The present application is concerned with methods for identifying and selecting formulation compositions suitable for delivery of an antimicrobial peptide to skin to produce an antimicrobial effect on the skin, and the antimicrobial formulations therefrom, such as emulsions, for use in skincare, such as skin creams.

Antimicrobial formulations for use in skincare have existed for many years, however the effectiveness of many such formulations has been shown to be limited, and many contain harsh chemicals.

An alternative approach to skincare formulations is to utilise natural products as the active agents, such as simple peptides, and a number of such formulations have indeed been produced. However, the effectiveness of many of these formulations is questionable, as is whether it is the peptide that provides the antimicrobial effect.

Antimicrobial Peptides are effective, fast-acting microbicidal agents with broad spectrum activity against a wide variety of gram positive and gram negative bacteria and enveloped viruses. Some antimicrobial peptides also have immune-modulation and/or wound healing properties. Antibacterial activity can be both bacteristatic and bactericidal.

Peptides are usually between 9 and 100 amino acids in length, and can be straight chain, circular, or covalently linked multimers. In some cases antimicrobial peptides exert their antimicrobial activity through non-covalently linked multimers of identical or near-identical peptides that interact with target membranes to form pores through the membrane that depolarize the membrane with respect to ionic and/or osmotic gradients. In some cases antimicrobial peptides interact with target membranes to structurally destabilize the membrane such that it disintegrates partially or completely.

The properties of antimicrobial peptides make them potentially suitable for topical use for the control of bacteria on or in the skin.

The present invention generally aims to produce improved formulations for delivery of antimicrobial peptides for use in skincare products, and in particular to enhance the antimicrobial effectiveness of such peptides in skincare formulations.

Accordingly in a first aspect, the present invention provides an antimicrobial formulation comprising an antimicrobial peptide, and at least one reagent selected from sunflower oil, petrolatum, cetostearyl alcohol, and lanolin.

In one embodiment, the formulation additionally comprises water, and in a preferred embodiment the formulation is an emollient, which may be an emulsion, preferably a stable emulsion, i.e. one that is at least stable for a few hours, but preferably for days, weeks, or even months.

One embodiment of the first aspect is a formulation comprising an antimicrobial peptide, sunflower oil, petrolatum, cetostearyl alcohol, and water, wherein the formulation is an emulsion.

Another embodiment of the first aspect is a formulation comprising an antimicrobial peptide,, sunflower oil, petrolatum, lanolin, and water, wherein the formulation is an emulsion.

Petrolatum is a major constituent of Vaseline®, and thus one reagent could be Vaseline®. Sunflower oil is a mixture of triglycerides, often including palmitic acid, stearic acid, oleic acid, and linoleic acid, and thus in one embodiment the at least one reagent could be one or more of these ingredients.

Cetostearyl alcohol is a mixture of fatty alcohols, mainly cetyl and stearyl fatty alcohols, and is a known emulsion stabiliser, and thus in one embodiment the at least one reagent could be cetyl or stearyl alcohol, or a mixture thereof, or could be an alternative emulsion stabiliser.

Lanolin is a wax (wool wax or wool grease), and is a mixture of long chain waxy esters, and is a known emulsion stabiliser and thus in one embodiment the at least one reagent could be lanolin, or could be an alternative emulsion stabiliser.

The formulation should preferably comprise minimal or no salt, or at most only a low concentration of salt, such as a concentration of 10 mM or 100 mM. In bulk solution the a-helical content of antimicrobial peptides has been shown to be stabilized by increasing salt concentration. The antibacterial activity of antimicrobial peptides is attenuated or abrogated by increasing salt concentration.

Particular embodiments of the first aspect are a formulation consisting of at least one antimicrobial peptide, water and at least one reagent selected from the list consisting of sunflower oil, petrolatum, cetostearyl alcohol and lanolin. In one embodiment, the at least one reagent consists of sunflower oil, petrolatum and cetostearyl alcohol, wherein the relative percentage of water, sunflower oil, petrolatum and cetostearyl alcohol may be about 50.0 (v/v)/ 37.5 (v/v)/ 12.5 (v/v)/ 2.0 (w/v), respectively, or may be dilutions thereof, such as 80% v/v formulation, such as in water, which may be due to introducing the antimicrobial peptide (in solution) into the formulation. In another particular embodiment, the at least one reagent consists of sunflower oil, petrolatum and lanolin, wherein the relative percentage of water, sunflower oil, petrolatum and lanolin may be about 50.0 (v/v)/ 37.5 (v/v)/ 12.5 (v/v)/ 2.0 (w/v), respectively, or may be dilutions thereof, such as 80% v/v formulation, such as in water.

The Applicant has shown that the amount of a-helical conformation of an

antimicrobial peptide in an emollient is critical to the effectiveness of the antimicrobial activity of that peptide in the emollient. In particular the amount of oc-helix content should often be less than 35%, based on circular dichroism analysis.

The formulations of the first aspect have the effect of abrogating peptide oc-helix content, with the effect that the antimicrobial activity is enhanced.

The Applicant has also shown that the addition of a non-ionic detergent such as Tween® 20 has the effect of stabilizing or increasing α-helical content or structure of antimicrobial peptides in a similar manner to increasing salt concentration, and thus should be avoided for use in the emollient. In a preferred embodiment the

formulation does not comprise a non-ionic detergent, and in particular does not comprise Tween® 20.

In a second aspect, the present Invention provides a method of selecting a

formulation composition suitable for delivery of an antimicrobial peptide to skin to produce an antimicrobial effect on the skin, comprising producing a mixture of reagents potentially suitable for such a composition, adding the antimicrobial peptide, and evaluating the ot-helix content of the peptide in the formulation. Evaluation of the a-helix content of the peptide could be performed using circular dichroism. Evaluation of the a-helix content of the peptide could be performed indirectly by assessing features that are dependent on a-helix content of the peptide, such as recognition or non-recognition irvan Enzyme Linked Immunosorbent Assay (ELISA) that is affected by a-helix content of the peptide.

The method of the second aspect enables formulation compositions for maintaining and/or enhancing antibacterial activity in skincare formulations, such as emollients, to be identified, based on the discovery by the Applicant that the a-helix content is related to the effectiveness of the peptide when delivered to the skin. Indeed the a- helical content or structure of the antimicrobial peptide in the emollient preparation should be attenuated. In particular, the a-helix content is most likely less than 40%, less than 35%, less than 30%, or less than 20%, and may be less than 10%.

Attenuation of antimicrobial peptide a-helicity in a skincare formulation, such as an emollient suitable for washing and/or moisturizing skin, allows for effective and easy topical application of antimicrobial peptides to human or animal skin for the control of bacteria and bacterial infections of the skin. This includes uses for the treatment of skin infections, the treatment and prevention of acne, the treatment and prevention of athlete's foot, and the provision and maintenance of hand and skin hygiene, in a moisturizing and/or non-astringent emollient formulation.

The non-ionic detergent Tween® 20 is shown to stabilize or increase a-helical content or structure of antimicrobial peptides in a similar manner to increasing salt concentration, and thus should be limited or avoided in any such formulation. Indeed the Applicant has shown that the bactericidal activity of antimicrobial peptides is inhibited or abrogated by formulation in the non^ionic detergent Tween® 20, providing confirmation of the role of attenuating a-helical content or structure of the antimicrobial peptide in maintaining or improving antibacterial activity of the antimicrobial peptide in skincare formulations.

The Applicant has further shown that antimicrobial peptides segregate into the aqueous phase of emulsified emollient preparations, and that attenuation of antibacterial activity in commercially available emollients correlates with an aqueous phase interferent.

Indeed detection of antimicrobial peptide LL37 in ELISA is attenuated by the aqueous phase of many commercially available emollients.

In addition to attenuation by addition of Tween® 20, attenuation of antibacterial activity of antimicrobial peptide LL37 in emollients correlates with attenuation of detection of LL37 in the aqueous phase of some commercial emollients in capture ELISA.

All of the above demonstrates a link between the stabilization of pre-contact (with skin and/or bacteria) a-helical secondary structure and attenuation of antibacterial activity of the antimicrobial peptide.

The present invention will now be described with reference to the following non- limiting examples and drawings in which

Figure 1 illustrates the enhancement of antimicrobial peptide a-helical structure by increasing concentrations of Tween® 20 by Circular Dichroism spectroscopy, for Magainin-2B (A), and LL37 (B); Figure 2 illustrates the enhancement of antimicrobial peptide a-helical structure by increasing concentrations of Tween® 20 by Circular Dichroism spectroscopy, for Magainin-2B (A), and LL37 (B);

Figure 3 illustrates the attenuation of antimicrobial peptide antibacterial activity by Tween® 20. E. coli bacteria were incubated with either LL37 (A) or Magainin-2B (B);

Figure 4 illustrates attenuation of antimicrobial peptide antibacterial activity by the salts NaF and NaCI;

Figure 5 illustrates attenuation of antimicrobial peptide antibacterial activity by commercially available emollients;

Figure 6 illustrates maintenance of antimicrobial peptide antibacterial activity in emollient Prep C, emollient Prep L, and emollient Prep N;

Figure 7 illustrates maintenance of antimicrobial peptide antibacterial activity in emollient Prep C, and attenuation of antimicrobial peptide antibacterial activity by addition of Tween® 20;

Figure 8 illustrates attenuation of Enzyme Linked Immunosorbent Assay (ELISA) detection of antimicrobial peptide LL37 by commercially available emollients;

Figure 9 illustrates attenuation of Enzyme Linked Immunosorbent Assay (ELISA) detection of antimicrobial peptide LL37 by Tween® 20;

Figure 10 illustrates attenuation of antimicrobial peptide antibacterial activity by the aqueous phase of a commercially available emollient preparation; and Figure 11 illustrates attenuation of antimicrobial peptide antibacterial activity by the aqueous phase of a commercially available emollient preparation.

Examples

Having regard to Figure 1 , Far-UV CD spectra were recorded for Magainin-2B (A) and LL37 (B) in the absence (solid lines) and presence (dashed lines) of 0.4%

Tween® 20 at 37 °C. Insets show the degree of a-helicity at 37 °C derived from

[Θ]222 plotted as a function of the molar detergent-to-peptide ratios.

Having regard to Figure 2, the degree of α-helicity at 37 °C derived from [Θ]222 is plotted as a function of the percentage concentration of Tween® 20 (v/v).

Having regard to Figure 3, E. coli bacteria were incubated with either LL37 (A) or Magainin-2B (B) at a concentration of 100 g/ml for 10 minutes in L-broth. Peptides (0) and no-peptide controls (■) were adjusted to either 0.5% Tween® 20 or an equal amount of diluent (H2O) before addition of bacteria. After incubation with

antimicrobial peptide for 10 minutes, samples were serially diluted in L-broth to halt the assay, and the dilutions inoculated onto L-agar plates and incubated overnight at 37 ^° C. After this, colonies were enumerated for 50% colony forming units using the Reed-Muench method. Asterisk indicates where no colonies were recovered (100% kill).

Having regard to Figure 4, E. coli or S. aureus bacteria were incubated with either LL37 or Magainin-2B at a concentration of 100 Mg/ml for 10 minutes in H ₂ 0. Peptides and no-peptide controls were adjusted to either 500 (■), 250 (0), or 0 (H) mM NaF or 500 (■), 250 (0), or 1 1 (H) mM NaCI before addition of bacteria. After incubation with antimicrobial peptide for 10 minutes, samples were serially diluted in L-broth to halt the assay, and the dilutions inoculated onto L-agar plates and incubated overnight at 37 ^° C. After this, colonies were enumerated for 50% colony forming units using the Reed-Muench method. Asterisk indicates where no colonies were recovered (100% kill).

Having regard to Figure 5, E. coli or S. aureus bacteria were incubated with LL37 at a concentration of 100 Mg/ml for 10 minutes in 80% (v/v) E45™ emollient wash, 80% (v/v) aqueous cream, or 80% (v/v) Nivea™ lotion. Antimicrobial peptide (0) and no- peptide control (■) were blended into emollient formulation or H ₂ 0 before addition of bacteria. After incubation with antimicrobial peptide for 10 minutes, samples were serially diluted in L-broth to halt the assay, and the dilutions inoculated onto L-agar plates and incubated overnight at 37 ^° C. After this, colonies were enumerated for 50% colony forming units using the Reed-Muench method. Asterisk indicates where no colonies were recovered (100% kill).

Having regard to Figure 6, £. coli bacteria were incubated with either LL37 (0) or CaLL (H) antimicrobial peptides at a concentration of 100 pg/ml, or no-peptide (PBS) control (■) for 10 minutes in Preparation C (A), Prep L (B), Prep N (C), or H ₂ 0 (D). Antimicrobial peptides and no peptide control were blended into the emollient preparations before addition of bacteria (final concentration of emollient 80% v/v). After incubation with antimicrobial peptide for 10 minutes, samples were serially diluted in L-broth to halt the assay, and the dilutions inoculated onto L-agar plates and incubated overnight at 37 ^° C. After this, colonies were enumerated for 50% colony forming units using the Reed-Muench method. Asterisk indicates where no colonies were recovered (100% kill). In all preparations, the antimicrobial activity of both peptides was clearly maintained (i.e. not attenuated or abrogated), or even enhanced, as no colonies were recovered from use of either peptide in any of the formulations.

Having regard to Figure 7, E. coli bacteria were incubated with LL37 at a concentration of 100 g/ml for 10 minutes in 80% (v/v) Preparation-C (S), or 80% ι

(v/v) Preparation-C adjusted to 0.08% (1 ^st panel) or 0.04% (2 ^nd panel) (v/v) Tween® 20 (■). Antimicrobial peptide and no-peptide (vehicle) control were blended into emollient formulation before addition of bacteria. After incubation with antimicrobial peptide for 10 minutes, samples were serially diluted in L-broth to halt the assay, and the dilutions inoculated onto L-agar plates and incubated overnight at 37 ^° C. After this, colonies were enumerated for 50% colony forming units using the Reed- Muench method. Asterisk indicates where no colonies were recovered (100% kill). The antimicrobial activity of peptide LL37 was clearly maintained (i.e. not attenuated or abrogated), or even enhanced, in Preparation C as no colonies were recovered, but that both 0.08% and 0.04 % Tween®20 attenuated the antimicrobial activity, which the Applicant has shown is in direct correlation with the degree of oc-helicity being increased in the presence of Tween®20.

Table 1 : Preparation C Formulation is a strong emulsion containing:

Cetostearyl alcohol 40 mg 2% w/v

Sunflower oil 750 ul 37.5% v/v

Vaseline® 250 ul 12.5% v/v

H ₂ 0 1000 ul 50% v/v Having regard to Figure 8, LL37 was blended with commercially available emollients to a concentration of 100 Mg/ml peptide and 90% (v/v) emollient. The no peptide controls were emollient similarly blended with PBS. Preparations were fractionated into distinct phases by centrifugation, and the aqueous phase harvested for ELISA analysis. Samples were applied to a commercial antibody-capture ELISA specific for LL37 after serial dilution in the manufacturer supplied diluent. As a positive control, a portion of the negative control sample was "spiked" with LL37 to a concentration equivalent to that expected if 100% of the LL37 in the LL37 experimental sample was found in the aqueous phase. E45™ emollient wash does not contain H ₂ 0. In samples prepared with E45™ emollient wash, the aqueous phase derives from the aqueous solution of antimicrobial peptide (or PBS) used to make the preparation. Data is presented as means of duplicate samples.

Having regard to Figure 9, LL37 at a fixed concentration of 1 ug/ml was pre-incubated with varying concentrations of Tween® 20 and applied to a commerciaj antibody- capture ELISA specific for LL37. As a positive control, LL37 was applied to the ELISA in H ₂ 0 in the absence of Tween® 20. As a negative control, PBS with no peptide was applied at a 1 :2000 dilution. Data is presented as means and standard deviations of triplicate samples.

Having regard to Figure 10, aqueous cream was fractionated into separate phases by centrifugation. The aqueous phase was harvested and used as an interferent in an assay of antimicrobial peptide antibacterial activity. E. coli bacteria were incubated with LL37 or CaLL antimicrobial peptides at concentrations of 3.2 mg/ml or 100 Mg/ml for 10 minutes. Interferent was added to peptides and controls at a concentration of 1 % (v/v) prior to addition of bacteria. After incubation with antimicrobial peptide for 10 minutes, samples were serially diluted in L-broth to halt the assay, and the dilutions inoculated onto L-agar plates and incubated overnight at 37 ^° C. After this, colonies were enumerated for 50% colony forming units using the Reed-Muench method. Asterisk indicates where no colonies were recovered (100% kill). Clearly the aqueous phase of aqueous cream interferes with the antimicrobial activity of antimicrobial peptides in a similar fashion to Tween®20, and it is thus believed that this is as a result of an ingredient within the aqueous cream stabilizing or increasing a-helical content or structure of the antimicrobial peptides, which the Applicant has shown to directly affect the antimicrobial activity of peptides. The Applicant believes that the other commercially available emollients and creams tested also affect the degree of oc-helicity in a similar fashion, with the result of reducing the antimicrobial activity of the peptides.

Having regard to Figure 1 1 , aqueous cream was fractionated into separate phases by centrifugation. The aqueous phase was harvested and used as an interferent in an assay of antimicrobial peptide antibacterial activity. E. coli bacteria were incubated with LL37 or CaLL antimicrobial peptides at a concentration of 100 _. ug/ml for 10 minutes. Interferent was added to peptides and controls at concentrations ranging from 80% to 0.008% (v/v) prior to addition of bacteria. After incubation with antimicrobial peptide for 10 minutes, samples were serially diluted in L-broth to halt the assay, and the dilutions inoculated onto L-agar plates and incubated overnight at 37 ^° C. After this, colonies were enumerated for 50% colony forming units using the Reed-Muench method. Clearly high concentrations (100%; 10%) of the interferent affected the antimicrobial activity of the peptides, but also concentrations as low as 1% interferent eradicated the antimicrobial activity. Lower concentrations had less of an effect on the antimicrobial activity.

Other formulations tested during the analysis were Prep L, Prep N (which was not fully emulsified), Prep SFE (which was a weak emulsion that collapsed after about 2 hours), and Prep 1 , which were all shown to maintain, or even enhance, the antimicrobial activity of the peptides tested.

Table 2: Prep L formulation is a strong emulsion containing: lanolin 40 mg 2% w/v sunflower oil 750 ul 37.5% v/v

Vaseline® 250 ul 12.5% v/v

H ₂ O 1000 ul 50% v/v

Table 3: Prep N formulation contains: sunflower oil 750 ul 37.5% v/v

Vaseline® 250 ul 12.5% v/v

H ₂ O 1000 ul 50% v/v

Table 4: Prep SFE formulation contains: sunflower oil 1000 ul 50% v/v H ₂ 0 1000 ul 50% v/v '

Table 5. Prep 1 formulation contains:

sunflower oil 250 ul 37.5% v/v

Vaseline® 500 ul 12.5% v/v glycerol 50 ul 50% v/v