FORMULATIONS COMPRISING INGENOL 3-(3,5-DIETHYLISOXAZOLE-4-

CARBOXYLATE)

TECHNICAL FIELD

The invention relates to formulation comprising ingenol 3-(3,5-diethylisoxazole-4-carboxylate). All documents mentioned in the text of this description are incorporated herein by reference. BACKGROUND OF THE INVENTION

Ingenol-3-angelate (PEP005, ingenol mebutate) is a diterpene-ester of the ingenol family which is isolated from various Euphorbia species, particularly from Euphorbia peplus (e.g. see

WO99/08994). Ingenol-3-angelate has been authorised in the USA, EU, Brazil, Australia and Canada for the treatment of actinic keratosis (also known as solar keratosis) under the brand name PICATO®.

Ingenol-3-angelate is believed to have a dual mode of action: 1) induction of cell death by direct cytoxicity or induction of apoptosis and 2) an immunostimulatory effect dominated by neutrophil recruitment and activation (Rosen, R.H., et al., J Am Acad Derm (2011); Ersvaer, E., et al., Toxins, (2010), 2, 174-194). Nanomolar concentrations of the agent cause activation and modulation of protein kinase C (PKC) classical and novel isoforms, with particular importance of PKCdelta. Through activation of PKCdelta the agent induces apoptosis in susceptible cells (Hampson, P., et al., Blood, (2005), 106, 1362-1368; Cozzi, SJ.,et al., Cancer Res, (2006), 66, 10083-10091). Rapid cytotoxicity on cancer cells is observed at high micromolar concentrations (Ogbourne, S.M., et al., Cancer Res (2004), 64, 2833-2839). Through activation of various PKC isoforms the agent also induces pro-inflammatory effects, including release of pro-inflammatory mediators (Challacombe, J.M., et al., J Immunol (2006), 177, 8123-8132, activation of vascular endothelium (Hampson, P., et al., Cancer Immunol Immunother, (2008), 57, 1241-1251);

chemoattraction of neutrophils through induction of interleukin 8 in keratinocytes and

development of specific anti-cancer immune responses by CD8+ cells through adjuvant properties in animal models (Le, T.T., et al., Vacccine, (2009), 27, 3053-3062).

However, angelic acid and angelic acid esters, such as those present in ingenol-3-angelate, are prone to isomerisation of the double bond to form the tiglate ester, particularly at basic pH

[Beeby, P., Tetrahedron Lett. (1977), 38, 3379-3382, Hoskins, W.M., J. Chem. Soc. Perkin Trans. 1, (1977), 538-544, Bohlmann, F. et. al., Chem. Ber. (1970), 103, 561-563].

Furthermore, ingenol-3-acylates are known to be unstable as they rearrange to afford ingenol-5- acylates and ingenol-20-acylates [Sorg, B. et. al, Z. Naturforsch., (1982), 37B, 748-756].

Other ingenol derivative have therefore been investigated as alternatives to ingenol-3-angelate. Ingenol-3-acylates, mainly of long-chain saturated and unsaturated aliphatic fatty acids, have been isolated from various Euphorbia species [H. Gotta, Z. Naturforschung, (1984), 39b, 683-94; K. Abo, Fitoterapia, (1988), 244-46, S. Zayed, J. Cancer Res. Clin. Oncol. (2001), 127, 40-47]. Furthermore, a small number ingenol-3-acylates have been prepared by semi-synthesis (B. Sorg et. al., Z. Naturforsch., (1982), 37b, 748-56). Some of these ingenol derivatives have been described and tested to be strong irritants and strong tumor-promoting agents. [B. Sorg et. al., Z. Naturforsch., (1982), 37b, 748-56; B. Sorg et. al., Carcinogenesis, (1987), 8, 1-4].

Besides the aliphatic ingenol esters, aromatic esters of ingenol are known. Milliamine C, an ingenol-3-anthraniloate derivative has been described (Marston, A. Planta Medica, (1983), 47, 141-47). Also ingenol-3-benzoate has been described (Sorg, B.; Z Naturforschung , (1982), 37b, 748-56). Various ingenol-3-acylates, including heteroaromatic and heterocyclic 3-O-acyl ingenol derivatives, and ingenol-3-carbamates have been disclosed in WO2012/083953.

STATEMENTS OF INVENTION

The present invention relates to the ingenol derivative, ingenol 3-(3,5-diethylisoxazole-4- carboxylate). The invention is based, in part, on the surprising discovery that formulations of ingenol 3-(3,5-diethylisoxazole-4-carboxylate) are more stable than similar formulations of ingenol mebutate, and are more efficacious than ingenol mebutate in eliminating dermal tumours in a mouse model.

In one aspect, the invention provides a formulation comprising: (a) ingenol 3-(3,5- diethylisoxazole-4-carboxylate); (b) a penetration enhancer; (c) a solvent; (d) a gellant; and (e) a buffer, wherein the formulation has a pH of no greater than 4.5 and no less than 2.5. In some embodiments of the formulation of the invention: (a) the penetration enhancer is present in an amount of between about 0.01% to about 40% w/w of the formulation; (b) the solvent is present in an amount of between about 0.5% to about 40% w/w of the formulation; (c) the gellant is present in an amount of between about 1% to about 20% w/w of the formulation; and/or (d) the ingenol 3-(3,5-diethylisoxazole-4-carboxylate) is present in an amount of between about 0.001% to about 0.5% w/w of the formulation. In some embodiments, (a) the penetration enhancer is isopropyl alcohol; (b) the solvent is benzyl alcohol; (c) the gellant is a hydroxyalkyl cellulose, such as hydroxyethyl cellulose; and/or (d) the buffer is citrate buffer. In some embodiments, the formulation has a pH of between 3 and 4. In some embodiments the formulation comprises a molar concentration of citric acid and sodium citrate that is obtainable from between about 0.55% to about 0.75% w/w citric acid monohydrate and about 0.14% sodium citrate dihydrate. In some embodiments, the formulation comprises between about 0.55% to about 0.75% w/w citric acid monohydrate and about 0.14% sodium citrate dihydrate. In some embodiments, the formulation comprises: (a) 30% w/w isopropyl alcohol; (b) 0.9% w/w benzyl alcohol; (c) 1.5% w/w hydroxyethyl cellulose; and/or (d) 0.56% w/w citric acid monohydrate and 0.14% w/w sodium citrate dihydrate, wherein the formulation has a pH of between 3 and 4. In further embodiments, the formulations of the invention comprise water in an amount of about 67% w/w to bring the total of all components to 100% w/w. In some embodiments, the formulation retains at least about 90% of the ingenol 3-(3,5-diethylisoxazole-4-carboxylate) after 12 months storage at 25 °C or 24 months storage at 2-8 °C.

In a second aspect, the invention provides a method for treating a dermal disease or condition, comprising topical administration of a formulation of the invention. In one embodiment, the dermal disease or condition is actinic keratosis. In another embodiment, the dermal disease or condition is a skin cancer, such as non-melanoma skin cancer, malignant melanoma, Merkel cell carcinoma, squamous cell carcinoma or basal cell carcinoma.

In a further aspect, the invention provides a process for preparing a formulation of the invention, comprising: (i) dissolving ingenol 3-(3,5-diethylisoxazole-4-carboxylate) in (a) a solvent, or (b) a mixture of a solvent and a penetration enhancer; and (ii) mixing the result of step (i) with a buffer and the other components of the formulation, wherein the gellant is added after the buffer or is premixed with the buffer. In one embodiment, the process comprises: (i) dissolving ingenol 3- (3,5-diethylisoxazole-4-carboxylate) in a mixture of a solvent and a penetration enhancer; and (ii) mixing the result of step (i) with a buffer and the other components of the formulation, wherein the gellant is added after the buffer. In another embodiment, the process comprises: (i) dissolving ingenol 3-(3,5-diethylisoxazole-4-carboxylate) in a mixture of a solvent and a penetration enhancer; and (ii) mixing the result of step (i) with a buffer and the other components of the formulation, wherein the gellant is premixed with the buffer. In some embodiments of the process, the solvent is benzyl alcohol and the penetration enhancer is isopropyl alcohol.

In a further aspect, the invention provides a method of treating a subject diagnosed with actinic keratosis, said method comprising topically applying an effective amount of the formulation of the invention to a treatment area for two days. In one embodiment, the method is for treating a subject diagnosed with actinic keratosis on (a) the balding scalp, or (b) the trunk (except chest) or the extremities.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows Kaplan-Meyer survival curves for (A) ingenol 3-(3,5-diethylisoxazole-4- carboxylate), (B) ingenol-3-angelate, and (C) vehicle control, in a B16 melanoma mouse model, with 'death event' defined as tumour exceeding 250 mm ³ or ulcerating tumour. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a formulation comprising: (a) ingenol 3-(3,5-diethylisoxazole-4- carboxylate); (b) a penetration enhancer; (c) a solvent; (d) a gellant; and (e) a buffer, wherein the formulation has a pH of no greater than 4.5 and no less than 2.5. Formulations of the invention are described in more detail below.

Ingenol 3-(3,5-diethylisoxazole-4-carboxylate)

The invention provides formulations of ingenol 3-(3,5-diethylisoxazole-4-carboxylate) or a pharmaceutically acceptable salt, ester or in vivo hydrolysable ester thereof. Ingenol 3-(3,5- diethylisoxazole-4-carboxylate) has the following structure:

and can be made according to the methods disclosed in WO 2012/083953.

In vivo hydrolysable esters include esters, acetals, ketals, or other derivatives which undergo a biotransformation in vivo before exhibiting their pharmacological effects.

Typically, ingenol 3-(3,5-diethylisoxazole-4-carboxylate) (or an ester or salt derivative thereof) is present in the formulation in an amount of between about 0.001% to about 0.5% by weight of the formulation, such as between about 0.01% to about 0.1% e.g. in an amount of about

0.0005%, 0.001%, 0.0025%, 0.005%, 0.01%, 0.015%, 0.025%, 0.05%, 0.075%, 0.1%, 0.125%, 0.15%, 0.2%, 0.25% or 0.5% by weight of the formulation. In two exemplary embodiments the formulation includes of ingenol 3-(3,5-diethylisoxazole-4-carboxylate) in an amount of about 0.05% or 0.015% by weight of the formulation.

Penetration enhancer

Skin penetration means the flux of the active ingredient into the different layers of the skin, i.e. the stratum corneum, epidermis and dermis, after application of the formulation to the skin. Skin permeation means the flux of an active ingredient through the skin into the systemic circulation or, in case of in vitro studies, the receptor fluid of the Franz cell apparatus used in such experiments, after application of a formulation to the skin.

Human skin, in particular the outer layer, the stratum corneum, provides an effective barrier against penetration of microbial pathogens and toxic chemicals. While this property of skin is generally beneficial, it complicates the dermal administration of pharmaceuticals in that a large quantity, if not most, of the active ingredient applied on the skin of a patient suffering from a dermal disease may not penetrate into the viable layers of the skin where it exerts its activity. In the formulations of the invention, skin penetration is facilitated by the inclusion of a penetration enhancer. Suitable penetration enhancers include sulfoxides, azones, pyrrolidines, alkanols, and mono-di- or polyglycols, such as dimethylsulfoxide (DMSO), laurocapram,

2-pyrrolidone, decanol, isopropyl alcohol and propylene glycol. Other penetration enhancers are menthol, eucalyptol, nicotinamide, ethyl acetate, and Eugenol. The inventors have found that a particularly suitable penetration enhancer is isopropyl alcohol.

In one embodiment, the formulation includes a penetration enhancer in an amount of from about 0.01% to about 40% by weight of the formulation, such as from about 0.1% to about 30%, e.g. about 10%, about 15%, about 20%, about 25%, about 28%, about 30% or about 32% by weight of the formulation.

In a typical embodiment, the penetration enhancer is present in an amount of about 30% by weight of the formulation. The inventors have found that a particular suitable penetration enhancer is isopropyl alcohol in an amount of about 30% by weight of the formulation.

Solvent

The formulations of the invention include a solvent. The solvent at least partially dissolves ingenol 3-(3,5-diethylisoxazole-4-carboxylate). Suitable solvents can be selected from the group consisting of lower alcohols, such as n-propanol, isopropanol, n-butanol, 2-butanol and benzyl alcohol, and diols such as propylene glycol. In some embodiments, the solvent may also act as the penetration enhancer of the invention (e.g. IPA can act as both the solvent and the penetration enhancer). The formulation may include more than one solvent, e.g. two or three solvents. For example, the formulation may include benzyl alcohol and isopropanol.

The solvent may be present in an amount of from about 0.5% to about 40% by weight of the formulation, such as from about 5% to about 30%, e.g. about 10%, about 15%, about 20%, about 25%, or about 30% by weight of the formulation. In a typical embodiment, the formulation comprises is benzyl alcohol as a solvent in an amount of about 0.9% w/w of the formulation and isopropyl alcohol as a penetration enhancer (and solvent) in an amount of about 30% w/w of the formulation. Gellant

The formulations of the invention comprise a gellant (a gelling agent). Gellants provide stiffness to a solution or a colloidal dispersion. The resulting gels do not flow at low shear stress and generally exhibit plastic flow behaviour.

In some embodiments, the gelling agent can act as an occlusive agent, e.g. it can form a layer on the surface of the skin on application of the formulation. This layer can form a hydration barrier sufficient to result in reduction of trans-epidermal water loss, thereby improving in skin hydration.

Suitable gellants will be known to the skilled person and include hydroxyalkyl cellulose polymers (e.g. hydroxymethyl cellulose, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose

(hypromellose) and hydroxypropylmethyl cellulose), carboxymethyl cellulose,

methylhydroxyethyl cellulose and methylcellulose, carbomer (e.g. Carbopol®), and carrageenans. In some embodiments, the emulsifier is hydroxypropyl cellulose, such as that available under the trade name Klucel® and METHOCEL®. A particularly suitable gellant is hydroxyethyl cellulose, such as that available under the trade name Natrosol® (e.g. Natrosol® 250 HX, Natrosol® PLUS CS, Grade 300 etc.) and METHOCEL®. The formulation may include more than one gelling agent, such as two or three gelling agents.

Typically, the gellant is present in an amount of from about 1% to about 20% by weight of the formulation, such as from about 0.5% to about 5% (e.g. about 1%, 3%, 5%, 10%, 15% or 20% w/w). In one embodiment, the gellant (e.g. hydroxyethyl cellulose) is present in an amount of about 1.5% by weight of the formulation. In another embodiment, the formulation comprises about 1.5% w/w hydroxyethyl cellulose. Buffers

The formulations of the invention include an aqueous buffer. The use of buffer means that fluctuations in pH can be minimised and thus the pH of the formulation can be kept more readily within the desired pH range (e.g. a pH of no greater than 4.5 and no less than 2.5, such as between 3.2 to 4).

Suitable buffer solutions that can be used in the formulations of the invention include, for example, citrate buffer, phosphate buffer, acetate buffer and citrate -phosphate buffer. A citrate buffer is particularly suitable.

The pH of the formulation will depend on the amount of buffer and the pH of the buffer used. Typically, the formulations of the invention comprise from about 2.5% to about 90% buffer solution by weight of the formulation, e.g. 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% buffer solution by weight of the formulation. The pH of the buffer solution will typically be between about 2 to about 4.5, e.g. pH 2, 2.5, 3, 3.5, 4, or 4.5. A buffer having a pH of from about 2 to about 3 is particularly suitable, because this pH range may permit the formulation to be stored at room temperature (25°C) for extended periods. For instance, in some embodiments the pH of the buffer is 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0. Typically, the buffer will be a citrate buffer having a pH of from about 2 to about 3. For example, a citrate buffer can be made by mixing citric acid and sodium citrate with water. Formulations comprising a molar concentration of citric acid and sodium citrate that is obtainable from between about 0.55% to about 0.75% w/w citric acid monohydrate and about 0.14% sodium citrate dihydrate have been found to be particularly suitable. The skilled person is aware of available hydrate salts of citric acid and sodium citrate (e.g. anhydrides, mono- and di-hydrates). In one embodiment, the formulation comprises between about 0.55% to about 0.75% w/w citric acid monohydrate (e.g. about 0.56% w/w) and about 0.14% w/w sodium citrate dihydrate. In some embodiments, the formulation comprises about 0.56% citric acid monohydrate and about 0.14% sodium citrate dihydrate. Methods of making buffers of the type disclosed herein are well known to the skilled person pH of the formulation

The formulations of the invention are typically acidic, because it has been found that ingenol 3- (3,5-diethylisoxazole-4-carboxylate) is more stable under these conditions.

The formulation of the invention has a pH of no greater than 4.5 and no less than 2.5, such as between 3 and 4, between 3.2 and 4, or between 3.2 and 3.8. For instance, in some embodiments the pH of the formulation is 2.5, 2.6, 2.8, 3.0, 3.2, 3.4, 3.5, 3.6, 3.8, 4.0, 4.2, 4.4 or 4.5.

As discussed above, the formulations of the invention comprise an aqueous buffer and a solvent. For formulations (solutions or suspensions) that are only partially aqueous, attempts to measure pH will only yield an "apparent pi I", rather than an absolute pi I. because the measured pH relates only to the ionic component. A reference to pH in the present application refers to the pH value as measured for a particular formulation, i.e. an absolute pH value where the formulation is fully aqueous and an apparent pH where the formulation is partially aqueous. Both apparent pH and absolute pH can be measure using an pH electrode. Suitable means of measuring pH are known to the skilled person.

General properties of the formulation

In some embodiments the formulations are colourless. In other embodiments they include a coloured substance, which can make it easy to see where the formulation has been applied. The formulations of the invention are typically transparent. In some embodiments, the formulations include suspended ingenol 3-(3,5-diethylisoxazole-4-carboxylate) solids. In these embodiments, the formulations are typically transparent except for the suspended ingenol 3-(3,5- diethylisoxazole-4-carboxylate) solids. In other less preferred embodiments, the formulations are turbid in appearance.

The formulations of the invention are suitable for topical administration, particularly in humans. For instance, the formulations have good spreadability, i.e. the formulations can readily be spread (e.g. using fingers) after application to the skin to provide a uniform layer. The formulations also have excellent extrudability. In some embodiments, the formulations can be applied topically and do not leave a visible residue. The volatile components of the formulations may also substantially evaporate to dryness after a certain period of time following topical application. Typically, the volatile components of the formulation will evaporate after a therapeutically effective amount of the ingenol 3-(3,5-diethylisoxazole-4-carboxylate) has penetrated into the skin (e.g. after about 1 minute, 2 minutes, 5 minutes, 10 minutes, 20 minutes, etc. following topical administration to a subject).

Optional additional components

Typically, the formulations of the invention do not contain any additional components, other than ingenol 3-(3,5-diethylisoxazole-4-carboxylate), a penetration enhancer, a solvent, a gellant; and a buffer (apart from possible trace amounts of impurities and/or degradation products of these components). However, in some embodiments, the formulation of the invention may contain additional components, non-limiting examples of which are provided below.

The formulations may include a viscosity-increasing ingredient. For example, when the formulation comprises a substantial amount of aqueous buffer solution (e.g. above about 60% by weight of the formulation), it may be necessary to add one or more viscosity-increasing ingredients (e.g. in an amount of e.g. about 5% by weight of the formulation) in order to form a gel. The viscosity-increasing ingredient may therefore function as the gelling agent. However, there may be no requirement for the additional of a viscosity-increasing ingredient if other components in the formulation are capable of acting as a gelling agent.

In some embodiments, the viscosity-increasing ingredient is an inorganic substance such as fumed silica (e.g. available under the trade name Aerosil®, such as Aerosil® 200P, which is a high purity amorphous anhydrous colloidal silicon dioxide). The viscosity-increasing ingredient may be a mixture of acrylamide acryloyldimethyl taurate copolymer, isohexadecane and polysorbate 80, such as that available under the trade name SEPINEO P600. The viscosity- increasing ingredient may be a mixture of a fatty alcohol and an alkylpolyglucoside, such as that available under the trade name SEPINEO SE68.

The amount of viscosity-increasing ingredient may vary (according to the viscosifying power of the ingredient), but the formulation may include from about 0.5% to about 40% viscosity- increasing ingredient by weight of the formulation. When the viscosity-increasing ingredient is microcrystalline wax it is typically present in an amount of from about 0.5% to about 10% by weight of the formulation. Where the viscosity-increasing ingredient is SEPINEO P600, it is typically included in an amount of from about 1% to about 10% by weight of the formulation, e.g. about 2.5% by weight of the formulation. Where the viscosity-increasing ingredient is SEPINEO SE68, it is typically included in an amount of from about 2% to about 30% by weight of the formulation, e.g. about 5% by weight of the formulation. Aerosil® is typically included in an amount of from about 1% to about 10% by weight of the formulation, e.g. 1%, 2%, 5% or 10% by weight of the formulation. In certain embodiments of the invention the formulation includes a hydrophilic non-ionic surfactant and/or a lipophilic non-ionic surfactant.

The term "hydrophilic surfactant" means an oil-in-water surfactant with a hydrophilic-lipophilic balance (HLB) value of 9-18, and "lipophilic surfactant" means a water-in-oil surfactant with an HLB value of 1.5-9. By way of an example, polysorbate 80 has an HLB value of 15 and is therefore a hydrophilic surfactant, whereas sorbitan trioleate has an HLB value of 1.8 and is therefore a lipophilic surfactant. The HLB of mixed surfactants is calculated according to their relative weightings (by volume) e.g. a 1 : 1 mixture by volume of polysorbate 80 and sorbitan trioleate has a HLB of 8.4.

In one embodiment, the formulation includes a hydrophilic non-ionic surfactant in an amount of from about 1 % to about 40% by weight of the formulation, optionally from about 2% to about

15% by weight of the formulation. In some embodiments, the formulation includes a hydrophilic non-ionic surfactant in an amount of from about 2% to about 10% by weight of the formulation, such as from about 2.5% to about 5% by weight of the formulation.

The hydrophilic non-ionic surfactant may be a polyethylene glycol ester of a vegetable oil containing at least 20 moles of ethylene oxide groups/mole of glyceride. Suitable polyethylene glycol esters are typically selected from polyoxyethylene castor oil derivatives (e.g. PEG 20, 30, 35, 38, 40, 50 and 60 castor oil or PEG 20, 25, 30, 40, 45, 50, 60 and 80 hydrogenated castor oil), PEG 20 and 60 corn glycerides, PEG 20 and 60 almond glycerides, PEG 40 palm kernel oil, sodium laurate sulfate, sucrose esters (e.g. sucrose stearate, sucrose distearate, sucrose cocoate or sucrose monolaurate), PEG cocoglyceride, PEG 8 caprylocaprate, polyglyceryl esters and linolenamide DEA. In some embodiments, the hydrophilic non-ionic surfactant is sucrose distearate, such as that available under the trade name Sisterna® SP30.

In certain embodiments, the hydrophilic non-ionic surfactant may be a mixture of acrylamide acryloyldimethyl taurate copolymer, isohexadecane and polysorbate 80, such as that available under the trade name SEPINEO P600. The hydrophilic non-ionic surfactant may be an alkylpolyglucoside, such as that available under the trade name SEPINEO SE68.

In some embodiments, the formulation includes a keratinolytic agent, such as an a-hydroxy acid or β-hydroxy acid. The use of a keratinolytic agent may improve penetration of the active substance, meaning that formulations comprising a keratinolytic agent are particularly useful for treating hyperkeratotic actinic keratosis.

Suitable keratinolytic agents for use in the formulations of the invention include retinoids, adapalene, tars, shale oil, allantoin, aluminium oxide, azelaic acid, benzoyl peroxide, lactic acid, salicylic acid, alcali and alkali earth sulfide, monochloroacetic acid, urea, and resorcin. Particular retinoids that may be suitable include retinol, retinaldehyde, retinoic acid, isotretinoin, adapalinen and tazarotene. Further keratinolytic agents include ammonium glycolate, ammonium lactate, betaine salicylate, calcium lactate, calcium thioglycolate, glycolic acid, lactic acid, phenol, potassium lactate and sodium lactate.

In one embodiment, the formulation includes an a-hydroxy acid selected from glycolic acid, lactic acid, mandelic acid, malic acid, citric acid and tartaric acid. In another embodiment, the formulation includes a β-hydroxy acid such as salicylic acid. In some embodiments, the keratinolytic agent is salicylic acid.

The keratinolytic agent (e.g. α-hydroxy acid or β-hydroxy acid) may be present in an amount of from about 0.1% to about 20% by weight of the formulation, e.g. about 0.5%, 1.0%, 2.5%, 5.0%, 7.5%, 10%, 15% or 20% by weight of the formulation. In some embodiments, the formulation includes salicylic acid in an amount of from about 0.1 % to about 20% by weight of the formulation, e.g. about 0.5%, 1.0%, 2.5%, 5.0%, 7.5%, 10%, 15% or 20% by weight of the formulation. Particularly suitable formulations

The inventors have found that certain formulations are particularly stable.

In one embodiment, the formulation comprises: (a) ingenol 3-(3,5-diethylisoxazole-4- carboxylate); (b) a penetration enhancer; (c) a solvent; (d) a gellant; and (e) a buffer, wherein the formulation has a pH of no greater than 4.5 and no less than 2.5, and wherein (i) the penetration enhancer is isopropyl alcohol, (ii) the solvent is benzyl alcohol, (iii) the gellant is a hydroxyalkyl cellulose, such as hydroxyethyl cellulose, and/or (iv) the buffer is citrate buffer. Typically, all the features (i) to (iv) are included. In these formulations, the formulation may in particular comprise (A) isopropyl alcohol in an amount of about 30% by weight of the formulation, (B) benzyl alcohol in an amount of about 0.9% by weight of the formulation, (C) hydroxyethyl cellulose in an amount of 1.5% by weight of the formulation, and/or (D) citric acid monohydrate in an amount of 0.55% to 0.75% by weight of the formulation and sodium citrate dehydrate in an amount of 0.14% by weight of the formulation. In one embodiment, all the features (A) to (D) are included. Said formulations may further comprise water in an amount of about 67% w/w (i.e. to bring the total of all components in the formulation to 100% w/w). In a some embodiments, the formulation may comprise: (a) 30% w/w isopropyl alcohol; (b) 0.9% w/w benzyl alcohol; (c) 1.5% w/w hydroxyethyl cellulose; and/or (d) 0.56% w/w citric acid monohydrate and 0.14% w/w sodium citrate dihydrate, wherein the formulation has a pH of between 3 and 4. In some embodiments, all the features (a) to (d) are included. The formulations may further comprise water in an amount of about 67% w/w (i.e. to bring the total of all components in the formulation to 100% w/w).

In another embodiment, the invention provides a formulation as described above, wherein the ingenol 3-(3,5-diethylisoxazole-4-carboxylate) is present in an amount between about 0.01% to 0.1 % by weight of the formulation.

In an embodiment of the formulations described above, the pH is between 3 and 4 inclusive. In another embodiment of the formulations described above, the pH is between 3.2 and 4 inclusive.

Stability of the formulations

The inventors have found that formulations of the invention exhibit favorable stability properties. In some embodiments, the formulation is chemically stable, where chemically stable (or chemical stability) means that 10% or less of the ingenol 3-(3,5-diethylisoxazole-4-carboxylate) is degraded after 12 months storage at 25°C or 24 months storage at 2-8°C. In some embodiments, 6% or less of the ingenol 3-(3,5-diethylisoxazole-4-carboxylate) is degraded after 12 months storage at 25°C or 24 months storage at 2-8°C. An approximation of chemical stability can be obtained by subjecting the formulation to stability studies at 25°C for 6 months or by subjecting the formulation to accelerated stability studies at 40°C for 3 months. If less than about 5% of the ingenol 3-(3,5-diethylisoxazole-4-carboxylate) has degraded after 6 months at 25°C, then a shelf- life of 12 months at room temperature is expected, i.e. 10% or less of the ingenol 3-(3,5- diethylisoxazole-4-carboxylate) will be expected to degrade over a storage period of 12 months at 25°C. If 10% or less of the ingenol 3-(3,5-diethylisoxazole-4-carboxylate) has degraded after 3 months at 40°C then a shelf-life of 12 months at 25°C is expected, i.e. 10% or less of the ingenol 3-(3,5-diethylisoxazole-4-carboxylate) will be expected to degrade over a storage period of 12 months at 25°C. These studies are carried out according to ICH Humidity Guidelines, at conditions of 25°C ±2760% RH ±5% and/ or 40°C ±2 75% RH ±5%, in hermetically sealed containers, as exemplified by the experiments provided in the Examples. In embodiments of the invention, the formulation of ingenol 3-(3,5-diethylisoxazole-4-carboxylate) retains at least about 90%, 95%, 97% or 99% of the ingenol 3-(3,5-diethylisoxazole-4-carboxylate) after 12 months storage at 25 °C or 24 months storage at 2-8 °C.

In some embodiments, the formulation is physically stable, where physically stable (or physical stability) means that the formulation retains its macroscopic and microscopic appearance over the shelf-life of the product. For example, visual inspection of the formulations can be used to assess physical stability (e.g. presence of precipitates, particularly precipitates of ingenol 3-(3,5- diethylisoxazole-4-carboxylate)). In some embodiments, the formulation is chemically stable and physically stable. Medical treatments and uses

The invention provides a method for treating a dermal disease or condition, comprising topical administration of a formulation of the invention. The invention also provides a formulation of the invention for use in treating a dermal disease or condition. The methods and uses are suitable for treating a dermal disease or condition in mammals, in particular humans.

Topical administration means that the formulations are applied cutaneously i.e. to the external skin on the body.

The uses and methods are useful for the topical treatment of dermal diseases or conditions including actinic keratosis, seborrheic keratosis, skin cancer, warts, keloids, scars, photo-aged or photodamaged skin, and acne. In particular, the uses and methods are particularly useful for the topical treatment of actinic keratosis. The uses and methods may, for instance, be useful for the cutaneous (topical) treatment of non-hyperkeratotic, non-hypertrophic actinic keratosis.

The uses and methods may be used for the topical treatment of skin cancers such as non- melanoma skin cancer, malignant melanoma, Merkel cell carcinoma, squamous cell carcinoma or basal cell carcinoma (including superficial basal cell carcinoma and nodular basal cell carcinoma).

The uses and methods may be used for the topical treatment of warts, e.g. human papilloma virus (HPV) infections on the skin, genitals and mouth.

The uses and methods may be used for the topical treatment of photodamaged skin such as fine lines, wrinkles and UV-ageing. UV-ageing is often manifested by an increase in the epidermal thickness or epidermal atrophy, most notably by solar elastosis, the accumulation of elastin containing material just below the dermal-epidermal junction. Collagen and elastic fibres become fragmented and disorganised. At a cosmetic level this can be observed as a reddening and/or thickening of the skin resulting in a leathery appearance, skin fragility and irregular pigmentation, loss of tone and elasticity, as well as wrinkling, dryness, sunspots and deep furrow formation. The uses and methods may be useful for reducing or minimizing scar tissue or improving cosmesis or functional outcome in a wound. For instance, the uses and methods may be useful for improving functional outcome in a wound which is cutaneous, chronic or diabetes associated, e.g. when the wound includes cuts and lacerations, surgical incisions, punctures, graces, scratches, compression wounds, abrasions, friction wounds, chronic wounds, ulcers, thermal effect wounds, chemical wounds, wounds resulting from pathogenic infections, skin graft/transplant, immune response conditions, oral wounds, stomach or intestinal wounds, damaged cartilage or bone, amputation sides and corneal lesions.

Therefore, in some embodiments, the uses and methods are cosmetic. Typically, the uses and methods are lesion specific, i.e. they are focused on a lesion being treated and do not extend to any larger degree to the surrounding skin. In other embodiments, however, the uses and methods can extend to a larger area than the lesions, and this can usefully lead to treatment of emerging lesions or sub-surface pre -lesions. Also, it can be convenient to apply a formulation to an area which includes several lesions, rather than applying it to each individual lesion in that area. The lesions could be of any size (i.e. surface area), e.g. greater than about 30 000 mm ² , greater than about 20 000 mm ² , greater than about 10 000 mm ² , greater than about 5000 mm ² , greater than about 1000 mm ² , greater than about 500 mm ² , greater than about 250 mm ² , or greater than about 150 mm ² . Typically, the lesion size is about 30 000 mm ² , about 20 000 mm ² , about 10 000 mm ² , about 5000 mm , about 1000 mm , about 500 mm , about 250 mm , about 150 mm , about 100 mm , about 75 mm , about 50 mm , about 25 mm or about 10 mm .

In the treatment of, for example, actinic keratosis on the face and/or scalp of a subject, a formulation of the invention may be applied on the face and scalp to the affected skin area (treatment area) once a day for 2 or 3 consecutive days. In the treatment of, for example, actinic keratosis on the trunk and/or extremities of a subject, a formulation of the invention may be applied on the trunk and extremities to the affected skin area (treatment area) once a day for 2 or 3 consecutive days. Immediately following application to the treatment area, subjects should wash their hands.

The formulations of the invention are typically packaged in hermetically sealed containers, e.g. a unit dose tube. A unit dose tube would typically contain about 0.5g of the formulation. One unit dose tube (tube with screw cap or individual packets) may be used for one treatment area. Treatment regimens for actinic keratosis

From Picato® and other topical agents used in the treatment of actinic keratosis, it is well known that differences in treatment efficacy exists between different anatomical regions. Regions like scalp, trunk (except chest) and extremities are more difficult to treat than face. From Picato® studies have shown that local skin reactions after a given dose are milder on difficult to treat anatomical regions than on anatomical regions more easily treated with the compound. According to the FDA label for Picato®, the size of the treatment area is limited to about 25 cm ² (2 inches x 2 inches).

The present invention provides a topical treatment regimen with the ingenol 3-(3,5- diethylisoxazole-4-carboxylate) for actinic keratosis (AK), which is of short duration and applicable to a large skin area, ie. full face or up to 250cm ² on the body or balding scalp. In particular, regimens applicable to, and optimised for use on, a large skin area on trunk (except chest) or extremities and the balding scalp are provided. The treatment is simple by the two day regimen. The dosage may vary, such that body locations which are considered difficult to treat may be treated with a higher dosage strength. The treatment is directed against treating nonhyperkeratotic actinic keratosis. The treatment is optimized towards acceptable side-effects in terms of measured local skin reactions (LSR).

The invention provides a method of treating a subject diagnosed with actinic keratosis, said method comprising topically applying an effective amount of a formulation of the invention to a treatment area for two days. In one embodiment, the method is for treating a subject diagnosed with actinic keratosis on the balding scalp. In another embodiment, the method is for treating a subject diagnosed with actinic keratosis on the trunk (except chest) or the extremities.

In some embodiments, the method comprises applying an effective amount of the formulation of the invention to a treatment area for two days to achieve reduction in the number of the actinic keratosis in the treated area. The lesions are not of atypical clinical appearance such as for example hypertrophic, hyperkeratotic or cutaneous horns and/or recalcitrant diseases, such as nonresponding to cryotherapy on two previous occasions. In one embodiment, the two day treatment is two consecutive days. In another embodiment, the treatment area is up to about 250 cm ² . Additional information and methods relating to treatment regimens for actinic keratosis, including the specific regimens discussed below, can be found in PCT/IB2014/002951,

PCT/IB2014/002940, and PCT/IB2014/002970.

Treatment regimens for the balding scalp

In embodiments of the method relating to treating a subject diagnosed with actinic keratosis on the balding scalp, a dosage strength of the formulation of between about 0.01% and about 0.1%, such as between about 0.037% and about 0.05% (e.g. 0.018%, 0.025%, 0.037%, 0.05%, 0.075% or 0.1%) is particularly suitable. For example, the invention provides a method of treating a subject diagnosed with actinic keratosis on the balding scalp, said method comprising topically applying an effective amount of a formulation of the invention to the balding scalp for two consecutive days, wherein said method provides a reduction in the number of actinic keratosis lesions in the treated area on the balding scalp, wherein the treated area is of a size up to about

250 cm ² , and wherein the dosage strength of the ingenol 3-(3,5-diethylisoxazole-4-carboxylate) is between about 0.037% and about 0.05%. In some embodiments, the amount of ingenol 3-(3,5- diethylisoxazole-4-carboxylate) applied is between about 0.162 mg ingenol 3-(3,5- diethylisoxazole-4-carboxylate)/per day/250 cm ² treatment area and about 0.9 mg ingenol 3-(3,5- diethylisoxazole-4-carboxylate)/per day/250 cm ² treatment area.

Treatment regimens for the trunk (exempt the chest) and extremities

In embodiments of the method relating to treating a subject diagnosed with actinic keratosis on the trunk (exempt the chest) and extremities, a dosage strength of the formulation of between about 0.01% and about 0.1% w/w, such as between 0.018% to about 0.1% (e.g. about 0.018%, about 0.025%, about 0.037%, about 0.05%, about 0.075% or about 0.1%) is particularly suitable. For example, the invention provides a method of treating a subject diagnosed with actinic keratosis on the trunk (except chest) or extremities, said method comprising topically applying an effective amount of a formulation of the invention to the trunk (except chest) or extremities for two consecutive days, wherein said method provides a reduction in the number of actinic keratosis lesions in the treated area, wherein the treatment area is of a size up to about 250 cm ² , and wherein the dosage strength of the ingenol 3-(3,5- diethylisoxazole-4-carboxylate) is between about 0.018% and about 0.1%.

Further treatment regimens

In some embodiments, a dosage strength of the formulation of between about 0.0015% to about 0.1 %, such as between about 0.006% and about 0.018% or between about 0.01 % to about 0.1 % (e.g. about 0.003, 0.006%, about 0.012%, about 0.018%, about 0.025%, about 0.037%, about 0.05%, about 0.075% or about 0.1%) is particularly suitable (e.g. when treating a large surface area). For example, the invention provides a method of treating a subject diagnosed with actinic keratosis, said method comprising topically applying an effective amount of a formulation of the invention to a treatment area for two consecutive days, wherein, said method provides a reduction in the number of actinic keratosis lesions in the treatment area, wherein, the treatment area is of a size up to about 250 cm ² , and wherein, the dosage strength of the formulation is between about 0.006% and about 0.018%. In some embodiments, the amount of the formulation of the invention topically applied is about 4.5 μΐ _^ at a concentration of about 0.0015%, about 0.003%, about 0.006%, about 0.012%, about 0.018%, about 0.025%, about 0.037%, about 0.05%, about 0.075% or about 0.1%/per day/250 cm ² treated area.

Process for preparing a formulation of the invention

The invention provides a process for preparing the formulation of the invention, comprising:

(i) dissolving ingenol 3-(3,5-diethylisoxazole-4-carboxylate) in (a) a solvent, or (b) a mixture of a solvent and a penetration enhancer; and (ii) mixing the result of step (i) with a buffer and the other components of the formulation, wherein the gellant is added after the buffer or is premixed with the buffer. In some embodiments, the process comprises: (i) dissolving ingenol 3-(3,5- diethylisoxazole-4-carboxylate) in a mixture of a solvent and a penetration enhancer; and (ii) mixing the result of step (i) with a buffer and the other components of the formulation, wherein the gellant is added after the buffer. In other embodiments, the process comprises: (i) dissolving ingenol 3-(3,5-diethylisoxazole-4-carboxylate) in a mixture of a solvent and a penetration enhancer; and (ii) mixing the result of step (i) with a buffer and the other components of the formulation, wherein the gellant is premixed with the buffer. The other components of the formulation added in step (ii) of the process include any additional components of the

formulation and any penetration enhancer, ingenol 3-(3,5-diethylisoxazole-4-carboxylate) or solvent not already added in step (i). Typically, dissolving ingenol 3-(3,5-diethylisoxazole-4- carboxylate) involves complete dissolution, but partial dissolution is also encompassed. In some embodiments, the solvent is benzyl alcohol and the penetration enhancer is isopropyl alcohol.

Definitions

The term "comprising" encompasses "including" as well as "consisting" e.g. a formulation "comprising" X may consist exclusively of X or may include something additional e.g. X + Y. The word "substantially" does not exclude "completely" e.g. a formulation which is

"substantially free" from Y may be completely free from Y. Where necessary, the word

"substantially" may be omitted from the definition of the invention.

The term "about" in relation to a numerical value x is optional and means, for example, x+10%. Unless specifically stated otherwise, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc. EXAMPLES

The invention is further illustrated by the followings examples. It will be appreciated that the examples are for illustrative purposes only and are not intended to limit the invention as described above. Modification of detail may be made without departing from the scope of the invention.

EXAMPLE 1 - FORMULATION STABILITY STUDIES

STUDY A

Components of the formulations

Preparation of formulations

Buffer stock solution: Weigh out water, citric acid monohydrate and sodium citrate dihydrate and mix. Adjust the pH to 2.8 with either citric acid or NaOH solutions of e.g. 0.5-1 M, if necessary. Buffer stock solution should be stored at 2-8°C.

Hydroxyethyl cellulose stock: Weigh out buffer stock solution in suitable container. Slowly add hydroxyethyl cellulose, while homogenisating at about 2000 rpm and increase to about 5000 rpm. Mix until homogeneity (no lumps). Hydroxyethyl cellulose stock solution should be stored at 2-8°C.

Alcohol phase:

• Charge appropriate amount of ingenol 3-(3,5-diethylisoxazole-4-carboxylate) in a small glass bottle with screw cap (preventing alcohol evaporation)

• Add appropriate amount of benzyl alcohol

• Add isopropyl alcohol (IP A), saving about 20% IPA (the remaining IPA is to be used for cleaning when alcohol phase is mixed with hydroxyethyl cellulose stock solution)

Final formulation:

• Prepare glass beaker for homogenisation on water with ice cooling

• Add the desired amount of hydroxyethyl cellulose stock solution corresponding to batch size

• Slowly add alcohol phase while stirring

• Rinse alcohol phase bottle with the remaining IPA. Cover beaker with parafilm to prevent IPA evaporation

• Continue stirring until homogeneity and make sure ice is present in the water bath

• Cover formulation to prevent evaporation and store at 2-8°C.

Testing chemical stability and physical stability

A number of formulations of the invention were tested for chemical stability. Each formulation was mixed with a solvent mixture of acetonitrile and phosphoric acid. Following mixing, organic impurities were identified using reversed phase HPLC with UV detection at 220 nm. The visual appearance of the formulations was also monitored.

Stability of ingenol 3-(3,5-diethylisoxazole-4-carboxylate)

n.a. ~ not analysed

n.a. ~ not analysed The level of the main degradation product for ingenol 3-(3,5-diethylisoxazole-4-carboxylate) is 1.6 % area after 6 months storage at 25°C/60%RH.

Accelerated stability testing at 40°C for 3 months indicates that 10% or less of the ingenol 3-(3,5- diethylisoxazole-4-carboxylate) is expected to degrade after storage for 12 months at room temperature (25°C). Similarly, the level of degradation after storage for 6 months at 25°C is indicative of a shelf life of 12 months at room temperature (i.e. 10% or less degradation after 12 months at room temperature).

STUDY B

Components of the formulations

Preparation of formulations

Buffer stock solution: Weigh out and mix sufficient water, citric acid monohydrate and sodium citrate dihydrate to prepare a 1% citrate buffer solution. Adjust the pH to 2.5 and 3.0,

respectively, with either citric acid or NaOH solutions of e.g. 0.5-1 M, if necessary. Buffer stock solution should be stored at 2-8°C.

Hydroxyethyl cellulose stock: Weigh out buffer stock solution in suitable container. Slowly add hydroxyethyl cellulose, while homogenisating at about 2000 rpm and increase to about 5000 rpm. Mix until homogeneity (no lumps). Hydroxyethyl cellulose stock solution should be stored at 2-8°C.

Alcohol phase:

• Charge appropriate amount of ingenol 3-(3,5-diethylisoxazole-4-carboxylate) in small glass bottle with screw cap (preventing alcohol evaporation) • Add appropriate amount of benzyl alcohol

• Add isopropyl alcohol (IP A), saving about 20% (remaining IPA to be used for cleaning when alcohol phase is mixed with hydroxyethyl cellulose stock solution)

Final formulation:

• Prepare glass beaker for homogenisation on water with ice cooling.

• Add the desired amount of hydroxyethyl cellulose stock solution corresponding to batch size

• Slowly add alcohol phase while stirring.

• Rinse alcohol phase bottle with the remaining IPA. Cover beaker with parafilm to prevent IPA evaporation

• Continue stirring until homogeneity and make sure ice is present in water bath.

• Cover formulation to prevent evaporation and store at 2-8°C.

Testing chemical stability and physical stability

A number of formulations of the invention were tested for chemical stability. This testing required mixing the formulation with a solvent mixture of acetonitrile and phosphoric acid.

Following mixing, organic impurities were identified using reversed phase HPLC with UV detection at 220 nm. The visual appearance of the formulations was also monitored.

Stability of ingenol 3-(3,5-diethylisoxazole-4-carboxylate)

n.a. ~ not analysed

The level of the main degradation product for ingenol 3-(3,5-diethylisoxazole-4-carboxylate) is 3.1 % area after 3 months storage at 40°C/75%RH.

n.a. ~ not analysed

The level of the main degradation product for ingenol 3-(3,5-diethylisoxazole-4-carboxylate) is 5.2 % area after 3 months storage at 40°C/75%RH.

Accelerated stability testing at 40°C for 3 months indicates that 10% or less of the ingenol 3-(3,5- diethylisoxazole-4-carboxylate) is expected to degrade after storage for 12 months at room temperature (25°C).

STUDY C

Components of the formulations

mg/g

Components 13058270 13343270

ingenol 3-(3,5-diethylisoxazole-4-carboxylate) 0.75 (0.075) 1 (0.1)

benzyl alcohol 9 (0.9) 9 (0.9)

isopropyl alcohol 300 (30) 300 (30)

citric acid monohydrate 7.3 (0.73) 7.3 (0.73)

sodium citrate dihydrate 1.4 (0.14) 1.4 (0.14)

water 666.6 (-67) 666.3 (-67)

hydroxyethyl cellulose HX 15 (1.5) 15 (1.5) Preparation of formulations

Buffer stock solution: Weigh out water, citric acid monohydrate and sodium citrate dihydrate and mix. Adjust the pH to 2.8 with either citric acid or NaOH solutions of e.g. 0.5-1 M, if necessary. Buffer stock solution should be stored at 2-8°C.

Hydroxyethyl cellulose stock: Weigh out buffer stock solution in suitable container. Slowly add hydroxyethyl cellulose, while homogenisating at about 2000 rpm and increase to about 5000 rpm. Mix until homogeneity (no lumps). Hydroxyethyl cellulose stock solution should be stored 2-8°C.

Alcohol phase:

• Charge appropriate amount of ingenol 3-(3,5-diethylisoxazole-4-carboxylate) in small glass bottle with screw cap (preventing alcohol evaporation)

• Add appropriate amount of benzyl alcohol

• Add isopropyl alcohol (IP A), saving about 20% (remaining IPA is to be used for cleaning when alcohol phase is mixed with hydroxyethyl cellulose stock solution)

Final formulation:

• Prepare glass beaker for homogenisation on water with ice cooling.

• Add the desired amount of hydroxyethyl cellulose stock solution corresponding to batch size

• Slowly add alcohol phase while stirring.

Rinse alcohol phase bottle with the remaining IPA. Cover beaker with parafilm to prevent IPA evaporation

• Continue stirring until homogeneity and make sure ice is present in water bath.

• Cover formulation to prevent evaporation and store at 2-8°C.

Testing chemical stability and physical stability

A number of formulations of the invention were tested for chemical stability. This testing required mixing the formulation with a solvent mixture of acetonitrile and phosphoric acid.

Following mixing, organic impurities were identified using reversed phase HPLC with UV detection at 220 nm. The visual appearance of the formulations was also monitored.

Stability of ingenol 3-(3,5-diethylisoxazole-4-carboxylate)

The level of the main degradation product for ingenol 3-(3,5-diethylisoxazole-4-carboxylate) 1.7 % area after 6 months storage at 25°C/60%RH.

The level of the main degradation product for ingenol 3-(3,5-diethylisoxazole-4-carboxylate) is 3.6 % area after 3 months storage at 40°C/75%RH.

Accelerated stability testing at 40°C for 3 months indicates that 10% or less of the ingenol 3-(3,5- diethylisoxazole-4-carboxylate) is expected to degrade after storage for 12 months at room temperature (25°C). Similarly, where measured, the level of degradation after storage for 6 months at 25°C is indicative of a shelf life of 12 months at room temperature (i.e. 10% or less degradation after 12 months at room temperature).

EXAMPLE 2 - COMPARATIVE STABILITY DATA FOR INGENOL MEBUTATE

Comparative stability data for ingenol mebutate formulations were prepared in a similar to Example 1.

Stability of 0.015% ingenol mebutate formulation (EE4252)

Stability of 0.05% ingenol mebutate formulation (EE4405)

Comparison of these data with those in Example 1 shows that formulations of ingenol 3-(3,5- diethylisoxazole-4-carboxylate) (at 0.05% and 0.015% w/w of the active pharmaceutical ingredient) are > 2-fold more stable after storage for 6 months at 25 °C than similar formulations of ingenol mebutate.

EXAMPLE 3 - SKIN PENETRATION AND PERMEATION STUDIES

To investigate the skin penetration and permeation of ingenol derivative from formulations of the invention, an in vitro skin diffusion test was conducted.

Full thickness skin from pig ears was used in the study. The ears were kept frozen at -18°C before use. On the day prior to the experiment the ears were placed in a refrigerator (5±3°C) for slow defrosting. On the day of the experiment, the hairs were removed using a veterinary hair trimmer. The skin was cleaned for subcutaneous fat using a scalpel and two pieces of skin were cut from each ear and mounted on Franz diffusion cells in a balanced order.

Flow-through Franz-type diffusion cells with an available diffusion area of 3.14 cm ² and receptor volumes ranging from 11.1 to 12.6 ml were used in substantially the manner described by T.J. Franz, "The finite dose technique as a valid in vitro model for the study of percutaneous absorption in man", in Current Problems in Dermatology, 1978, J.W.H. Mall (Ed.), Karger, Basel, pp. 58-68. The specific volume was measured and registered for each cell. A magnetic bar was placed in the receptor compartment of each cell. After mounting the skin, physiological saline (35°C) was filled into each receptor chamber for hydration of the skin. The cells were placed in a thermally controlled water bath which was placed on a magnetic stirrer set at 400 rpm. The circulating water in the water baths was kept at 35±1°C resulting in a temperature of about 32°C on the skin surface. After half an hour the saline was replaced by receptor medium, 0.04 M isotonic phosphate buffer, pH 7.4 (35°C), containing 4% bovine serum albumin and left for hydration another hour. The inlet and outlet ports of the receptor chamber were connected to stainless steel HPLC tubing. The cells were connected to a 12-channel peristaltic pump, and the receptor fluid was pumped continuously through each cell and collected in vials placed at a fraction collector. A controller was used to program independently the duration of each fraction. Sink conditions were maintained at all times during the period of the study, i.e. the concentration of the active compounds in the receptor medium was below 10% of the solubility of the compounds in the medium.

The in vitro skin penetration and permeation was tested in 6 replicates (i.e. n=6). Each test formulation was applied to the skin membrane at 0 hours in an intended dose of 4 mg/cm ² . A glass spatula was used for the application, and the residual amount of the formulation was determined so as to give the amount of the formulation actually applied on the skin.

The skin penetration and permeation experiment was allowed to proceed for 21 hours. Samples were then collected from the following compartments:

About 6 ml of the receptor fluid was sampled from each cell every third hour until 21 hours post application. The sample collection of the first 45 minutes was discarded due to the lag time of the system. The recipient fluid remaining in the diffusion cell at the end of the study corresponded to the 21 hour sample.

The stratum corneum was collected by tape stripping 10 times using D-Squame ^® tape (diameter 22 mm, CuDerm Corp., Dallas, Texas, USA). Each tape strip was applied to the test area using a standard pressure for 5 seconds and removed from the test area in one gentle, continuous move.

For each repeated strip, the direction of tearing off was varied. The viable epidermis and dermis was then sampled from the skin by taking a full biopsy of 3.14 cm ² of the applied area for analysis. The skin surrounding the test area was discarded.

The concentration of ingenol 3-(3,5-diethylisoxazole-4-carboxylate) in the samples was determined by LC-MS/MS. Results

These studies allowed the amount of ingenol 3-(3,5-diethylisoxazole-4-carboxylate) found in the stratum corneum, epidermis and dermis and receptor fluid to be calculated, as a percentage of the applied dose.

EXAMPLE 4 - TESTING THE EFFECT OF INGENOL 3-(3,5-DIETHYLISOXAZOLE-4- CARBOXYLATE) IN A B 16 MELANOMA MODEL

Female C57BL/6JBomTac mice were injected intradermally in the right flank with 0.5 x 10 ⁶ viable B 16 melanoma cells suspended in 50 μΐ RPMI-1640 glutaMAX. Animals were either not treated or dosed topically once daily for 2 days on day 3 and day 4 after injection of B16 cells with a volume of 20 μΐ of ingenol 3-(3,5-diethylisoxazole-4-carboxylate) in the indicated vehicle applied to the treatment field (tumour and tissue surrounding the tumour) with a diameter of approximately 1.6 cm. Mice were subsequently monitored for tumour growth and tumours were measured daily with a calliper. Kaplan Meier survival curves (with tumour volume > 250 mm ³ as surrogate death event) were generated (Figure 1). Animals that died or were euthanized during the study period due to compromised well-being were included in the data analysis as censored objects. Animals with ulcerating tumours which were evidently in a growing stage (considerable increase in tumour size over several days) or animals with metastasis were recorded as death events and are included in the statistical analysis. The vehicle consisted of: 30% w/w isopropyl alcohol; 0.9% w/w benzyl alcohol; 1.5% w/w hydroxyethyl cellulose; 0.56% w/w citric acid monohydrate and 0.14% w/w sodium citrate dihydrate, made up to 100% with water and having a pH of between 3.2 and 4. Results

0.1% ingenol 3-(3,5-diethylisoxazole-4-carboxylate) cured or delayed tumor growth significantly in the B16-F0 melanoma mouse model, when compared to untreated animals.

EXAMPLE 5 - COMPARING THE EFFECT OF INGENOL 3-(3,5-DIETHYLISOXAZOLE-4- CARBOXYLATE) AND INGENOL MEBUTATE IN A B 16 MELANOMA MODEL

A comparison of the effect of ingenol 3-(3,5-diethylisoxazole-4-carboxylate) and ingenol mebutate was performed essentially as described in Example 4. B16 melanoma cells were injected intradermally at day 0 and the selected compounds were formulated at 0.1% in the vehicle (as described in Example 4) and applied topically to the skin overlaying the injection site at day 3 and day 4. Kaplan-Meyer survival curves are shown in Figure 2 with 'death event' defined as tumour exceeding 250 mm ³ or ulcerating tumour.

Median survival times and Log-rank tests:

*Not possible to calculate median survival time since less than half of the tumours in the treatment group have exceeded 250 mm ³ .