae ATCC 8308 0 4+

Salmonella typhosa ATCC 6539 0 4+

Proteus vulgaris ATCC 13315 0 4+ 25

Serratia marcescens ATCC 8100 0 4+

Enterobacter cloacae ATCC 23355 0 4+

Pseudomonas aeruginosa ATCC 9027 0 4+

Pseudomonas aeruginosa ATCC 15442 0 4+

Pseudomonas aeruginosa ATCC 13388 0 4+ 30

Pseudomonas aeruginosa ATCC 14502 0 4+

Pseudomonas stutzeri 0 4+

Candida albicans ATCC 10231 0 4+

Saccharomyces cerevisiae 0 4 +

Aspergillus niger ATCC 9642 0 4+

Penicillium chrysogenum ATCC 9480 0 4+

Trichophyton mentagrophytes 0 4+ 35 8104627 i '; 'v - 12 -

Example V

Example I was repeated with mixtures of chlorhexidine diacetate (CDA) and methyl p-hydroxybenzoate # chlorhexidine gluconate (CG) and methyl p-hydroxybenzoate # chloro-5 hexidine dihydrochloride (CDC) and methyl p-hydroxybenzoate # and chlorhexidine diacetate (CDA) and propyl p-hydroxybenzoate. The results are shown in Table E below.

10 8104627 - 13 - *

TABLE

Chlorhexidine acetate and

Micro-organism 0.0125% CDA + 0.0063% CDA + 0.2% methyl 0.2% methyl_

Pseudomonas aeruginosa ATCC 9027 0 0 "" ATCC 15442 0 0 "" ATCC 13388 0 0 "" ATCC 14502 0 0

Chlorhexidine gluconate and 0.0125% CG + 0.0063% CG + 0.2% methyl 0.2% methyl__

Pseudomonas aeruginosa ATCC 9027 0 0 "" ATCC 15442 0 0 "" ATCC 13388 0 0 "" ATCC 14502 0 0

Chlorhexidine dihydrochlori 0.0125% CDC + 0.0063% CDC + 0.2% methyl 0.2% methyl_

Pseudomonas aeruginosa ATCC 9027 0 0 "" ATCC 15442 0 0 "ATCC 13388 0 0" "ATCC 14502 0 0 Chlorhexidine diacetate and 0.0125% CDA + 0.0063% CDA + 0.04% propyl 0.04% propyl

Pseudomonas aeruginosa ATCC 9027 0 0 ”" ATCC 15442 0 0 "" ATCC 13388 0 0 "" ATCC 14502 8104627

E

methyl p-hydroxybenzoate 0.0032% CDA + 0.0125% CDA + 0.0063% CDA + 0.0032% CDA + Blank 0.2% methyl 0.1% methyl 0.1% methyl 0.1% methyl 0 0 0 0 4+ 0 0 0 0 4+ 0 0 0 0 4+ 0 0 0 0 4+ methyl p-hydroxybenzoate 0.0032% CG + 0.0125% CG + 0.0063% CG + 0.0032% CG + Blank 0.2% methyl_0.1% methyl 0.1% methyl_0.1% methyl_ 0.0 0 0 4+ 0 0 0 0 4+ 0 0 0 0 4+ 0 0 0 0 4+ de and methyl p-hydroxybenzoate 0 0.0032% CDC + 0.0125% CDC + 0.0063% CDC + 0.0032% CDC + Blank 0.2% Methyl 0.1% Methyl 0.1% Methyl 0.1% Methyl 0 0 0 0 4+ 0 0 0 0 4+ 0 0 0 0 4+ 0 0 0 0 4+ propyl p-hydroxybenzoate 0.0032% CDA + 0.0125% CDA + 0.0063% CDA + 0.0032% CDA + Blank 0.04% propyl 0.02% propyl 0.02% propyl 0.02% propyl 0 0 0 0 4+ 0 0 0 0 4+ 0 0 0 0 4+ 0 0 0 0 4+. 8 1 0 4 6 2 7

r. y- «V

- 14 -

The results with chlorhexidine diacetate, chlorhexidine gluconate and chlorhexidine dihydrochloride separately have been reported previously. The results with methyl p-hydroxybenzoate and propyl p-hydroxybenzoate separately are shown in Table 5 below.

TABLE F

Microorganism Methyl p-hydroxybenzoate 0.2% 0.1% Blanco

Pseudomonas aeruginosa ATCC 9027 1+ 4+ 4+ 10 "" ATCC 15442 2+ 4+ 4+ "" ATCC 13388 3+ 4+ 4+ "" ATCC 14502 2+ 4+ 4+

Propyl p-hydroxybenzoate 0.05% _ 0.025% Blank 15 Pseudomonas aeruginosa ATCC 9027 3+ 4+ 4+ "" ATCC 15442 3+ 4+ 4+ "" ATCC 13388 2+ 4+ 4+ "'* ATCC 14502 3+ 4+ 4+

Example VI

The assay was performed on Pseudomonas aeruginosa ATCC 13388 because this microorganism is known to be resistant to conventional antimicrobial agents.

2 g

About 10-10 CFU / ml of said microorganism was added to 100 ml of 0.85 wt% saline with different concentrations of chlorhexidine gluconate, eutectic mixture of isopropyl, isobutyl and n-butyl p-hydroxy benzoates with a weight ratio of about 4: 2.95: 2.96, and combinations thereof. As a blank, only 0.85 wt% saline was used. 1 ml of each test solution was pipetted into sterilized plastic Petri dishes (100 x 15 mm) after 1, 2, 3 and 20 hours, respectively. In addition, 20 ml of Tripticase soy agar containing 0.07 wt% lecithin and 0.5 wt% Polysorbate 80 as preservative activators were added to each Petri dish. The dishes were gently rotated to mix the 8104627-15 content and left to cool. The plates were then inverted and incubated for 48 hours at 37 ° C. The results were expressed with figures having the following meanings: 5 0 no growth 1+: sparse growth 2+: medium growth 3+: heavy growth 4: liquefaction (such as in the blank Bactericidal activity occurred in tests in which no provocative microorganisms could be detected, and was retained throughout the duration of the test. The results are shown in Table G below, the eutectic mixture being designated as Parabens.

15 TABLE G

Chlorhexidine gluconate - Parabens synerglsme Bacterial activity against Pseudomonas aeruginosa after 1 hour_2 hours 3 hours_20 hours 20 0.05% CG 0 0 0 0 0.025% CG 2 + 0 * 0 0 0.0125% CG 3+ 3+ 2+ 2+ 0, 10% Parabens 2+ 2+ 2+ 4+ 0.05% Parabens 4+ 4+ 4+ 4+ 25 0.025% CG - 0.05% Parabens 00 00 0.0125% CG - 0.025% Parabens 1 + 0 02+ 0.0063% CG + 0.0125% Parabens 2+ 2+ 1+ 2+

Blank 4+ 4+ 4+ 4+

Example VII

The assay was performed on the fungi Aspergillus niger ATCC 9642 and Candida albicans ATCC 10231. About 104-105 CFU / ml Aspergillus spores or Candida cells were added to 100 ml of 0.85 wt% saline with various salts. concentrations of chlorhexidine gluconate, an eutectic mixture 8 (04527 - 16) of isopropyl, isobutyl and n-butyl p-hydroxybenzoates with a weight ratio of about 4: 2.95: 2.96, and with combinations thereof. 0.85 wt.% Saline solution used without further matter: 1 ml of each test solution was introduced into sterilized Petri dishes (100 x 15 mm) of plastic after 1, 5, 2, 3 and 20 hours, respectively, using a pipette. Petri dish 20 ml of Trypticase soy agar containing 0.07 wt% lecithin and 0.5 wt% Polysorbate 80 as preservative inactivators was added.

The dishes were rotated gently to mix the contents. After 10 cooling, the plates were turned and incubated at 30 ° C for 72 hours. The growth results were valued with figures having the following meanings: 0: no growth 1+: sparse growth 15 2+: medium growth 3+: heavy growth 4+ : liquefaction (as with the blank).

Fungicidal activity occurred in tests in which provocative microorganisms could not be detected, and was maintained throughout the test.

The results are shown in Tables H and I below with the eutectic mixture designated as Parabens.

25 8104627 - 17 - TABLE Η

Chlorhexidine gluconate - Parabens synergy Fungicidal activity against Candida albicans after 1 hour 2 hours 3 hours 20 hours 5 0.0125% CG 1 + 00 0 0.0063% CG 2+ 1+ 1+ 0 0.0032% CG 3+ 2+ 2 + 1+ 0.0016% CG 4+ 3+ 3+ 1+ 0.05% Parabens 000 0 10 0.025% Parabens 1+ 1+ 1+ 0 0.0125% Parabens 4+ 4+ 4+ 4+ 0.0063 % Parabens 4+ 4+ 4+ 4+ 0.0063% CG - 0.0063% Parabens 000 0 0.0032% CG - 0.0125% Parabens 0000 15 0.0016% CG - 0.0125% Parabens 2+ 1 + 1+ 0 0.0016% CG - 0.0063% Parabens 3+ 2+ 2+ 1+

Blank 4+ 4+ 4+ 4+

TABLE I

20 Chlorhexidine Gluconate - Parabens Synergy

Fungicidal activity against Aspergillus niger after 1 hour 2 hours 3 hours 20 hours 0.0125% CG 4+ 4+ 4+ 3+ 0.0063% CG 4+ 4+ 4+ 3+ 25 0.05% Parabens 2+ 1+ 1+ 0 0.025% Parabens 4+ 4+ 4+ 4+ 0.0125% CG - 0.05% Parabens 1 + 0 0 0 0.0063% CG - 0.05% Parabens 1 + 00 0 0.0032% CG - 0.05% Parabens 1 + 1 + 0 0 30 0.0016% CG - 0.05% Parabens 2+ 2+ 1+ 0 0.0125% CG - 0.025% Parabens 2+ 2+ 2+ 1+ 0, 0125% CG - 0.0125% Parabens 4+ 4+ 4+ 3+

Blank 4+ 4+ 4+ 4+ 35 8104627

Claims (16)

An antimicrobial agent consisting of a liquid mixture of one or more chlorhexidine compounds and one or more lower alkyl esters of p-hydroxybenzoic acid, characterized in that the antimicrobial agent is about 0.0032-0.05 wt% chlorhexidine dihydrochloride, chlorhexidine diacetate and / or chlorhexidine gluconate, and contains about 0.0063-0.2 wt% of one or more lower alkyl esters of p-hydroxybenzoic acid. Antimicrobial agent according to claim 1, 10, characterized in that the agent contains chlorhexidine diacetate. Antimicrobial agent according to claim 1, characterized in that the agent contains chlorhexidine dihydrochloride. Antimicrobial agent according to claim 1, characterized in that the agent contains chlorhexidine gluconate. Antimicrobial agent according to claim 1, characterized in that the agent contains methyl p-hydroxybenzoate and propyl p-hydroxybenzoate. Antimicrobial agent according to claim 1, characterized in that the agent contains methyl p-hydroxybenzoate. Antimicrobial agent according to claim 1, characterized in that the agent contains propyl p-hydroxybenzoate. Antimicrobial agent according to claim 1, characterized in that the agent contains a eutectic mixture of three or more lower alkyl esters of p-hydroxybenzoic acid. Antimicrobial agent according to claim 8, characterized in that the eutectic mixture consists of a mixture of isopropyl p-hydroxybenzoate, isobutyl p-hydroxybenzoate and n-butyl p-hydroxybenzoate. Antimicrobial agent according to claim 9, 30, characterized in that the ratio of isopropyl p-hydroxybenzoate, isobutyl p-hydroxybenzoate and n-butyl p-hydroxybenzoate in the eutectic mixture is about 4: 2.95: 2.96 . 11. Antimicrobial agent according to claim 1, characterized in that the concentration of chlorhexidine compound is about 0.0032% by weight. 8104627 s - 19 - Antimicrobial agent according to claim 5, characterized in that the concentration of methyl p-hydroxybenzoate is about 0/05 wt.% And the concentration of propyl p-hydroxybenzoate is about 0/0125 wt.%. Antimicrobial agent according to claim 9, characterized in that the concentration of the eutectic mixture is about 0.0063% by weight. 14. A method of preparing an antimicrobial agent consisting of a liquid mixture of one or more chlorhexidine compounds and one or more lower alkyl esters of p-hydroxybenzoic acid by combining the constituents, characterized in that an antimicrobial agent is prepared according to one of claims 1-13. 15. Method for controlling bacteria, yeasts and fungi with an antimicrobial agent consisting of a liquid mixture of one or more chlorhexidine compounds and one or more lower alkyl esters of p-hydroxybenzoic acid, characterized in that an antimicrobial agent is used as antimicrobial agent agent according to any one of claims 1-13 or prepared by the method according to claim 14. 16. Antimicrobial agents and methods as well as antimicrobial agents obtainable and obtained therefrom as described in the description and examples. 25 8104627