Field of the invention Embodiments of the present invention relates to an antimicrobial composition, comprising chlorhexidine and pentane-l,5-diol, and further to the use of such a composition to combat microorganisms.

Background Microorganisms may be combated in different ways depending on the reasons for combating them. The approach is different depending on if the purpose is to prevent growth of microorganisms, inhibit further growth or reduce and eliminate the microorganism. Additionally the approach is different depending on where the microorganism is located such as in a liquid, on a surface, within a mammalian body etc.

US 2004/0191274 Al discloses topical compositions, which may be used as an antiseptic and/or disinfectant. The topical composition disclosed comprises at least one Cl to C4 alcohol; at least one anti-microbial, such as chlorhexidine; and at least one emollient, such as polyethylene glycol. CN 1130020, Tawil, G. G. et al Alexandria Journal of Pharmaceutical Sciences,

1988, 2(1), 58-60 and Frank M. von et al Pharmaceutische Industrie, 1991, 53(5) 512- 516 all disclose combinations of chlorhexidine and propyleneglycol. Frank M. von et al concludes that no synergy can be found between chlorhexidine and propyleneglycol in combination. On the contrary, previous findings of Tawil, G.G. et al states the propyleneglycol should potentiate the bactericidal effect of chlorhexidine. However,

Tawil, G.G. et al present no data for propyleneglycol when tested alone.JP 2003267862 discloses a combination of chlorhexidine and 1,3-butandiol.

WO 07/063065 discloses the use of a composition comprising pentane-l,5-diol to reduce and/or eliminate odour from a mammal, such as odour from urine, menstruation, faeces, liquids from leg ulcers, decubital ulcers, blood and perspiration. Furthermore, WO 07/063065 discloses an absorption product comprising pentane-1,5 diol to reduce and/or eliminate odour from a mammal, such as odour from urine, menstruation, faeces, liquids from leg ulcers, decubital ulcers, blood, and perspiration. There are various compositions and compounds, which are known to possess anti-bacterial properties. One example of a compound known to possess anti-bacterial properties is propane- 1 ,2-diol (propylene glycol), which is a diol widely used to combat microorganisms in dermatology. Although not as widely used as propylene glycol, also pentane-l,5-diol have been used to combat microorganisms in dermatology. According to WO 04/1 12765, the antibacterial effect of pentane-l,5~diol is believed to be shared by other low-molecular weight aliphatic diols Further, use of pentane- 1,5 -diol is disclosed to have effect against multiresistant strains of bacteria.

Chlorhexidine is a chemical antiseptic. It is bactericidal to both gram-positive and gram-negative microbes. Further, it is bacteriostatic. The mechanism of action is believed to be membrane disruption, and not ATPase inactivation as previously thought. Chlorhexidine is used for general skin cleansing, such as in surgical scrubs for preoperative skin treatment. It has a long duration of action and a low toxicity. Skin sensitivity to chlorhexidine has been reported. Also, chlorhexidine may be neurotoxic at high levels, whereby it would be beneficial to decrease the level of chlorhexidine, while still maintaining its effect.

Multi-resistance of bacteria to antibiotics is becoming more and more common. In the health sector there is today a mounting concern worldwide regarding the future use of traditional antimicrobials. Alternative methods and approaches have to be used to manufacture affective antimicrobial agents. Accordingly, there is an increasing medical need to identify new antimicrobial compositions, which can be effectively used to inactivate microorganisms. In particular there is a growing list of microorganisms, such as bacteria, virus and fungi, which become resistant to antibiotics. Additionally, there is an increasing population of individuals becoming allergenic against a variety of antibiotics or preservative components used in antibiotic preparations, leading to needs for novel compositions which can be used as alternatives to conventional technologies.

In addition to finding novel antibiotics, it would also be highly desirable to identify novel synergistic mixtures of known antimicrobial compounds. Several known antibiotics may give rise to adverse side effects at doses applied when administered alone.

Summary

Accordingly, the present invention seeks to mitigate, alleviate, circumvent or eliminate one or more of the above-identified deficiencies by providing a composition comprising between 0.000001 and 5 wt % chlorhexidine and between 1 and 75 wt % pentane-l,5-diol. Further, various embodiments of the present invention relates to the use of a composition comprising chlorhexidine and a diol for inactivating microorganisms selected from the group consisting of gram positive and gram negative bacteria, fungi, including yeasts, moulds and dermatophytes and virus, and especially Staphylococcus aureus and P. acnes.

Advantageous features of the invention are defined in the dependent claims.

Detailed description of example embodiments

In the context of the present description and the appended claims, "C3-C8 alkanediol" is intended to mean an organic compound comprising a linear or branched, saturated hydrocarbon with three to eight carbon atoms, which organic compound has two hydroxyl groups being attached to different carbon atoms. Examples of such C3-C8 alkanediol encompasses, but are not limited to, propane- 1,2-diol, propane~l,3-diol, butane- 1,2-diol, butane- 1,3 -diol, butane- 1,4-diol, butane-2,3-diol, 2-methylpropane-l,2- diol, 2-methylpropane- 1 ,3-diol, 2-hydroxymethyl- 1 -propanol, pentane- 1 ,2-diol, pentane-2,3-diol, 2-hydroxymethyl-l-butanol, 2-methylbutane- 1,2-diol, 3- methylbutane- 1,2-diol, 2-methylbutane- 1,3 -diol, 3-methylbutane-l,3-diol, 2- methylbutane- 1,4-diol, hexane- 1,2-diol, hexane- 1,6-diol, 2-methylpentane-l,5-diol, 3- methylpentane-l,5-diol, 2-methyl-2,4-pentane, heptane- 1,7-diol, heptane- 1,2-diol; octane- 1,2-diol and octane- 1,8-diol.

In the context of the present description and the appended claims, chlorhexidine is intended to include various forms of chlorhexidine. Such forms includes, but are not limited to chlorhexidine in its free from, salts of chlorhexidine, such as the dihydrochloride, the diacetate or the D-diglyconate, solvates of chlorhexidine and solvated salts of chlorhexidine.

One embodiment of the present invention relates to a composition, such as pharmaceutical composition, comprising chlorhexidine and a C3-C8 alkanediol. such as a C3-C5 diol. In one specific embodiment the C3-C8 alkanediol is selected from pentane- 1,5 -diol, propane- 1,2-diol, butane- 1,3 -diol, pentane- 1,2-diol, hexane- 1,2-diol, hexane- 1 ,6-diol and 2-methyl-pentane-2,4-diol. In one other specific embodiment the C3-C8 alkanediol is selected from propane- 1 ,2-diol and pentane- 1,5-diol.

In yet one embodiment, according to the examples below, the C3-C8 alkanediol is pentane- 1,5-diol.

It was envisaged by the present inventor that a synergistic antimicrobial effect is achieved when chlorhexidine is combined with a C3-C8 alkanediol, such as pentane- 1,5-diol. Thus, it was envisaged that a composition, comprising both chlorhexidine and a C3-C8 alkanediol, such as pentane-l,5-diol, have higher antimicrobial effect than compositions comprising the same concentrations of either chlorhexidine or C3-C8 alkanediol, such as pentane-l,5-diol. Specifically, the synergistic effect between chlorhexidine and pentane-l,5-diol is shown in the examples below. Although, WO 07/063065 discloses that pentane-1, 5- diol, when used to reduce odour from a mammal, may be combined with an antiseptic agent, it is silent about any possible synergistic effect. Furthermore, WO 07/063065 is silent about suitable concentrations to be used of said components in a pharmaceutical composition. According to WO 07/063065, at least 0.1 wt% pentane-l,5-diol should be employed. However, as disclosed herein, such a concentration is to low to have any synergistic effect when combined with chlorhexidine.

However, it is not unlikely that a synergistic effect also is obtained by the combination of chlorhexidine and another C3-C8 alkanediol than pentane-l,5-diol, but pentane-1 ,5-diol is preferred as it is well known with a preferable toxicological profile, low risk for sensibility and skin irritation. Due to the synergistic antimicrobial effect it may be possible to use less of each component and still obtain the desired effect. Further, by using less of each component, each component may give rise to fewer side effects and less severe side effects than used alone. A composition comprising both chlorhexidine and pentane-1, 5-diol is more effective to combat microorganisms than a composition only comprising one active component.

Without being bound to any theory it may be assumed that the mechanism of action of chlorhexidine, i.e. the disruption of lipid membrane, is aided by the specific hydrophobicity of C3-C8 alkanediols. Especially, the hydrophobicily of pentan-1, 5-diol appears to be significantly suitable in this respect. Further, the solubilizing properties of C3-C8 alkanediols may be important in this context. Alternatively, the antimicrobial effect of C3-C8 alkanediols is improved by the simultaneous disruption of lipid membrane caused by chlorhexidine. Another theory is that the mechanism of action of the antimicrobial effect of C3-C8 alkanediols is the dissolution of the lipid membranes, thereby enhancing the ability of chlorhexidine to disrupt said lipid membrane, and vice versa. Perhaps, the dissolution effect and hydrophobicity of pentane-1, 5-diol cooperates with chlorhexidine in a way maximizing the synergistic effect there between.

A pharmaceutical composition comprising chlorhexidine and pentane-1 , 5-diol may be used in therapy. When used in therapy it may be administered topically. Such a topically administered composition may be used to treat various infections, such as infections affecting the skin or mucous membranes. Examples of such skin infections include impetigo, secondary infected atopic dermatitis and other dermatosis with secondary infections. Chlorhexidine and pentane-l,5-diol may also be used to treat fungal skin and mucous membrane infections such as tinea pedis, tine corporis, tinea unguium, cutaneous and mucous membrane Candida infections etc. and viral infections such as herpes infections (e. g. herpes labialis), papilloma and pox skin or mucous membrane infections

A pharmaceutical composition, comprising chlorhexidine and a pentane-1,5- diol, may also be used to inactivate microorganisms selected from the group consisting of gram positive and gram negative bacteria, fungi, including yeasts, moulds and dermatophytes and virus. Examples of such microorganisms include, but are not limited to, Staphylococcus aureus, P. acnes, Streptococci, gram negative rodes, Candida albicans, Candida glabrata, Malassezia, the mould Aspergillus fumigatm and the dermatophytes Trichophyton rubrum, T. menlagrophytes, Epidermophyton floccusum, Microsporum canis, papilloma virus, herpes virus and pox virus. A composition, comprising chlorhexidine and pentane-l,5-diol, has been shown to be especially effective in affecting, i.e. killing off, combating or preventing, Staphylococcus aureus and P. acnes, as disclosed in the examples below.

A composition for topical administration may take the form of- or being integrated in - a liquid, semi-liquid or solid disinfectant preparation, a bacteriostatic or bacteriocidal solution, lotion, cream, soap, shampoo, ointment, paste, wet towel, hygiene dish, patch, diaper or similar personal hygiene protection device.

If intended for application to the skin or mucous membranes, the composition according to embodiments of the invention may comprise one or several of tonicity adjustment agent or moisturizing agent, such as sodium chloride, urea and lactic acid, w-absorbing agent, colorant, such as calcium carbonate and zinc oxide, and fragrant, such as an aetheric oil. The composition of the invention may also comprise a cationic, neutral, or anionic detergent, in particular a salt of a fatty acid.

Preferably, a composition comprising chlorhexidine and a C3-C8 alkanediol, and preferably pentane-1 ,5-diol, also comprises a carrier. The carrier may be an aqueous carrier or a lipid carrier, which further may comprise a thickening agent. Such thickening agents may be selected from cellulose derivatives, in particular methyl cellulose, hydroxymethyl cellulose, hydroxymethyl-propyl cellulose. According to a further embodiment of the invention, the carrier comprises a salt of a fatty acid. Another embodiment of the invention relates to pharmaceutical compositions as disclosed above, which further includes one or more pharmaceutically acceptable adjuvants or excipients. Such pharmaceutical compositions may be prepared in a manner known in the art and are sufficiently storage-stable and suitable for administration to humans and animals.

A pharmaceutical composition as disclosed herein to be administered topically may take the form of ointments, lotions, pastes, creams, gels, talc, sprays, solutions and emulsions as readily understood to the one skilled in the art. Such ointments, lotions, creams and gels may comprise excipients, such as animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicons, bentonites, silicic acid, talc and zinc oxide or mixtures of these substances. Talc and sprays may comprise excipients, such as lactose, talc, silica acid, aluminium hydroxide, calcium silicate and polyamide powders or mixtures thereof. Sprays may also comprise propellants such as chloroflourohydrocarbons. Solutions and emulsions may comprise excipients, such as solvents, solubilising agents and emulsifiers such as water, ethyl alcohol, isopropylalcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene alcohol, dimethylformamide, oils such as cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, glycerol formal, tetrahydrofurfuyl alcohol, polyethylene glycols and fatty acid esters such as sorbitan or mixtures thereof.

A pharmaceutical composition as disclosed herein may be administered to a patient in a pharmaceutically effective dose. By "pharmaceutically effective dose" is meant a dose that is sufficient to produce the desired effects in relation to the condition for which it is administered. The exact dose is dependent on the, activity of the compound, manner of administration, nature and severity of the disorder, age and body weight of the patient, and different doses may be needed. The administration of the dose can be carried out both by single administration in the form of an individual dose unit or by several smaller dose units or by multiple administrations of subdivided doses at specific intervals. The "patient" for the purposes of the present invention includes both humans and other mammal. Thus the methods are applicable to both human therapy and veterinary applications.

One further embodiment of the present invention relates to a composition, such as a pharmaceutical composition, comprising at least 0.000001 wt%, such as at least 0,00005 wt% chlorhexidine and at least 1 wt% of a C3-C8 alkanediol, such as pentane- 1,5-diol.

Another embodiment of the present invention relates to a composition, such as a pharmaceutical composition, comprising 0.000001 wt% to 5 % wt%, such as 0,00005 to 5 wt% or 0,00005 to 1 wt%, chlorhexidine and 1 to 75 wt%, such as 1 to 25 wt%, 2 to 15 wt%, or 3 to 15 wt%, of a C3-C8 alkanediol, such as pentane-l,5-diol.

Another embodiment of the present invention relates to a composition, such as a pharmaceutical composition, comprising at least 0,00001 wt% chlorhexidine and at least 3 wt% of pentane-l,5-diol. Although, lower concentrations may be used, the antimicrobial effect is more pronounced at or above these concentrations.

Another embodiment of the present invention relates to a composition, such as a pharmaceutical composition, comprising at least 0,0001 wt% chlorhexidine and at least 5 wt% of pentane-l,5-diol.

As higher concentrations may increase the risks of side effects, another embodiment of the present invention relates to a composition, such as a pharmaceutical composition, comprising between 0,0001 and 5 wt%, such as 0,001 and 2.5 chlorhexidine and between 3 and 25 wt%, such as between 5 and 15 wt%, of pentane- 1,5-diol.

Another embodiment of the present invention relates to a pharmaceutical composition comprising at least 0,001 wt% chlorhexidine and a C3-C8 alkanediol, such as pentane-l,5-diol.

Another embodiment of the present invention relates to a pharmaceutical composition comprising chlorhexidine and at least 2 wt% of a C3-C8 alkanediol, such as pentane-l,5-diol. Another embodiment of the present invention relates to a pharmaceutical composition comprising chlorhexidine and at least 5 wt% of a C3-C8 alkanediol, such as pentane-l,5-diol.

When combined together, chlorhexidine and pentane-l,5-diol give rise to synergistic antibacterial effects as shown in the examples below. These synergistic effects make the use of lower amounts of the respective compounds possible, while still obtaining the desired effect. A combination of chlorhexidine and pentane-l,5-diol is effective in preventing growth of various microorganisms. Such microorganisms include bacteria, fungi and virus. Such bacteria include, but are not limited to, S. aureus and P. acnes. Similarly to chlorhexidine, also a composition comprising chlorhexidine and a" C3-C8 alkanediol may be used to used for general skin cleansing, such as in surgical scrubs for pre-operative skin treatment. The disinfection achieved by use of such a composition will be more effective than if only chlorhexidine is used. Furthermore, the well-known water binding effect of diols make them perfect moisturizers to the skin and will therefore make such a treatment less harsh when applied to the skin.

Furthermore, a composition comprising chlorhexidine and a C3-C8 alkanediol may be used in other types of disinfections and cleaning. Without limitations such disinfections may include treatment of various surfaces, such as floors, walls, table tops, bench tops, table leafs and surgical instruments,. Such disinfections may also be combined with autoclavation or may even replace it. In one embodiment such disinfections includes disinfection of skin but other embodiment relating to disinfection excludes disinfections of parts of a human or animal body. Such disinfections excluding disinfections of parts of a human or animal body is to be distinguished from a therapeutic method.

In an embodiment wherein a composition comprising chlorhexidine and a C3- C8 alkanediol is used to disinfect and/or clean a surface not part of an animal or human body, the content of C3-C8 alkanediol may be 1-95 wt% and the content of chlorhexidine 0.000001 % to 10 wt%. Although the present invention has been described above with reference to specific illustrative embodiments, it is not intended to be limited to the specific form set forth herein. Rather, the invention is limited only by the accompanying claims and other embodiments than the specific above are equally possible within the scope of these appended claims. In the claims, the term "comprises/comprising" does not exclude the presence of other species or steps. Additionally, although individual features may be included in different claims, these may possibly advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and/or advantageous. In addition, singular references do not exclude a plurality. The terms "a", "an", "first", "second" etc do not preclude a plurality.

Examples

The examples given below are only intended to further illustrate the invention and are by no means intended to limit the scope of the invention as defined by the appended claims. Example 1, MIC of pentane-l,5-diol and chlorhexidine against S. aureus The minimal inhibitory concentration (MIC) of pentane-l,5-diol and chlorhexidine against S. aureus was determined using bacteria concentrations of 6 x 10 ⁶ (20 μl applicated onto DST-agar plates). The results are depicted below in table 1 and 2.

Table 1. MIC of pentane-l,5-diol against S. aureus and P. acnes

Pentane-1 ,5-dιol MIC

(wt %) (S aureus)

5 +++ ^a

6 ++

7 ++

8 + 9

10 20 30 Control (no pentane-1 ,5-dιol) +++ a: +++: Same growth as on the control plate without any chlorhexidine and/or pentane-l,5-diol ++: Reduced growth compared to the control

+: Very poor growth -: No growth

Table 2. MIC of chlorhexidine digliiconate against S. aureus

Chlorhexidine (wt %) MIC

0,00001 ++H

0,00005 H-

0,0001 -

0,0005 -

0,001 -

0,005

0,01 -

0,02 -

0,05 0,1 0,5 1 Control (no chlorhexidine) +++

Example 2, MIC of pentane-1, 5-diol in combination with chlorhexidine against S. aureus

The minimal inhibitory concentration (MIC) of pentane-l,5-diol combined with chlorhexidine against S. aureus was determined using bacteria concentrations of 6 x 10 ⁶ (20 μl applicated onto DST-agar plates). The results are depicted below in table 3.

Table 3. MIC of pentane-1 ,5-dioI combined with chlorhexidine dighiconate against S. aureus

Chlorhexidine Chlorhexidine hlorhexidine

IVIIw J I\V/|I|p C I w IVIIO

0,0001 % and 0,00005% and 0,00001 % and

9% Pentane-1 ,5-dιol 9% Pentane-1 ,5-dιol 9% Pentane-1 ,5-dιol

8% Pentane-1 ,5-dιol 8% Pentane-1 ,5-dιol 8% Pentane~1 ,5-dιol

7% Pentane-1 ,5-dιol 7% Pentane-1 ,5-dιol 7% Pentane-1 ,5-dιol +

6% Pentane-1 ,5-dιol 6% Pentane-1 ,5-dιol 6% Pentane-1 ,5-dιol

5% Pentane-1 ,5-dιol 5% Pentane-1 ,5-dιol 5% Pentane-1 ,5-dιol +++

As seen from table 1-3 there is clearly a synergistic antibacterial effect in combining pentane-1, 5 -diol with chlorhexidine digluconate. While neither chlorhexidine in concentrations at or below 0,00005% nor pentane-1, 5-diol in concentrations at or below 8 % is effective agains S. aureus, a combination of 0,00005 % chlorhexidine with 5% or more of pentane-1, 5-diol is. Further, even such low concentrations of chlorhexidine and pentane-1, 5-diol as 0,00001 % and 7 %, respectively, are effective when combined.

Example 3, MIC of pentane-1, 5-diol and chlorhexidine against P. acnes The minimal inhibitory concentration (MIC) of pentane-1, 5-diol and chlorhexidine against P. acnes was determined using bacteria concentrations of 6 x 10 ⁶ (20 μl applicated onto blood agar plates). The results are depicted below in table 4 and 5.

Table 4. MIC of pentane-l,5-diol against P. acnes Pentane-1 ,5-dιol (wt % ) MIC

1 +++

3 ++

5 +

6 -

7 -

10 -

20 -

30 -

Control (no pentane-1 ,5-dιol) +++

Table 5. MIC of chlorhexidiiie diglueoeate against P. acnes

Chlorhexidine (wt %) MIC

0,00001 +++

0,00005 ++

0,0001 0,0005 0,001 0,005 0,01 0,02 0,05 0,1 0,5 1 Control (no chlorhexidine) +++

Example 4, MIC of pentane-1, 5-diol in combination with chlorhexidine against P. acnes

The minimal inhibitory concentration (MlC) of pentane-1, 5-diol combined with chlorhexidine against P. acnes was determined using bacteria concentrations of 6 x

10 ⁶ (20 μl applicated onto blood agar plates). The results are depicted below in table 6.

Table 6. MIC of pentane-1, 5-diol combined with chlorhexidine digluconate against P. acnes

Chlorhexidine Chlorhexidine Chlorhexidine

MIC MIC MIC

0,0001 % and 0,00005% and 0,00001 % and 6% Pentane-1 ,5-dιol 6% Pentane-1 ,5-djol - 6% Pentane-1 ,5-dιol +

5% Pentane-1 ,5-dιol 5% Pentane-1 ,5-dιol - 5% Pentane-1 ,5-dιol ++

3% Pentane-1 ,5-dιol 3% Pentane-1 ,5-dιol - 3% Pentane-1 ,5-dιol +++

1 % Pentane-1 ,5-dιol 1 % Pentane-1 ,5-dιol ++ 1 % Pentane-1 ,5-dιol +++

As seen from table 4-6 there is clearly a synergistic antibacterial effect in combining pentane-1, 5-diol with chlorhexidine digluconate. While neither chlorhexidine in concentrations at or below 0,00005% nor pentane-1, 5-diol in concentrations at or below 5 % is effective agains P. acnes, a combination of 0,00005 % chlorhexidine with 3% or more of pentane-1, 5-diol is.

Example 5, MIC of pentane-1, 5-diol in combination with chlorhexidine against S. pyogenes A CCUG 4208 The minimal inhibitory concentration (MIC) of pentane-1, 5-diol combined with chlorhexidine against S. pyogenes A CCUG 4208 was determined using bacteria concentrations of 6 x 10 ⁷ (20 μl applicated onto blood agar plates). The results are depicted below in table 7 and 8.

Table 7, MIC of chlorhexidine digluconate and 1,5-pentanediol against S. pyogenes

1,5-pentanediol (%) MIC chlorhexidine digluconate (%) MIC

2 +++ 0.00001 +++

4 ++ 0.00005 +++

6 + 0.0001 +

7 0.0005 -

8 0.001 -

9 0.005 _

10 0.01 -

15 0.05 _

0.1

0.5 -

The positive control (agar plate without substance) showed a MIC value corresponding to +++. Table 8. MIC of pentane-l,5-diol combined with chlorhexidine digluconate against S. pyogenes

1,5-pentandiol MIC

#1 Chlorhexidinne digluconate 0,00001% ++ #2 Chlorhexidinne digluconate 0,00005% + #3 Chlorhexidinne digluconate 0,0001 % -

1,5-pentandiol (4% MIC

#4 Chlorhexidine digluconate 0,00001%

#5 Chlorhexidinne digluconate 0,00005%

#6 Chlorhexidinne digluconate 0,0001

1,5 -pentandiol (6%) MIC

#7 Chlorhexidine digluconate 0,00001 %

#8 Chlorhexidinne digluconate 0,00005%

#9 Chlorhexidinne digluconate 0,0001 %

As seen from table 7 and 8 there is clearly a synergistic antibacterial effect in combining pentane-l,5-diol with chlorhexidine digluconate. While neither chlorhexidine in concentrations at or below 0,0005% nor pentane-l,5-diol in concentrations at or below 6 % is effective agains S. pyogenes, a combination of 0,00001 % chlorhexidine with 4% or more of pentane-l,5-diol is.

Example 6 MIC of pentane-l,5-diol in combination with chlorhexidine Chlorhexidine digluconate (CHG) solution, 20% water, C9394-25ML, (Sigma, St. Louis, USA) and PD, (BASF AG, Ludwigshafen, Germany) were used in the study and the following reference strains were tested; C. albicans (CCUG 5594), S. aureus (CCUG 17621) and P. acnes (CCUG 1794).

Minimum inhibitory concentration of CHG and pentane-l,5-diol (FD) were determined by agar dilution assay. A series of dilutions of each compound were prepared in blood agar medium for P. acnes, and in Diagnostic Sensitivity Test (DST) agar (Oxoid, Basingstoke, UK) for C. albicans and S. aureus. Dilution series with each agent were made" with freshly prepared agar at 48-50 ⁰ C. Final concentration ranges were as follows: CHG solution, 0.00001 to 1 % (w/v) and PD, 1-30% (v/v). The prepared plates were solidified and used immediately.

For each organism an inoculum was prepared by growing organisms on blood agar. Colonies were suspended in phosphate-buffered saline (PBS) and suspensions were adjusted to approximately 6x10 ⁶ CFU/ml for C. albicans and P. acnes and 6x10 ⁵ CFU/mL for S. aereus. The plates were inoculated with 20 μl spots and incubated for one day for C. albicans and S. aureus and three days for P. acnes. MICs were then determined as the lowest concentration of the compound inhibiting growth of the isolates.

First the MICs of CHG and PD, respectively, were determined for each microorganism. Then, a range of combined CHG and PD concentrations were tested on each micro-organism to determine if there was synergistic activity. The definition of synergistic effect is: "An effect that is greater than the sum of the effects of the two drugs, such as the equation: 1+1...". Each isolate was tested twice.

CHG was effective against all micro-organisms (Table 9). MICs of CHG were 0.0005 (% w/v) against C. albicans and 0.0001 (% w/v) against S. aureus and P. acnes. PD demonstrated an activity against C. albicans, S. aureus and P. acnes of 5, 9 and 6, % v/v, respectively. MIC of CHG was much lower than PD with a factor of Ix 10 ^"4 for C. albicans, 6xlO ^"5 for S. aureus and 9xlO ^"5 for P. acnes (Table 9).

When CHG and PD were combined, in concentrations lower than their respective MICs, their antimicrobial activity increased. MICs of CHG was reduced from 0.0005 to 0.00005 (%, w/v) and MICs of PD from 5 to 3 (%, v/v) for totally preventing growth of C. albicans . MICs of CHG was reduced from 0.0001 to 0.00001 (%, w/v) and MICs of PD from 9 to 8 (%, v/v) for S. aureus. MICs of CHG was reduced from 0.0001 to 0.00005 (%, w/v) and MICs of PD from 6 to 3 (%, v/v) for totally preventing growth of P. acnes (Table 9).

Table 9. Minimum Inhibitory concentration (MIC) of chlohexidine diglueonate (CHG) (w/v %) and pentane-l,5-diol (PD) (v/v %) alone and in combination against

C. albicans, S. aureus and P. acnes by agar dilution method. Ratio indicates reduction of the MIC of CHG and PD alone and in combination. Strains MIC alone MIC Ratio (alone/combined) combined CHG PD CHG PD CHG/CHG+PD PD/PD+CHG

C. albicans 0.0005 5 0.00005 3 10 2

CCUG5594

S. aureus 0.0001 9 0.00001 8 10 1

CCUGl 7621

P. acnes 0.0001 6 0.00005 2 2

CCUGl 794