WO2018109456A1

WO2018109456A1 - Antimicrobial compositions

Info

Publication number: WO2018109456A1
Application number: PCT/GB2017/053717
Authority: WO
Inventors: Michael Kerr; Teresa ALLEN
Original assignee: Provita Eurotech Ltd
Priority date: 2016-12-12
Filing date: 2017-12-12
Publication date: 2018-06-21
Also published as: GB201621050D0

Abstract

There is provided an antimicrobial biocidal composition comprising a permeability enhancer and a cationic membrane-active agent which allow a cationic membrane-active agent to be used at a sub-minimal inhibitory concentration to achieve a biocidal effect.

Description

Antimicrobial Compositions Field of Invention The present invention relates to the fields of microbiology, medicine and infection control. In particular, it concerns microbiocidal compositions comprising a permeability enhancer and a cationic membrane-active agent, wherein the cationic membrane-active agent is provided at a minimal inhibitory concentration (MIC) or sub-minimal inhibitory concentration.

Background

Antimicrobial compositions are used both in prophylactic treatments for example, mouthwashes and the like, and medicine for example, to treat wound infection and the like, and as components of other formulations, for example cosmetic formulations.

It has been suggested that some commonly used antimicrobial agents present toxicity concerns when in use at concentrations in which they are known to be bactericidal. For example, there are concerns over the toxicity of chlorhexidine when used in the range of 0.05 to 4.0% (w/v). It would be advantageous if such agents could be provided at lower concentrations such that toxicity concerns are avoided, whilst still providing microbiocidal activity. Summary of the Invention

The inventors have determined a cationic membrane-active agent, for example chlorhexidine, cetylpyridinium and salts thereof, or benzalkonium and salts thereof, or Poly(hexamethylene biguanide) hydrochloride (PHMB), when provided at a minimal inhibitory concentration (MIC) or even a sub-minimal inhibitory concentration (sub-MIC), can provide a bactericidal effect when the cationic membrane-active agent is provided in combination with a permeability enhancer additive. A minimal inhibitory concentration (MIC) of an agent is a concentration at which bacteria are not killed, but at which the growth or reproduction of the bacteria is slowed.

Methods for determining a cidal concentration of an agent, a minimal inhibitory concentration and a sub minimal inhibitory concentration of an agent are well known in the art. The most commonly employed methods are the tube dilution method and agar dilution methods. The tube dilution test is the standard method for determining levels of microbial resistance to an antimicrobial agent. Serial dilutions of a test agent are made in a liquid microbial growth medium which is inoculated with a standardized number of bacterial organisms and incubated for a prescribed time. The lowest concentration (highest dilution) of test agent preventing appearance of organism growth is considered to be the minimal / minimum inhibitory concentration (MIC). At this dilution the test agent is considered to be

bacteriostatic. The minimal bactericidal concentration (MBC) or the minimum lethal

concentration (MLC) of an antibacterial is defined as the maximum dilution of the product that will kill a test organism. This can be determined by sub-culturing the last clear (no appearance of growth) MIC tube onto growth medium and examining for bacterial growth by serial dilution. MLC cannot be done without testing for MIC.

Accordingly, a first aspect of the present invention provides a composition comprising a cationic membrane-active agent and at least one permeability enhancer additive capable of enhancing the antimicrobial effect of the cationic agent, wherein the agent is provided at a concentration equal to or less than the minimal inhibitory concentration of the agent when the agent is provided alone and the additive is provided at a concentration less than the microbiocidal concentration of the additive when it is provided alone. In embodiments there is provided a microbiocidal composition comprising a cationic membrane-active agent and at least one permeability enhancer additive wherein the enhancer is provided at less than a minimal bactericidal

concentration and wherein the cationic membrane-active agent is provided in the composition at a concentration equal to or less than a static or sub-static concentration (minimal inhibitory concentration).

Accordingly, there is provided a synergistic composition which provides for cidal activity, when the components of the composition are provided in combination, when each of the components, when provided alone, at the concentration they are provided in the composition, would not provide cidal activity.

Suitably the permeability enhancer additive may be provided at less than 25 % w/v. Suitably, the permeability enhancer additive may be provided at less than 15 % w/v, suitably less than 10 % w/v, suitably less than 5% w/v.

In embodiments, the composition may be a bactericidal composition. Optionally, the bactericidal composition can show activity against candidal challenges. In embodiments, the composition can be cidal in relation to Candida.

In embodiments, the composition can show cidal activity against bacteria typically found on skin, in particular Staphylococcus, in particular Staphylococcus epidermidis.

In embodiments, using standard methods for determining levels of microbial resistance to an antimicrobial agent, serial dilutions of a cationic membrane agent can be made in a liquid microbial growth medium which is inoculated with a standardized number of organisms and incubated for a prescribed time, wherein the standardized number of organisms can be 1x 10⁸ and the prescribed time can be 4 hours. A static concentration is a minimal inhibitory concentration and a sub static concentration is a concentration less than a minimal inhibitory concentration.

In embodiments, a minimial inhibitory concentration (MIC) (%w/v), when provided alone, can be

Chlorhexidine 2.0 x 10^"4

N-methylpyrrolidone (NMP) 12.5

2-pyrollidone (PYD) 6.25

Ethanol 6.25

(MIC concentration in relation to S. epidermis)

In embodiments a minimal bactericidal concentration (%w/v), when provided alone, can be

Chlorhexidine 1.0x 10 ^"3

N-methylpyrrolidone (NMP) 25

2-pyrollidone (PYD) 25

Ethanol 25

(provided in relation to S. epidermis) In embodiments the permeability enhancer additive can be selected from an aprotic solvent and/or an alcohol. Aprotic solvents are typically not ionized at physiological pH. Suitably, an alcohol that may be used in a composition of the present invention includes, but is not limited to, an aliphatic alcohol, in particular with inherent antimicrobial properties, which may be short chain, linear or branched, suitably from 1 to about 8 carbon atoms, suitably about 1 to 4 carbon atoms. For example, the alcohol may be selected from methanol, ethanol, n- propanol, n-butanol, isobutanol, n-pentanol, isopentanol, hexanol, and isoamyl alcohols or combinations thereof. In embodiments, the aprotic solvent can be selected from at least one of N- methylpyrrolidone, 2-pyrrolidone and dimethylacetamide. Optionally an alcohol may be selected from at least one of ethanol and glycol. In embodiments, a permeability enhancer additive can be selected from, but is not limited, to N-methylpyrrolidone, 2-pyrrolidone, dimethylacetamide, other aprotic solvents and pharmaceutical solvents, such as glycols, in particular dipropylene glycol.

In embodiments of the composition, the cationic membrane agent can be selected from at least one of chlorhexidine, cetylpyridinium or salts thereof, benzalkonium or salts thereof, and Poly (hexamethylene biguanide)

hydrochloride (PHMB).

In particular embodiments of the composition the agent can be chlorhexidine or a salt thereof. Suitably, in embodiments, chlorhexidine can be provided at a concentration of between 0.0001 and 0.01 % w/v, preferably 0.0002% w/v and 0.001 % w/v, more preferably 0.0002% to 0.00099% and most preferably chlorhexidine can be provided at a concentration of 0.0002%.

Suitably, in embodiments when the cationic membrane agent is selected from benzalkonium or cetylpyridinium it can be provided at a concentration of between 0.0001-0.005% w/v.

In particular embodiments of the composition, the additive can be selected from at least one of N-methylpyrrolidone, 2-pyrrolidone, ethanol, or combinations thereof, for example N-methylpyrrolidone and ethanol or 2-pyrrolidone and ethanol. In embodiments, the additive can be present in the composition at a concentration in the range 5% v/v to 25% v/v, 5% v/v to 15% v/v, 5% v/v to 10% v/v.

In particular embodiments of a composition of the invention, the cationic membrane agent can be chlorhexidine at a concentration of about 0.0002% (v/v) or less, and the permeability enhancer additive can be N-methylpyrrolidone at a concentration in the range of about 5% to 20% (v/v). Optionally no further agents with antimicrobial activity may be provided in a composition, or may only be provided at a concentration which is not cidal when they are provided alone. In embodiments a composition of the invention can comprise, i) a cationic, membrane-active agent at a static or substatic concentration, ii) an additive at less than 25% w/v, and iii) an alcohol at a concentration from 1 % (v/v) to 15% (v/v). In such embodiments, the cationic membrane-active agent can be selected from chlorhexidine, benzalkonium and cetylpyridinium and salts thereof. In

embodiments an additive can be selected from at least one of N- methylpyrrolidone, 2-pyrrolidone, dimethylacetamide, dimethylformamide and DMSO.

In embodiments, a composition can comprise a permeability enhancer additive of N-methylpyrrolidone at about 10% (v/v) or more, an alcohol at a concentration of about 5 % (v/v) to 15% v/v and chlorhexidine at a static or substatic

concentration.

In alternative embodiments, the composition can comprise i) chlorhexidine at a static or substatic concentration, ii) pyrrolidone at a concentration in the range 5 to 20% (v/v), and iii) alcohol at a concentration of 5% to 15% (v/v). As noted above, a static or substatic concentration of chlorhexidine in relation to an organism could be readily determined by a person of skill in the art, for example the MIC of chlorhexidine for S. epidermis was determined to be

2.0 x10^"4. Suitably, a composition of the invention may further comprise dispersion media, surfactants, antioxidants, preservatives, salts, drug stabilisers, gels, binders excipients, fragrance, dyes, flavouring agents or like and combinations thereof. The composition may be provided as a liquid, paste, cream, lotion, ointment semi-solid or solid form, for topical administration. In embodiments, the composition can be provided in an absorbent material, for example cotton.

In embodiments, the composition can be provided in an aerosol composition wherein said aerosol composition may optionally include a propellant, for example, a compressed gas such as nitrogen, air or the like. In embodiments the composition can be provided in a solution, mouthwash, gel or spray.

According to a second aspect of the invention there is provided a method for cleaning a surface comprising contacting the surface with a composition of the invention.

In embodiments, a surface can be a medical device, for example, but not limited to a medical implant, or a dental implant or device.

In embodiments, the surface can be skin, for example the skin of cattle, of sheep or another domesticated animal or human skin. The method may comprise applying the composition to the skin with a swab wherein the swab comprises a composition of the invention. Alternatively, the composition may be applied to a surface by a brush, other cleaning apparatus and / or in combination with an ultrasound device.

According to a third aspect of the present invention there is provided a swab comprising a composition of the present invention.

According to a fourth aspect of the present invention there is provided a kit of parts comprising at least a permeability enhancer and a cationic membrane- active agent which can be combined to provide the composition of the first aspect.

According to a fifth aspect there is a method to prepare a composition of the first aspect, the method comprising providing a cationic membrane-active agent in combination with a permeability enhancer additive, wherein both the cationic membrane-active agent and the permeability enhancer additive are provided at a concentration equal to or less than a static concentration and bacterial concentration respectively, optionally wherein the membrane-active agent and additive are provided at a concentration equal to or less than a static or sub static concentration (MIC).

The swab may comprise any suitable absorbent material, for example cotton or the like. Brief Description of the Figures

Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying figures in which; Figure 1 illustrates table A showing minimal inhibitory concentrations and minimal bactericidal concentrations for Chlorhexidine, N-methylpyrrolidone (NMP), 2- pyrollidone (PYD) and ethanol;

Figure 2 illustrates table B, a composite table illustrating killing activity of formulations containing sub-minimal inhibitory concentration chlorhexidine digluconate (0.0002% (v/v)), N-methylprrolidone and ethanol against S.

epidermis, wherein initial inoculum / bioburden of ca.10⁶ cfu/m S. epidermis is killed rapidly with selected formulations producing no growth of survivors (cfu/ml) at 10 minutes contact;

Figure 3 illustrates table C showing a control test with 0.0002% Chlorhexidine;

Figure 4 illustrates table D showing a rate of kill for S.epidermis with 0.0002% Chlorhexidine and 15% NMP;

Figure 5 illustrates table E showing a rate of kill for S. epidermis with 0.0002% Chlorhexidine and 15%NMP and 10% ethanol; Figure 6 illustrates table F illustrating results of a formulation of 2-pyrrolidone (PYD) demonstrating a rate of kill for S. epidermis with 0.0002% Chorhexidine and 5% PYD; Figure 7 illustrates table G illustrating results of a formulation with 0.0001 % Chlorhexidine and 5% PYD; and

Figure 8 illustrates table H showing the rate of kill for S. epidermis with 0.0001 % Chlorhexidine and 5% PYD and 10% ethanol.

Detailed Description of the Invention

The present inventors have surprisingly shown that the formulations described herein provide bactericidal activity against bacterial and candidal challenges, whilst only utilising minimal inhibitory concentrations or sub-minimal inhibitory concentrations of cationic membrane-active agent. Without wishing to be bound by theory, the inventors suggest that the formulations of the present invention, and in particular the specific minimal inhibitory or sub-minimal inhibitory concentrations, can be used to kill pathogens such as bacteria. Chlorhexidine compositions comprising chlorhexidine acetate or chlorhexidine gluconate have been shown to have antiseptic activity in concentrations from about 0.01 % w/v to about 10% w/v of the total weight of the composition. Therefore, it would not be expected that very low concentrations, such as minimal inhibitory or sub-minimal inhibitory concentrations, would successfully kill pathogens. These results therefore overturn the prejudice that static concentrations of agents might not usefully be used to kill bacteria.

The cationic membrane-active agents of the present invention show increased antimicrobial activity when combined with a permeability enhancer. The permeability enhancer improves the penetration of the cationic membrane-active agent, allowing a cidal effect to be obtained with less of the cationic membrane- active agents (minimal inhibitory or sub-minimal inhibitory concentrations). As used herein, chlorhexidine refers to N,N""1 ,6-Hexanediylbis[N'-(4- chlorophenyl)(imidodicarbonimidic diamide)] or salts thereof. Salts of

chlorhexidine include chlorhexidine dihydrochloride, chlorhexidine diacetate, and chlorhexidine digluconate.

As used herein, "a" or "an" may mean one or more unless context demands otherwise. The word "comprising" (and any form of comprising such as

"comprise and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and

"include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open ended and do not exclude additional, unrecited elements or method step.

Throughout the specification, unless the context demands otherwise, the terms 'comprise' or 'include', or variations such as 'comprises' or 'comprising', 'includes' or 'including' will be understood to imply the includes of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

Preferred features and embodiments of each aspect of the invention are as for each of the other aspects mutatis mutandis unless context demands otherwise.

Examples

The present invention will now be described with reference to the following examples which are provided for the purpose of illustration and are not intended to be construed as being limiting on the present invention. It will be understood by those skilled in the art that various changes in the form and details may be made therein without departing from the scope of the present invention. Test solutions were prepared aseptically to include combinations of ethanol (0, 5, 10 and 15% (v/v)) and NMP or PYD (0, 5, 10 and 15% v/v) and dipropylene glycol with or without the inclusion of a MIC level of chlorexidine digluconate, benzalkonium chloride or cetylpyridinium chloride. Activity of Antimicrobial agents with Additives

Cultures of Staphylococcus epidermidis in Tryptone Soya Broth (TSB) were gown in an orbital incubator (aerobic atmosphere, 37°C, 150 rpm). The bacterial suspension was centrifuged (3000g, 15 minutes), washed twice with sterile water, and resuspended in sterile water to an OD₅₄₀ of 0.3 corresponding to a viable population of the order of 1 x 10⁸ colony forming units (cfu)/ml. The inoculum level was verified on each occasion by serial decimal dilution and viable count determination using the Miles and Misra (1938) (Miles, AA; Misra, SS, Irwin, JO (1938 Nov). "The estimation of the bactericidal power of the blood." The Journal of hygiene 38 (6): 732-49) drop plate technique onto agar plates. This culture was provided as a standardized microbial suspension.

The standardized microbial suspension and test solutions, including sterile water controls were equilibrated to ambient temperature prior to their incorporation into to test procedure.

A volume (0.1 ml) of standardized microbial suspension was added to 9.9ml of test solution and the suspension thoroughly mixed with the test solution utilising a vortex mixer (treated microbial suspension). Each test parameter was tested in triplicate (n=3).

The initial bioburden for the test procedure was therefore estimated to be of the order of 1x10⁶cfu/ml. The kill kinetics of the treated microbial suspension was followed for four hours or a suitable alternative time period. Sampling times for determining surviving microorganism were e.g. t = 0, 5, 10, 30, 60 and 240 minutes.

In sampling for surviving microorganisms, a 1 ml volume was taken from the test mixture and added to a volume (9ml) of diluents containing a neutralizer system to inactivate carry over of antimicrobial agents. Following immediate and thorough mixing, decimal serial dilution to the 10^"4 level was performed and the resulting suspensions plated out using the Miles and Misra (1938) plating technique, left to dry at room temperature and subsequently incubated at 37°C for 24-48h. Following incubation, the plates were examined for the presence of growth and colonies counted on appropriate plates. As illustrated by figure 1 the MIC of chlorhexidine against S. epidermis when provided alone was determined to be 2.0 x10^"4 (% w/v) and cidal activity required 1.0 x10^"3 (% w/v). As illustrated by Figure 3, after treatment of S. epidermis with 0.0002% w/v of chlorhexidine alone for 4 hours, a percentage of S. epidermis survived. However, as illustrated in Figure 4, inclusion of 15% NMP in the composition including 0.0002% chlorhexidine resulted in no survival of S.

epidermis after 20 minutes. An even faster kill rate was observed when the composition further included 10% ethanol. As noted, in each case the level of NMP and ethanol is less than the % w/v required for cidal activity when they are provided alone.

As illustrated in Figure 6, a composition comprising 0.0002% chlorhexidine and 5% PYD resulted in no survivors of S. epidermis after 20 minutes. Again, the concentration of each of chlorhexidine and PYD is less than the cidal % illustrated in Figure 1.

As illustrated in Figure 8, a composition comprising 0.0001 % chlorhexidine, 5% PYD and 10% ethanol resulted in no survivors of S. epidermis after 10 minutes.

Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in the text is not repeated in this text is merely for reasons of conciseness. Reference to cited material or information contained in the text should not be understood as a concession that the material or information was part of the common general knowledge or was known in any country.

Claims

A microbiocidal composition comprising a cationic membrane-active agent and at least one additive capable of enhancing the antimicrobial effect of the agent, wherein the agent is chlorhexidine provided at a concentration of between 0.0002% w/v and 0.00099% w/v.

A composition as claimed in any one of the preceding claims wherein the additive is selected from an aprotic solvent and/ or an alcohol.

A composition as claimed in claim 2 wherein the aprotic solvent is selected from at least one of N-methylpyrrolidone, 2-pyrrolidone, dimethylacetamide, dimethylformamide and DMSO.

A composition of any one of claims 1 to 3 wherein the alcohol is selected from at least one of ethanol, glycol, diglycerols, or triglycerols.

A composition as claimed in any one of the preceding claims wherein the additive is selected from at least one of N-methylpyrrolidone, 2- pyrrolidone, ethanol, and combinations of N-methylpyrrolidone and ethanol or 2-pyrrolidone and ethanol, and the additive is present in the composition at a concentration in the range 5% v/v to less than 25% v/v.

A composition as claimed in any preceding claim, wherein the agent is chlorhexidine at a concentration of about 0.0002% (w/v), and the additive is N-methylpyrrolidone at a concentration in the range 5% to 20% (v/v).

A composition as claimed in any preceding claim comprising, i)

chlorhexidine at a static or substatic concentration, ii) an additive selected from at least one of N-methylpyrrolidone, 2-pyrrolidone,

dimethylacetamide, dimethylformamide and DMSO at less than 25% w/v, and iii) an alcohol at a concentration from 1 % (v/v) to 15% (v/v).

8. A composition as claimed in claim 7, comprising N-methylpyrrolidone at about 10% (v/v) and alcohol at a concentration of about 5 % (v/v) to 15%.

9. A composition as claimed in claim 7 comprising i) pyrrolidone at a

concentration in the range 5 to 20% (v/v) and ii) alcohol at a concentration of 5% to 15% (v/v).

10. A method of cleaning a surface comprising contacting the surface with a composition of any one of claims 1 to 9.

1 1. A swab comprising a composition as claimed in any one of claims 1 to 9 and an absorbent material.

12. A kit comprising

a) a cationic membrane-active agent at a concentration equal to or less than a minimal inhibitory concentration;

b) a permeability enhancer additive at a concentration less than a bactericidal concentration;

c) a container in which to mix a) and b)

wherein the combination of a) and b) provides a bactericidal composition.

13. A method of manufacturing a composition of any one of claims 1 to 9 comprising the steps:

a) providing a cationic membrane-active agent at a concentration equal to or less than a minimal inhibitory concentration,

b) providing a permeability enhancer additive at a concentration less than a bactericidal concentration c) mixing a) and b) to provide a bactericidal composition.

14. A biocidal composition comprising a cationic membrane-active agent at a concentration equal to or less than a minimal inhibitory concentration, and at least one permeability enhancer additive at a concentration less than a bactericidal concentration optionally further comprising additional components at concentrations less than a bactericidal concentration wherein the combination of the cationic membrane-active agent and permeability enhancer provides a bactericidal effect.