WO2016071365A1

WO2016071365A1 - Topical pharmaceutical compositions of paclitaxel

Info

Publication number: WO2016071365A1
Application number: PCT/EP2015/075641
Authority: WO
Inventors: Hesham H.A. SALMAN; Maria LLORENTE DOMÍNGUEZ; Izaskum IMBULUZQUETA ITURBURUA; Irene ESPARZA CATALÁN; Luis Antonio RUIZ ÁVILA; Jordi ARMENGOL MANSILLA; Benjamín SANTOS LOBO
Original assignee: Spherium Biomed, S.L.; Bionanoplus, S.L.
Priority date: 2014-11-03
Filing date: 2015-11-03
Publication date: 2016-05-12

Abstract

The present invention relates to topical pharmaceutical microemulsions of paclitaxel comprising a plurality of core-shell nanocapsules based on half (C₁-C₄) alkyl esters of poly (methyl vinyl ether-co-maleic anhydride) (PVM/MA) copolymers. The invention also relates to a process for the preparation of said compositions and to their use in the prevention and/or treatment of several diseases, particularly actinic keratosis.

Description

TOPICAL PHARMACEUTICAL COMPOSITIONS OF PACLITAXEL

FIELD OF THE INVENTION The invention relates to pharmaceutical compositions of paclitaxel for topical application wherein paclitaxel is encapsulated in core-shell nanocapsules based on half esters of poly (methyl vinyl ether-co-maleic anhydride) (PVM/MA) copolymers. The invention also relates to a process for the production of said compositions and to their use in medicine, particularly in the prevention and/or treatment of actinic keratosis.

BACKGROUND OF THE INVENTION

Paclitaxel is a mitotic inhibitor used in cancer chemotherapy pertaining to the taxane family of drugs. It was discovered in 1962 when it was isolated from the bark of the Pacific yew tree, Taxus brevifolia and named "taxol". It is approved for the treatment of ovarian, breast, non-small cell lung carcinomas and AIDS-related Kaposi's sarcoma.

Paclitaxel has the drawback of being poorly soluble. The commercially available pharmaceutical formulation of paclitaxel contains paclitaxel dissolved in Cremophor EL (polyethoxylated castor oil) and dehydrated ethanol (1 : 1, v/v) to enhance the solubility of paclitaxel in water. However, a large number of studies have reported various side effects of Cremophor EL, such as hypersensitivity, neurotoxicity and neuropathy. Therefore, premedication is mandatory before paclitaxel administration. The premedication schedule includes corticosteroids, diphenhydramine or chlorpheniramine, H2 -receptor antagonists and antiemetics. Despite such premedication, minor reactions (flushing and rash) still occur in 41-44% of all patients and major, potentially life threatening, reactions in 1.5-3%.

The ability of paclitaxel to inhibit cell division also enables paclitaxel to address hyperproliferative pathologic processes such as psoriasis or actinic keratosis.

Actinic keratosis is a premalignant condition of the skin. It is more common in fair-skinned people and it is associated with those who are frequently exposed to the sun, as it is usually accompanied by solar damage. The lesions are considered as potentially pre-cancerous, since some of them progress to squamous cell carcinoma, so treatment is recommended. Untreated lesions have up to 20% risk of progression to squamous cell carcinoma.

Paclitaxel is usually administered intravenously. The topical use of paclitaxel alone is not possible due to the excessive irritating action of the drug. In order to solve this drawback, a few formulations of paclitaxel have been designed for topical administration.

WO 2009/001209 Al discloses pharmaceutical compositions for topical treatment of actinic keratosis based on a conjugate between hyaluronic acid and paclitaxel. This formulation does not contain Cremophor but it is an aqueous composition wherein the main component is water.

Paolino D. et al. (Paolino D. et al. 2012. European Journal of Pharmaceutics and Biopharmaceutics, 81 : 102-112) discloses paclitaxel-loaded ethosomes® potentially useful for the treatment of squamous cell carcinoma. Said ethosomes® are vesicles made of phospholipids, ethanol and water.

Khandavilli S. and Panchagnula R. (Khandavilli S. and Panchagnula R. 2007. Journal of Investigative Dermatology, 127(1): 154-162) discloses paclitaxel nanoemulsions containing Labrasol- vitamin E-TPGS (3: 1) and Labrafil M1944CS for dermal administration.

In summary, paclitaxel compositions of the state of the art have different problems such as low long-term stability, low encapsulation efficacy, poor drug solubilization, low percutaneous absorption, systemic absorption, a cost and complex production process which requires the use of toxic organic solvents or complex techniques.

Therefore, it is necessary to develop further topical compositions of paclitaxel free of Cremophor EL which are capable of solving all or some of the drawbacks related to the known compositions and that are effective in the treatment of hyperproliferative diseases such as actinic keratosis or squamous cell carcinoma. SUMMARY OF THE INVENTION The inventors have surprisingly found that polymeric micro emulsions comprising a half (C₁-C₄) alkyl ester of a poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA) copolymer, a non-volatile organic solvent capable of solubilizing the copolymer selected from the group consisting of propylene glycol and polyethylene glycol, triacetin, a surfactant selected from the group consisting of polysorbates, anionic surfactants, block copolymers based on ethylene oxide and propylene oxide, polyvinylic alcohol, and a mixture thereof, an oil selected from the group consisting of medium- chain triglycerides, oleic acid, thyme oil, clove oil and mixtures thereof, and paclitaxel can be formed without the need of adding water and are suitable for topical application. Said microemulsions are composed of a plurality of core-shell nanocapsules wherein the shell of the nanocapsule comprises the copolymer; and the core of the nanocapsule comprises paclitaxel, triacetin and the oil.

The microemulsions of the invention have demonstrated to have several advantages over other formulations of the prior art such as the spontaneous formation, ease of manufacturing and scale-up, thermodynamic stability and improved drug solubilization. Said microemulsions are capable of solving all or some of the drawbacks related to other compositions of paclitaxel, for example, low long-term stability, low encapsulation efficacy, poor drug solubilization, low percutaneous absorption, systemic absorption, a cost and complex production process which requires the use of toxic organic solvents or complex techniques.

The examples of the present invention shown that the microemulsions of the invention have high stability under accelerated storage conditions and allow the local delivery of paclitaxel to skin compartment. Particularly, the microemulsions of the invention have higher stability than other compositions of the state of the art.

In a first aspect, the invention relates to a topical pharmaceutical microemulsion comprising a plurality of core-shell nanocapsules, wherein said microemulsion comprises:

(i) paclitaxel,

(ii) a half ((C₁-C₄) alkyl ester of a poly (methyl vinyl ether-co-maleic anhydride) (PVM/MA) copolymer,

(iii) a non-volatile organic solvent capable of solubilizing component (ii) selected from the group consisting of propylene glycol and polyethylene glycol, (iv) triacetin,

(v) a surfactant selected from the group consisting of polysorbates, anionic surfactants, block copolymers based on ethylene oxide and propylene oxide, polyvinylic alcohol and a mixture thereof,

(vi) an oil selected from the group consisting of medium-chain triglycerides, oleic acid, thyme oil, clove oil and a mixture thereof

wherein said microemulsion does not comprise more than 2% w/w of water and wherein each core-shell nanocapsule comprises a core and a shell, said shell comprising component (ii) and said core comprising components (i), (iv) and (vi). In a second aspect, the invention relates to a process for producing a topical pharmaceutical microemulsion according to the invention comprising:

a) dissolving the half (d-C₄) alkyl ester of a PVM/MA copolymer in a non-volatile organic solvent selected from the group consisting of propylene glycol and polyethylene glycol until a homogeneous solution is obtained,

b) adding to the solution in a) a surfactant selected from the group consisting of polysorbates, anionic surfactants, block copolymers based on ethylene oxide and propylene oxide, polyvinylic alcohol and a mixture thereof, and an oil selected from the group consisting of medium-chain triglycerides, oleic acid, thyme oil and clove oil and stir until a homogeneous solution is obtained, and

c) adding to the solution in b) a solution of paclitaxel dissolved in triacetin and stir until a clear solution of nanocapsules is formed.

In a third aspect, the invention relates to a topical pharmaceutical microemulsion according to the invention for use in medicine.

In a further aspect, the invention relates to a topical pharmaceutical microemulsion according the invention for use in the prevention and/or treatment of a disease selected from the group consisting of actinic keratosis, squamous cell carcinoma, Kaposi's sarcoma and psoriasis.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides microemulsions based on half (C₁-C₄) alkyl esters of PVM/MA copolymers, methods for producing said microemulsions and applications thereof. PHARMACEUTICAL COMPOSITIONS OF THE INVENTION

(i) paclitaxel,

(iii) a non-volatile organic solvent capable of solubilizing component (ii) selected from the group consisting of propylene glycol and polyethylene glycol,

(iv) triacetin,

wherein said microemulsion does not comprise more than 2 %w/w of water and wherein each core-shell nanocapsule comprises a core and a shell, said shell comprising component (ii) and said core comprising components (i), (iv) and (vi).

The compositions of the invention are suitable for topical application. The term "topical" as used herein, relates to a preparation applied to the surface of a part of the body and is used to describe formulations that have effects only in a specific area of the body and formulated in such a manner that the systemic absorption of the medicament is minimal. Topical application includes application in the exterior of the body such as, without limitation, the skin, scalp and nails; and also the application to mucosae such as, without limitation, nasal or rectal mucosae.

In the context of the present invention, the terms "pharmaceutical composition" and "pharmaceutical microemulsion" are interchangeable and refer to a system made of spherical micro droplets with a diameter between 10 and 100 nm. The microemulsions of the invention comprise a plurality of core-shell nanocapsules. Microemulsions are isotropic, thermodynamically stable transparent or translucent systems of two immiscible solvents and a surfactant. In the context of the present invention, based on the macroscopical examination only transparent or slightly milky formulations were considered to be within the microemulsion range.

The term "pharmaceutical", as used herein, means that the microemulsions of the invention are pharmaceutically acceptable to the patient from a pharmacological/toxicological point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding composition, formulation, stability, patient acceptance and bioavailability.

The term "core-shell nanocapsule", in the context of the present invention, refers to a colloidal system of a polymeric particle with an average size between 10 and 100 nm approximately, formed by natural or synthetic polymers (in this case, formed by polymerization of a half (d-C₄) alkyl ester of a PVM/MA copolymer). The term "average size" or "mean size", as used herein, relates to the average diameter of a population of nanocapsules moving together in a non-aqueous medium. The average size of these systems can be measured by standard processes known by persons skilled in the art and which are described, by way of illustration, in the experimental part attached to the examples described below. The average size of the nanocapsules can be mainly affected by the amount and molecular weight of the copolymer, and by the amount of paclitaxel present in the nanocapsules of the invention (generally, the larger the amount or molecular weight of said components, the larger the average size of the nanocapsule), and by some parameters of the process for the production of said nanoparticles, such as the stirring speed, etc.

The core-shell nanocapsules of the present invention are nano -vesicular systems formed by an inner cavity (known as "core") which contains paclitaxel, triacetin and an oil selected from the group consisting of medium-chain triglycerides, oleic acid, thyme oil, clove oil and mixtures of said oils, said core surrounded by a polymeric wall or membrane (known as "shell") comprising the copolymer. The paclitaxel is confined to a reservoir or within a cavity ("core") surrounded by a polymer wall or membrane ("shell"). The person skilled in the art knows that the core of the core-shell vesicular nanocapsule may also contain other excipients. However, due to the large specific surface of these systems, the molecules of paclitaxel may be trapped or adsorbed in the surface of the nanocapsules. The shell may also comprise triacetin and surfactant. In an embodiment, the average size of each nanocapsule is comprised between 10-100 nm.

The term "w/w", in the context of the present invention, relates to the weight of each component relative to the total weight of the microemulsion unless other is stated.

The term "paclitaxel", as used herein, refers to a compound with chemical name (2α,4α,5 β,7β, 10β, 13α)-4, 10-Bis(acetyloxy)- 13- { [(2R,3 S)-3-(benzoylamino)-2-hydroxy- 3-phenylpropanoyl]oxy}-l,7-dihydroxy-9-oxo-5,20-epoxytax-l l-en-2-yl benzoate and having the chemical formula:

Paclitaxel is a mitotic inhibitor used in cancer chemotherapy and pertaining to the taxane family of drugs. Paclitaxel was first isolated from the bark of the Pacific yew, Tanus brevifolia, and named "taxol". The generic name changed to "paclitaxel" when it was developed commercially and Taxol™ is now the trademark name of the commercial product.

In a preferred embodiment, the microemulsions of the invention comprise from 0.01 to 2% w/w of paclitaxel, preferably from 0.05 to 1.8% w/w, more preferably from 0.1 to 1.5% w/w, more preferably from 0.5 to 1.3% w/w, more preferably from 0.8 to 1.2% w/w of paclitaxel wherein w/w is the weight of paclitaxel relative to the total weight of the microemulsion. In a more preferred embodiment, paclitaxel is present in a concentration of 1% weight relative to the total weight of the microemulsion.

The term "paclitaxel" also includes pharmaceutically acceptable salts thereof. The term "pharmaceutically acceptable" refers to those properties and/or substances which are acceptable to the patient from a pharmacological/toxicological point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding composition, formulation, stability, patient acceptance and bioavailability. The term "pharmaceutically acceptable salt" embraces salts with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include both inorganic acids, for example and without limitation hydrochloric, sulfuric, phosphoric, diphosphoric, hydrobromic, hydroiodic and nitric acid and organic acids, for example and without limitation citric, fumaric, maleic, malic, mandelic, ascorbic, oxalic, succinic, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic, cyclohexylsulfamic (cyclamic) or p-toluenesulphonic acid. Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides and organic bases, for example and without limitation alkyl amines, arylalkyl amines and heterocyclic amines.

Paclitaxel is soluble in triacetin. The term "triacetin", as used herein, is the triglyceride 1,2,3-triacetoxypropane (the triester of glycerol and acetic acid) and is also known as glycerin triacetate. Its chemical name is l,3-diacetyloxypropan-2-yl-acetate and its chemical formula is the following:

In a preferred embodiment, the microemulsion of the invention comprises from 10 to 20% w/w, preferably from 12 to 18% w/w, more preferably from 15 to 17% w/w, even more preferably from 16.4 to 16.8 % w/w of triacetin.

The pharmaceutical microemulsions of the invention are based on a half ((d-C₄) alkyl ester of a poly (methyl vinyl ether-co-maleic anhydride) (PVM/MA) copolymer. As used herein, the term "(d-d) alkyl" relates to a radical derived from a linear or branched alkane of 1 to 4 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, etc. The terms "half ((d-d) alkyl ester of a poly (methyl vinyl ether- co-maleic anhydride) (PVM/MA) copolymer" or "half ((d-d) alkyl ester of a PVM/MA copolymer" are used interchangeably here and refer to water-insoluble copolymers that are water-soluble when neutralized by bases in aqueous solution and having a structure of formula

wherein R is a C₁-C₄ alkyl, in which only one of the two carboxyl groups is esterified. These include the half ester form of different alkyl chain lengths (monoethyl ester, monobutyl ester and isopropyl ester). Said copolymers are commercialized by International Specialty Products (ISP) under trademark Gantrez® ES and include Gantrez® ES 225 (monoethyl ester), Gantrez® ES 425 (monobutyl ester) and Gantrez® ES335I (isopropyl ester) and are supplied as alcoholic solutions, for example, in ethanolic solutions [50% (w/v)].

Gantrez ES 225 Gantrez ES 425

In a preferred embodiment, the half (C₁-C₄) alkyl ester of a PVM/MA copolymer is selected from the group consisting of ethyl ester of a PVM/MA copolymer, isopropyl ester of a PVM/MA copolymer and n-butyl ester of a PVM/MA copolymer; more preferably n-butyl ester of a PVM/MA copolymer.

In a preferred embodiment, the microemulsion of the invention comprises fromO. l to 10% w/w of a half ((C₁-C₄) alkyl ester of a PVM/MA copolymer, preferably from 0.1 to 8% w/w, more preferably from 0.1 to 6% w/w, preferably from 0.1 to 5% w/w, preferably from 0.2 to 3%> w/w, more preferably from 0.5 to 2%> w/w, more preferably from 1 to 2% w/w, even more preferably from 1.0 to 1.4% w/w. In another embodiment the microemulsion of the invention comprises from 1 to 5% w/w.

In the pharmaceutical compositions of the invention the half ((C₁-C4) alkyl ester of a PVM/MA copolymer is dissolved in a non-volatile organic solvent. The term "non-volatile organic solvent capable of solubilizing a half ((d-d) alkyl ester of a PVM/MA copolymer", as used herein, refers to an organic liquid that does not evaporate easily or evaporates very slowly at room temperature (e.g., PG, PEG400, glycerol) that usually has low vapor pressure and higher boiling point than water. The non-volatile organic solvents useful in the present invention are capable of solubilizing a half ((d-d) alkyl ester of a PVM/MA copolymer. The non-volatile organic solvent is a solvent capable of solubilizing the specific quantity of (d-d) alkyl ester of a PVM/MA copolymer used in the microemulsion of the invention. Solvents useful in the present invention are those in which the polymer is soluble, freely soluble and very soluble according to the definition of the European Pharmacopoeia. Since the quantity of the (d-C₄) alkyl ester of a PVM/MA copolymer used in the microemulsion of the invention is small, the non-volatile organic solvent can also be a solvent in which the polymer is very slightly soluble, slightly soluble or sparingly soluble according to the definition of the European Pharmacopoeia.

In a preferred embodiment a non- volatile organic solvent is considered to be capable of solubilizing a half (d-C₄) alkyl ester of a PVM/MA copolymer when it dissolves 10 mg/ml of said copolymer. This can be assayed by routine methods known to those persons skilled in the art. In a preferred embodiment non- volatile oganic solvents that can be used in the present invention are selected from the group consisting of propylene glycol and polyethylene glycol.

In a preferred embodiment the microemulsion of the invention comprises from 10 to 30% w/w, preferably from 13 to 25% w/w, preferably from 15 to 25% w/w, more preferably from 20 to 25% w/w, even more preferably from 21.7 to 22.1% w/w of a non- volatile organic solvent capable of solubilizing the polymer selected from the group consisting of propylene glycol and polyethylene glycol. In a preferred embodiment the non-volatile organic solvent is propylene glycol. The term "propylene glycol", as used herein, relates to an organic compound with the chemical formula C₃H₈0₂ also called propane- 1,2-diol. It is a viscous colourless liquid which is nearly odourless but possesses a faintly sweet taste.

In another preferred embodiment, the non- volatile organic solvent is polyethylene glycol 400 (PEG-400). The formation of micro emulsions requires the use of a surfactant. The term "surfactant", as used herein, refers to a compound that lowers the surface tension or interfacial tension between two liquids or between a liquid and a solid. Surfactants may act as detergents, wetting agents, emulsifiers, foaming agents and dispersants. In a preferred embodiment, the microemulsion comprises from 40 to 60% w/w, preferably from 45 to 58% w/w, more preferably from 50 to 55%, even more preferably from 54.0 to 54.4% w/w of a surfactant. In a preferred embodiment the surfactant is selected from the group consisting of polysorbates, anionic surfactants, block copolymers based on ethylene oxide and propylene oxide, polyvinylic alcohol, and mixtures thereof. Exemplary surfactants that can be used in the present invention are, without limitation, non-ionic surfactants, for example, polysorbates (i.e., oily liquids derived from pegylated sorbitan esterified with fatty acids, e.g., lauric acid, palmitic acid, stearic acid, oleic acid, etc.; esters of plain (non-PEG-ylated) sorbitan with fatty acids are usually referred to by the name "Span"), polyoxyethylene derivative of sorbitan monolaurate (Tween® 20), polyoxyethylene derivative of sorbitan oleate (Tween® 80), etc., anionic surfactants, e.g., sodium dodecyl sulfate (SDS), etc., block copolymers based on ethylene oxide and propylene oxide commercialized as Pluronics® by BASF, polyvinylic alcohol (PVA), etc. Mixtures of surfactants can also be used. In a preferred embodiment, the surfactant is a surfactant or a combination of surfactants having a HLB comprised between 7 and 40.

In a preferred embodiment, the surfactant is a polysorbate, more preferably polysorbate 80. The term "polysorbate 80", also known as Tween 80, refers to a nonionic surfactant and emulsifier often used in foods and cosmetics. The synthetic compound is a viscous, water-soluble yellow liquid derived from polyethoxylated sorbitan and oleic acid. Its full chemical name is polyoxyethylene (20) sorbitan monooleate or (x)-sorbitan mono-9-octadecenoate poly(oxy-l,2-ethanediyl). Its chemical formula is:

In another preferred embodiment, the surfactant is Tween® 20.

In another embodiment the surfactant is a block copolymer based on ethylene oxide and propylene oxide, preferably Lutrol L-44.

The composition of the present invention requires the presence of an oil capable of forming microemulsions with the other components. Said oil is selected from the group consisting of medium-chain triglycerides, oleic acid, thyme oil, clove oil and mixtures thereof. In a preferred embodiment, the microemulsion of the invention comprises from 1 to 10% w/w, preferably from 1 to 8%, more preferably from 2 to 6%, more preferably from 2 to 5%, even more preferably from 3.7 to 4.2% w/w of an oil selected from the group consisting of medium-chain triglycerides, oleic acid, thyme oil, clove oil and mixtures thereof. In an embodiment the oil is capable of dissolving more than 10 mg/ml of paclitaxel.

In an embodiment, the oil is medium-chain triglycerides. The term "medium- chain triglycerides", as used herein, refers to triglycerides containing 6-12 carbon fatty acid esters of glycerol. The fatty acids found in medium-chain triglycerides are called medium-chain fatty acids. Like all triglycerides (fats and oils), medium-chain triglycerides are composed of a glycerol backbone and three fatty acids. In the case of medium-chain triglycerides, 2 or 3 of the fatty acid chains attached to glycerol are medium-chain in length. Their name is caproic or hexanoic acid (C6:0), caprylic or octanoic acid (C8:0), capric or decanoic acid (C10:0) and lauric or dodecanoic acid (C12:0). In a preferred embodiment, the oil is capable of dissolving more than 10 mg/ml of paclitaxel. Preferably, said oil is caprylic/capric acid triglyceride.

In another embodiment, the oil is oleic acid. The term "oleic acid", as used herein, refers to a monounsaturated omega-9 fatty acid, abbreviated with a lipid number of 18: 1 cis-9. Its chemical formula is (9Z)-Octadec-9-enoic acid. It occurs naturally in various animal and vegetable fats and oils. Olive oil is the oil that is predominantly composed of oleic acid.

In another embodiment, the oil is thyme oil. The term "thyme oil", as used herein, refers to the essential oil of common thyme (Thymus vulgaris), containing 20- 54% thymol. Thyme essential oil also contains a range of additional compounds, such as /?-cymene, myrcene, borneol and linalool.

In another embodiment, the oil is clove oil. The term "clove oil", as used herein, refers to the essential oil extracted from the clove plant, Syzygium aromaticum. It has the CAS number 8000-34-8. In the context of the present invention, clove oil includes any of the three types of clove oil known (bud oil, leaf oil and stem oil). Bud oil is derived from the flower-buds of S. aromaticum and it consists of 60-90%) eugenol, eugenyl acetate, caryophyllene and other minor constituents. Leaf oil is derived from the leaves of S. aromaticum and consists of 82-88%) eugenol with little or no eugenyl acetate, and minor constituents. Stem oil is derived from the twigs of S. aromaticum and consists of 90-95%> eugenol, with other minor constituents.

The microemulsion of the invention does not require the use of water as a component of the formulation. However, the microemulsion of the invention may contain non- significant quantities of water coming from the rest of the components of the formulation. Particularly, the microemulsion of the invention does not comprise more than 2% w/w of water wherein w/w is the weight of each component relative to the total weight of the microemulsion. The microemulsion of the invention may comprise between 0 and 2% w/w of water, preferably between 0 and 1.9% w/w of water, more preferably between 0 and 1.8% w/w, even more preferably between 0 and 1.7% w/w, even more preferably between 0 and 1.6% w/w, most preferably between 0 and 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1% w/w of water. In a preferred embodiment the microemulsion does not comprise water.

Said compositions do not require the use of other components to be useful for topical application. Therefore, in a preferred embodiment, the microemulsion consists of:

(i) paclitaxel,

(ii) a half ((d-C₄) alkyl ester of a poly (methyl vinyl ether-co-maleic anhydride) (PVM/MA) copolymer, (iii) a non-volatile organic solvent capable of solubilizing component (ii) selected from the group consisting of propylene glycol and polyethylene glycol,

(iv) triacetin,

(v) a surfactant selected from the group consisting of polysorbates, anionic surfactants, block copolymers based on ethylene oxide and propylene oxide, polyvinylic alcohol, and mixtures thereof,

(vi) an oil selected from the group consisting of medium-chain triglycerides, oleic acid, thyme oil, clove oil and a mixture thereof and

a quantity of water ranging from 0 to 2% w/w.

In another embodiment the microemulsion consists of:

(i) paclitaxel,

(ii) a half ((d-C₄) alkyl ester of a poly (methyl vinyl ether-co-maleic anhydride) (PVM/MA) copolymer,

(iv) triacetin,

(vii) ethanol and

a quantity of water ranging from 0 to 2% w/w.

In another embodiment the microemulsion of the invention comprises:

(i) from 0.01 to 2% w/w of paclitaxel,

(ii) from 0.1 to 10% w/w of a half (d-C₄) alkyl ester of a PVM/MA copolymer, preferably 0.1 to 8% w/w, more preferably 0.1 to 6% w/w, even more preferably 0.1 to 5% w/w,

(iii) from 10 to 30% w/w of a non- volatile organic solvent capable of solubilizing component (ii) selected from the group consisting of propylene glycol and polyethylene glycol,

(iv) from 10 to 20% w/w of triacetin (v) from 40 to 60% w/w of a surfactant selected from the group consisting of polysorbates, anionic surfactants, block copolymers based on ethylene oxide and propylene oxide, polyvinylic alcohol, and mixtures thereof and

(vi) from 1 to 10% w/w of an oil selected from the group consisting of medium-chain triglycerides, oleic acid, thyme oil, clove oil and a mixture thereof

wherein w/w is the weight of each component relative to the total weight of the microemulsion.

In another embodiment the microemulsion according to the invention comprises: (i) from 0.8 to 1.2% w/w of paclitaxel,

(ii) from 1.0 to 8% w/w of n-butyl ester of a PVM/MA copolymer, preferably from 1.0 to 6% w/w, more preferably from 1.0 to 5.2% w/w, even more preferably from 1.0 to 1.4% w/w

(iii) from 21.7 to 22.1 % w/w of propylene glycol,

(iv) from 16.4 to 16.8 % w/w of triacetin,

(v) from 3.7 to 4.2 % w/w of medium-chain triglycerides, and

(vi) from 54.0 to 54.4% w/w of polysorbate 80

In another embodiment the microemulsion according to the invention comprises:

(i) from 0.1 to 1.5% w/w of paclitaxel,

(ii) from 1.0 to 8% w/w of n-butyl ester of a PVM/MA copolymer, preferably from 1.0 to 6% w/w, more preferably from 1.0 to 5.2% w/w, even more preferably from 1.0 to

1.4% w/w

(iii) from 21.7 to 22.1 % w/w of propylene glycol,

(iv) from 16.4 to 16.8 % w/w of triacetin,

(v) from 3.7 to 4.2 % w/w of medium-chain triglycerides, and

(vi) from 54.0 to 54.4% w/w of polysorbate 80

In another embodiment the microemulsion according to the invention comprises:

(i) from 0.8 to 1.2% w/w of paclitaxel,

(iii) from 13 to 22.1 % w/w of propylene glycol,

(iv) from 16.4 to 16.8 % w/w of triacetin, (v) from 3.7 to 4.2 % w/w of medium-chain triglycerides, and

(vi) from 54.0 to 54.4% w/w of polysorbate 80

In another embodiment the microemulsion according to the invention comprises: (i) from 0.1 to 1.5% w/w of paclitaxel,

(iii) from 13 to 22.1 %> w/w of propylene glycol,

(iv) from 16.4 to 16.8 % w/w of triacetin,

(v) from 3.7 to 4.2 %> w/w of medium-chain triglycerides, and

(vi) from 54.0 to 54.4% w/w of polysorbate 80

More preferably the microemulsion of the invention consists of:

(i) from 0.8 to 1.2%> w/w of paclitaxel,

1.4 w/w,

(iii) from 21.7 to 22.1 %> w/w of propylene glycol,

(iv) from 16.4 to 16.8 % w/w of triacetin,

(v) from 3.7 to 4.2 %> w/w of medium-chain triglycerides,

(vi) from 54.0 to 54.4% w/w of polysorbate 80, and

(vii) from 1.0 to 1.4% w/w of ethanol.

More preferably the microemulsion of the invention consists of:

(i) from 0.1 to 1.5% w/w of paclitaxel,

1.4 w/w,

(iii) from 21.7 to 22.1 %> w/w of propylene glycol,

(iv) from 16.4 to 16.8 % w/w of triacetin,

(v) from 3.7 to 4.2 %> w/w of medium-chain triglycerides,

(vi) from 54.0 to 54.4% w/w of polysorbate 80, and

(vii) from 1.0 to 1.4% w/w of ethanol. More preferably the microemulsion of the invention consists of:

(i) from 0.8 to 1.2% w/w of paclitaxel,

(ii) from 1.0 to 8% w/w of n-butyl ester of a PVM/MA copolymer, preferably from 1.0 to 6% w/w, more preferably from 1.0 to 5.2% w/w, even more preferably from 1.0 to 1.4 w/w,

(iii) from 13 to 22.1 %> w/w of propylene glycol,

(iv) from 16.4 to 16.8 % w/w of triacetin,

(v) from 3.7 to 4.2 %> w/w of medium-chain triglycerides,

(vi) from 54.0 to 54.4%> w/w of polysorbate 80, and

(vii) from 1.0 to 1.4% w/w of ethanol.

More preferably the microemulsion of the invention consists of:

(i) from 0.1 to 1.5% w/w of paclitaxel,

(iii) from 13 to 22.1 %> w/w of propylene glycol,

(iv) from 16.4 to 16.8 % w/w of triacetin,

(v) from 3.7 to 4.2 %> w/w of medium-chain triglycerides,

(vi) from 54.0 to 54.4% w/w of polysorbate 80, and

(vii) from 1.0 to 1.4% w/w of ethanol.

Even more preferably the microemulsion of the invention consists of:

(i) from 0.8 to 1.2% w/w of paclitaxel,

(ϋ) from 1.0 to 5.2% w/w of n-butyl ester of a PVM/MA copolymer,

(iii) from 13.0 to 22.1% w/w of propylene glycol,

(iv) from 16.4 to 16.8 % w/w of triacetin,

(v) from 3.7 to 4.2 %> w/w of medium-chain triglycerides,

(vi) from 54.0 to 54.4%> w/w of polysorbate 80, and

(vii) from 1.0 to 5.2% w/w of ethanol.

Even more preferably the microemulsion of the invention consists of:

i) from 0.1 to 1.5% w/w of paclitaxel,

ϋ) from 1.0 to 5.2% w/w of n-butyl ester of a PVM/MA copolymer,

iii) from 13.0 to 22.1%) w/w of propylene glycol, (iv) from 16.4 to 16.8 % w/w of triacetin,

(v) from 3.7 to 4.2 % w/w of medium-chain triglycerides,

(vi) from 54.0 to 54.4% w/w of polysorbate 80, and

(vii) from 1.0 to 5.2% w/w of ethanol.

The microemulsions of the invention do not require the use of additional preservatives. Therefore, in another embodiment, the microemulsion does not contain additional preservatives. By "additional preservatives", as used herein, is understood as substances added to pharmaceutical products to prevent decomposition by microbial growth or by undesirable chemical changes. Additional preservatives include antimicrobial additives and antioxidants.

Although it is not required to add additional preservatives, the microemulsions of the invention may contain preservatives. Exemplary preservatives that can be used in the microemulsions of the invention include, without limitation, potassium sorbate, sorbic acid, thimerosal, benzalkonium chloride, parabens, etc.

Alternatively, the composition of the invention may contain excipients. The term

"excipient", as used herein, refers to an inactive substance that can be liquid, solid or semisolid, used as a medium or carrier for the active ingredients of a composition. Illustrative, non- limitative examples of excipients are liquid paraffin or melted lipids such as wax, cotton oil, hydrogenated vegetable oil, canola oil, coconut oil, etc. Said excipients are particularly useful in the production of core-shell vesicular nanocapsules and they may be found in the core of said nanocapsules.

The person skilled in the art knows that the microemulsions of the invention may be administered in the form of pharmaceutical compositions comprising paclitaxel as a sole active ingredient or in combinations with other active ingredients.

The pharmaceutical compositions of the invention can be administered by different topical routes such as, without limitation, buccal, nasal or rectal route. In a preferred embodiment they are applied on the skin. More preferably they are applied by massage.

PROCESS OF THE INVENTION In a second aspect, the invention relates to a process for producing a topical pharmaceutical microemulsion of the invention comprising:

a) dissolving the half (C1-C4) alkyl ester of a PVM/MA copolymer in a non- volatile organic solvent selected from the group consisting of propylene glycol and polyethylene glycol until a homogeneous solution is obtained,

b) adding to the solution in a) a surfactant selected from the group consisting of polysorbates, anionic surfactants, block copolymers based on ethylene oxide and propylene oxide, polyvinylic alcohol, and mixtures thereof, and an oil selected from the group consisting of medium-chain triglycerides, oleic acid, thyme oil and clove oil and stir until a homogeneous solution is obtained, and

The method of preparation of the microemulsion of the invention comprises the previous preparation of a drug solution mixing paclitaxel with triacetin in a suitable jacketed tank heating to 30°C and stir until complete paclitaxel dissolution. Once dissolved, the paclitaxel solution is cooled to room temperature (20-25°C) since the next steps of the process should be performed between 20 and 25°C. Therefore, in a preferred embodiment steps a), b) and c) of the process of the invention are performed at a temperature between 20 and 25°C.

The first step of the process of the invention involves dissolving the half (Cl-

C4) alkyl ester of a PVM/MA copolymer in a non-volatile organic solvent until a homogeneous solution is obtained. Said homogeneous solution is obtained by stirring. The person skilled in the art can decide if a homogeneous solution is obtained by macroscopical evaluation.

The second step of the process of the invention involves adding a surfactant and an oil selected from the group consisting of medium-chain triglycerides, oleic acid, thyme oil and clove oil to the solution of the first step containing the half (C1-C4) alkyl ester of a PVM/MA copolymer and the non-volatile organic solvent, and stir until a homogenous solution is obtained. The obtention of a homogenous solution can be monitored by the same method used above.

The third step of the process of the invention involves adding the appropriate amount of paclitaxel solution dissolved in triacetine previously obtained to the solution obtained in the second step of the process and stir until a clear solution is formed. The final mixture has to be stirred at least for an hour. Obtaining a clear solution indicates that nanocapsules have been formed.

All the specific embodiments disclosed in the context of the compositions of the invention are applicable to the process of the invention.

MEDICAL USES OF THE PHARMACEUTICAL COMPOSITIONS OF THE INVENTION

The pharmaceutical compositions of the invention can be applied for the treatment of all such diseases that can be topically treated with paclitaxel.

In a third aspect, the invention relates to a topical pharmaceutical microemulsion of the invention for use in medicine.

Paclitaxel has been disclosed to be useful in the treatment of several diseases, including actinic keratosis (WO 2009/001209 Al), squamous cell carcinoma (Paolino

D. et al. 2012. Eur J Pharm Biopharm, 81(1): 102-12), Kaposi's sarcoma (Saville MW. et al. 1995. Lancet, 346(8966):26-8), and psoriasis (Kilfoile BE et al. 2012. J Control

Release, 163(l): 18-24).

In a fourth aspect, the invention relates to a topical pharmaceutical microemulsion of the invention for use in the prevention and/or treatment of a disease selected from the group consisting of actinic keratosis, squamous cell carcinoma,

Kaposi's sarcoma and psoriasis.

In another aspect, the invention relates to the use of a topical pharmaceutical microemulsion of the invention for the manufacture of a medicament for the prevention and/or treatment of a disease selected from the group consisting of actinic keratosis, squamous cell carcinoma, Kaposi's sarcoma and psoriasis.

In another aspect, the invention relates to a method of prevention and/or treatment of a subject suffering from a disease selected from the group consisting of actinic keratosis, squamous cell carcinoma, Kaposi's sarcoma, and psoriasis comprising the administration to said subject of a topical pharmaceutical microemulsion of the invention. The term "prevention", as used herein, refers to the administration of the microemulsion of the invention in an initial or early stage of a disease, or to also prevent its onset.

The term "treatment" is used to designate the administration of the microemulsion of the invention to control disorder progression before or after the clinical signs had appeared. By control of the disorder progression it is meant to designate beneficial or desired clinical results including, but not limited to, reduction of symptoms, reduction of the length of the disorder, stabilization pathological state (specifically avoidance of further deterioration), delay in the disorder's progression, improvement of the pathological state and remission (both partial and total). In a particular embodiment of the invention the microemulsion of the invention is used to control the disorder progression once at least one of the disorder's clinical signs has appeared.

The term "medicament", as used herein, refers to a pharmaceutical microemulsion of the invention comprising paclitaxel. The medicament may be administered by any suitable topical route. It is prepared by conventional means with pharmaceutically acceptable excipients. Formulations for application on the skin are preferred.

The term "subject", as used herein, refers to any animal or human that is suffering from one of the diseases disclosed above. Preferably, the subject is a mammal. The term "mammal", as used herein, refers to any mammalian species, including but not being limited to domestic and farm animals (cows, horses, pigs, sheep, goats, dogs, cats or rodents), primates, and humans. Preferably, the mammal is a human being. In the context of the present invention, the mammal is suffering from a disease selected from the group consisting of actinic keratosis, squamous cell carcinoma, Kaposi's sarcoma, and psoriasis or in risk of suffering from one of said diseases.

In a preferred embodiment the disease is actinic keratosis. The term "actinic keratosis", as used herein, refers to a malignant neoplasm of epidermal keratinocytes triggered by exposure to ultraviolet radiation and is an early stage in the continuous process from atypical keratinocyte proliferation to the development of non-melanoma skin cancer (NMSC) or squamous cell carcinoma. In another embodiment the disease is squamous cell carcinoma. The terms "squamous cell carcinoma", "squamous cell cancer", "epidermoid carcinoma" and "squamous cell epithelioma" are used here interchangeably and refer to a cancer of a kind of epithelial cell, the squamous cell. These cells may be found on the epidermis but also in the lining of the digestive tract, lungs and other areas of the body. Squamous-cell carcinoma occurs as a form of cancer in several tissues, including without limitation, the lips, mouth, esophagus, urinary bladder, prostate, lung, vagina and cervix, among others. The composition of the invention is useful in the treatment of any of such cancers. In a preferred embodiment the squamous cell carcinoma to be treated is the squamous cell carcinoma of the skin which is a non-melanoma skin cancer.

In another embodiment the disease is Kaposi's sarcoma. The term "Kaposi's sarcoma", as used herein, refers to a tumor caused by infection with human herpesvirus 8 (HHV8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV) or KS agent. It is one of the AIDS-defining illnesses. Kaposi's sarcoma is a systemic disease that can present with cutaneous lesions with or without internal involvement. The erythematous to violaceous cutaneous lesions seen in Kaposi's sarcoma have several morphologies: macular, patch, plaque, nodular, and exophytic. The cutaneous lesions can be solitary, localized or disseminated.

In another embodiment the disease is psoriasis. The term "psoriasis", as used herein, refers to a common, chronic and relapsing/remitting immune-mediated skin disease characterized by red, scaly patches, papules and plaques, which usually itch. The skin lesions may vary in severity from minor localized patches to complete body coverage. There are five main types of psoriasis: plaque, guttate, inverse, pustular and erythrodermic. The microemulsion of the invention is useful for the treatment of any kind of psoriasis.

All the embodiments disclosed in the context of the compositions of the invention are applicable to the medical uses of the pharmaceutical compositions of the invention.

The invention is described below by means of several examples which do not limit, but rather illustrate the invention.

EXAMPLES 1. Materials

Glyceryl tricapryl-caprate (MCT), propylene glycol, natural clove essential oil, natural thyme essential oil and polysorbate 80 (Tween® 80) were purchased from Guinama or Panreac. Triacetin and Gantrez® ES (poly(methyl vinyl ether-alt-maleic acid monobutyl ester) (GES 425) were purchased from Sigma- Aldrich, Panreac or Quimivita. Cremophor EL® was purchased from Fagron. Oleic acid and ethanol absolute were purchased from Panreac. Paclitaxel was purchased from Teva Czech Industries S.R.O. or Yunnan Hande Biotech Co. Ltd. Labrasol and Labrafil were purchased from Gattefosse. Vitamin E-TPGS was purchased from Isochem.

2. Product description: paclitaxel solution for topical application

Two formulations consisting on a microemulsion were prepared: one containing water and 0.1% w/w of paclitaxel and the other prepared in absence of added water and containing 1% w/w of paclitaxel. Their composition is showed in Table I.

Table I. Composition of micro emulsions of paclitaxel with and without water as additional component. Microemulsion F3 is a slightly yellow viscous 1% (w/w) paclitaxel solution and corresponds to a microemulsion according to the invention (not containing water and having polysorbate 80 as the main component in w/w).

Microemulsion F5 is a microemulsion containing water as a main component in w/w.

The manufacturing process for each composition was the following:

2.1 Manufacturing process for the microemulsion according to the invention (F3)

100 g of product were prepared as follows:

First, a drug solution was prepared by mixing 1.14 g of paclitaxel with 18.86 g of triacetin in a suitable jacketed tank and heating to 30°C. Then, the mixture was stirred until complete drug dissolution. Once dissolved, it was left to cool down to room temperature (20-25°C). The next steps were performed between 20 and 25°C.

Then, mixture 1 was prepared in a separate container. In order to prepare said mixture 2.40 g of commercial Gantrez® ES-425 (50% wt solution in ethanol) were mixed with 21.90 g of propylene glycol and the mixture was stirred until a homogeneous solution was obtained. Since Gantrez® ES-425 is a high viscous and adhesive polymer, special attention was paid to avoid product losses.

After that, mixture 2 was prepared. In order to prepare said mixture 3.9 g of medium-chain triglycerides and 54.2 g of polysorbate 80 were added to the solution containing propylene glycol and Gantrez® ES-425 (mixture 1), and said mixture 2 was stirred until a homogeneous solution was obtained.

Finally, 17.6 g of drug solution were added to mixture 2 and the final mixture was stirred until a clear solution was obtained, and at least for one hour.

2.2 Manufacturing process for the microemulsion F5

The microemulsion F5 was prepared as follows:

First, a drug solution was prepared by mixing paclitaxel with triacetin in a suitable jacketed tank and heating to 30°C. Then, the mixture was stirred until complete drug dissolution. Once dissolved, it was left to cool down to room temperature (20- 25°C). The next steps were performed between 20 and 25°C. Said drug solution is added to the medium-chain triglycerides and the Gantrez® ES-425 (50% wt solution in ethanol) dissolved in propylene glycol. Then, the surfactant is added.

Then, the final mixture is diluted with water (70 μΐ of mixture per 150 μΐ of water).

3. Stability studies The two paclitaxel- loaded topical polymeric formulations described previously were submitted to stability studies under accelerated storage conditions over a six month period.

Three 7g different batches of each formulation (F5 and F3) were prepared by weight as previously described. Each freshly prepared batch was aliquoted in 7 glass vials (1 g of formulation per vial). The first one was processed for analysis to evaluate macroscopical characteristics (color, turbidity, viscosity, etc.) and also to evaluate the total and free (non-encapsulated) amount of paclitaxel. The remaining six vials were submitted to accelerated storage conditions in a climatic chamber (40°C ± 2°C/ 75 % RH ± 5% RH). Each month, one vial per batch was evaluated macroscopically and also processed for analysis.

The parameters evaluated were: paclitaxel content, encapsulation efficacy and macroscopical appearance.

Paclitaxel quantification was conducted in order to know the total amount of paclitaxel as well as the amount of paclitaxel outside the nanoparticles.

Determination of the total amount of paclitaxel

F5 was diluted 200 times. For that purpose, 25 mg of the formulation were transferred into 5 mL volumetric flasks and diluted up to volume with acetonitrile:H20 (90: 10).

F3 was diluted 2000 times. For that purpose, 5 mg of each formulation were transferred into a 10 ml volumetric flasks and diluted up to volume with acetonitrile:H20 (90: 10). All the solutions were prepared in triplicate and injected once onto the HPLC column. Determination of the free amount of paclitaxel (non-encapsulated)

F5 was filtered through a 10 kDa amicon tube by centrifugation for 45 minutes at 3000 xg and 5°C.

F3 was previously diluted with water before filtering (120 mg of formulation were mixed with 324 mg of water).

F5 was diluted 5 times with acetonitrile and formulation F3 filtrate was diluted 50 times with acetonitrile.

All of them were prepared in triplicate and injected once onto the HPLC column. Briefly, the chromatographic conditions were:

• Analytical column: Zorbax SB-C18 (150x4.6 mm) 5 μιη, 100 A with the following guard-column: Zorbax SB-C18 (12.5x4.6 mm)

• Mobile phase: acetonitrile:H20 (65:35)

• Flow rate: 1 mL/min

· Injection volume: 20 μΐ

• Autosampler temperature: 22 °C

• Column temperature: 30 °C

• Detection wavelength: 280 nm

For each analytical run, 9 freshly prepared paclitaxel quality control standards (3 Qmin, 3 Qmed and 3 Qmax) were measured in order to ensure the quality of the analysis. Only analytical runs with at least 8 quality control standards with less than 5% relative error from the nominal concentrations were accepted.

Each vial was processed in triplicate. Thus, three analyses per formulation batch and nine analyses per formulation were conducted.

It was considered that there were no changes in paclitaxel concentration when the relative error of the measured paclitaxel concentration in each formulation was lower than 5%.

The average of total and free amount of paclitaxel in each formulation was used to calculate the encapsulation efficiency using the following expression: Total amount of paclitaxel-Free amount of paclitaxel

Ό ) = X

Total amount of paclitaxel For macroscopical evaluation, each aliquot was analyzed to evaluate significant color changes and precipitation or aggregation processes. 3.1 F5 stability results

When freshly prepared (Time 0), the formulation was transparent and a colourless solution at a concentration of 0.1 % (w/w). After the first month of storage (40°C ± 2°C / 75 % RH ± 5% RH), the concentration of paclitaxel in all the batches remained constant and the macroscopical appearance of the formulation was similar to that observed for the freshly prepared one. However, 24 hours after the change of storage conditions from climatic chamber to room temperature, the macroscopical appearance of all the batches changed to a white suspension with drug precipitates. After a second month of storage, paclitaxel concentration was approximately a 10 % lower in all the batches, and the decrease in concentration reached 36 % at the end of the six months. Over this period of time (from two to six months) at (40°C ± 2°C / 75 % RH ± 5% RH) the effect on macroscopical characteristics was similar than that observed after the first month: all the batches were colourless and transparent when taken out from the climatic chamber, but changed to turbid white suspensions with drug precipitates after 24 hours at room temperature.

However, this behaviour in macroscopical characteristics was not observed for the batches stored at room temperature over the same period of time (six months). T

The chromatogram obtained for those batches that were stored for 1 month was similar to that obtained for freshly prepared ones. However, for batches submitted to accelerated storage conditions for more than two months, a new peak is observed at a retention time of 3.3 ± 0.1 min whose area increases over time, while paclitaxel peak area decreases. At the light of these results, it could be considered that this new and unknown peak with a slightly different spectrum than that of paclitaxel is a paclitaxel degradation product.

All these results allowed inventors to conclude that formulation F5 was not stable under accelerated storage conditions from the first storage month.

3.2 F3 stability results Freshly prepared formulations (Time 0) were transparent and slightly yellow solutions at a concentration of 1.0 % (w/w). Both macroscopical characteristics and paclitaxel concentration remained constant in all the batches over the six month period. Additionally, in this case no macroscopical changes were observed after the formulation was taken out from the climatic chamber.

Nevertheless, the analysis of the chromatograms revealed that the unknown peak found in F5 degraded formulations started to appear after five months of accelerated storage conditions for F3 formulation. Considering that the stability study of formulation F5 allowed concluding that that peak is related to paclitaxel degradation process, it could be considered that paclitaxel is starting to degrade in formulation F3 from the fifth month. However, this possible degradation peak of paclitaxel is only incipient and no significant due to the fact that paclitaxel concentration at the end of the study remained constant (relative error < 5%).

In view of these results it could be considered that formulation F3 remained stable over six month period of time under accelerated storage conditions, but first symptoms of degradation were observed from the fifth month.

3.3. Conclusions

Concerning formulation F5, clear symptoms of degradation appeared from the first month of storage under accelerated conditions. However, formulation F3 remained stable over the six month period of accelerated storage conditions despite small signs of degradation at the endpoint of the study.

Therefore, it can be concluded that the formulation that does not contain water (F3) is more stable than the formulation containing water (F5).

4. In vitro percutaneous absorption of paclitaxel through pig ear skin

In vitro percutaneous pig skin absorption of paclitaxel was tested in Franz cells for three different formulations: F3 (2M-C: 1 % w/w paclitaxel), F5 (2MC-(H) 0.1 % w/w paclitaxel) and a control formulation (1% w/w paclitaxel in cremophor EL:ethanol 1 : 1).

This kind of assay requires the analysis of the drug in three different specimens: receptor compartment (liquid that has been in contact with the skin along the study), skin wash fluids (fluids obtained as a result of the washing of the surface of the skin at the end of the study) and skin (through which the drug is absorbed).

Pig ears were obtained from the municipal slaughterhouse from animals slaughtered on the same day. After reception, ears were cleaned with water and soap and finally biopsied and cleared of adhering subcutaneous tissues with a scalpel. Then, the skin was dermatomized with a dermatome at 400 μιη. Prior to its use in Franz cells, the thickness of each skin was measured using a specific micrometer and only skins with thickness between 380 and 430 μιη were accepted for the present study.

Each dermatomized pig ear skin was mounted in horizontal position between the two parts of the cell demarcating two compartments, one on each side of the skin:

• Receptor Compartment: Fluid applied to the lower side of the skin, consisting of 7 mL fixed volume of PBS:EtOH (60:40), with a sampling port for sample collection.

• Donor compartment: Teflon cylinder (dosage wafer) with an accurately defined surface of 1.767 cm2, applied to the upper side of the skin.

Each cell has a water jacket that allows keeping the system at a constant temperature at all times. In the present study the system was kept at 32 ± 1 °C.

Inside each cell there is a small magnet and a helix, set up at a constant stirring speed of 400 rpm to homogenize the fluid in the receptor compartment.

Half an hour after the skin is mounted on the cell and conditioned, the integrity of the skin barrier and the water tightness of the experimental model were verified for each diffusion cell before the application of the studied products, by measurement of the Trans Epidermal Water Loss (TEWL). The measurement was performed directly on the donor compartment using an evaporimeter. Finally, formulations were administered on each cell.

For the present paclitaxel permeation study, a total of 21 cells were mounted corresponding to:

^• 6 cells with formulation F3: 2M-C (1 % w/w paclitaxel)

^• 6 cells with formulation F5: 2MC-(H) (0.1 % w/w paclitaxel)

· 3 cells with a control formulation (1 % w/w paclitaxel in cremophor:ethanol

1 : 1)

^• 6 blank cells for calibration and quality control standards preparation. Additionally, it is well known that when a topical formulation is administered onto the skin, it is necessary to apply a massage in order to increase drug permeation. For that reason, and despite not being included in the initial design of the present study, we considered of great interest to have an estimation of the effect of that massage after the application of both F3 and control formulations. For that purpose only three cells were used: control formulation was administered to one cell and F3 was administered to the remaining two cells.

Taking into account that the tested formulations showed a high viscosity, the administered dose was measured gravimetrically. Thus, approximately 30 mg of formulation (accurately controlled by weight in each case) were administered in the donor compartment and at different timepoints (0, 1, 3, 8, 12 and 24 h), one mL of the receptor compartment fluid was sampled automatically and replaced by an equivalent volume of fresh liquid. At the end of the study, the skin surface was washed in order to eliminate the remaining non absorbed drug, and skin and receptor compartment were collected and processed for analysis as follows:

• Receptor Compartment: The receptor compartment fluid was a mixture of PBS and ethanol in a 60:40 ratio, in order to guarantee sink conditions. (Paclitaxel solubility in this mixture is 95 μg/mL). For the analysis of paclitaxel in this fluid, samples were filtered through 0.2 μιη PTFE membranes and injected into the HPLC. The established calibration range for this specimen is 0.035 - 0.500 μg/mL.

• Wash fluid: After 24 hours in contact with the Franz cell receptor chamber, each skin was washed in order to remove the non-absorbed Paclitaxel remaining on the surface. For that purpose, the surface of each skin is wiped with 8 cotton buds as follows: 4 half cotton buds were dampened in ethanol and rubbed 4 times each onto the exposed area of the skin (the dry side of the cotton bud is used each time to dry the skin before the next wiping); 2 more half cotton buds are dampened in ethanol and rubbed 12 times each onto the entire surface of the skin. Another 2 cotton buds were also impregnated in ethanol and used to clean the dosage wafer of the cell that could have been in contact with the drug. All the cotton buds used were left to dry overnight in a Falcon tube. Once they are dry, eight mL of acetonitrile are added and the mixture is sonicated for 1 hour in a bath with ice to avoid temperature increments. The fluid obtained after the sonication process was called wash fluid. Finally, this wash fluid was filtered through 0.2 μηι PTFE membranes and injected into the HPLC. The established calibration range for this specimen is 5.1 - 70.0 μg/mL.

• Skin: Several studies were conducted in order to optimize the procedure of paclitaxel extraction from skin. The initial approach consisted in skin digestion in order to achieve a complete extraction, but all digestion methods required the use of aggressive solvents that degraded paclitaxel. For that reason other methods were tested (sonication with acetonitrile, methyltertbutilether and methanol). Finally, the selected protocol is: addition of 3 mL of methanol with 0.1% acetic acid to the skin collected from the cell and introduced in a Falcon tube. Then, the skin was kept at room temperature for 15 h. At the end of this time, the mixture is sonicated for 1 hour. Finally, this solution was filtered through 0.2 μιη PTFE membranes and injected to the HPLC. The established calibration range for this specimen is 0.1 - 1 μg/mL.

A freshly made calibration curve and 6 freshly made quality control standards per specimen (three different concentration levels in duplicate) were prepared per group of cells (6 cells + 2 control cells) in order to accurately quantify the amount of paclitaxel present in each of the specimens at each of the timepoints. In order to obtain accurate and reproducible results for the paclitaxel in vitro skin absorption study in all specimens (processed skin, wash and receptor compartment samples), a triple bioanalytical validation was needed.

Samples collected from Franz cell study (pig ear skin, wash fluids and receptor compartments) were processed and analyzed once each. The total amount of paclitaxel recovered in each compartment was calculated and transformed in percentage considering the initial amount of drug administered onto the skin.

Results

Table II shows the total amount of paclitaxel recovered per cell after 24 h (in the case of receptor compartments, no detectable paclitaxel levels were found at the different studied times).

F5 cell 2 ND 27.4 < 0.17 ND 85.3 < 0.9 86.0

F5 cell 3 ND 25.2 0.23 ND 82.5 1.2 83.8

F5 cell 4 ND 27.0 0.29 ND 79.8 1.4 81.4

F5 cell 5 ND 26.1 < 0.17 ND 82.5 < 0.9 83.1

F5 cell 6 ND 26.0 ND ND 83.1 ND 83.1

Average ± < 0.14 ±

26.7 ± 1.1 83.2 ± 2.2 < 0.8 ± 0.7 83.9 + 1.8 SD 0.12

F3 cell 1 ND 292.3 0.46 ND 81.4 0.2 81.6

F3 cell 2 ND 268.3 0.63 ND 81.3 0.3 81.6

F3 cell 3 ND 297.2 0.24 ND 86.2 0.1 86.3

F3 cell 4 0.3 276.5 2.71* 0.1 82.5 1.4* 84.0

F3 cell 5 ND 278.9 0.30 ND 84.5 0.2 84.7

F3 cell 6 ND 285.0 0.36 ND 83.8 0.2 84.0

Average ± 283.0 +

0.40 + 0.15 83.3 ± 1.9 0.2 ± 0.1 83.7 ± 1.8 SD 10.6

Control cell 1 ND 297.2 « 0.17 ND 88.1 < 0.1 88.2

Control cell 2 ND 326.7 ND ND 87.1 ND 87.4

Control cell 3 ND 289.5 1.02* ND 83.5 0.52* 84.0

Average ± 304.5 ± < 0.09 ±

86.3 ± 2.5 < 0.1 ± 0.1 86.5± 2.3 SD 19.7 0.12

Table II. Total amount of paclitaxel detected in the different compartments (skin, wash fluid and receptor compartment) after 24 h in contact with the different formulations).

N.D: Not detected

* Not included in the average calculation as it is considered and outlier.

** Note that values expressed as "Not detected" were considered as "zero" for the calculation of the average, and values expressed as <0.17 or <0.9 or <0.1 were considered as 0.17, 0.9 and 0.1 respectively for the calculation of the average. As it can be seen the mass balance of the study does not comply with the specified requirements in the "OECD guideline for the testing of chemicals, skin absorption: in vitro method" (number 428), where a mass balance between 90% and 110% is required. However, the mass balance was similar in all cases (between 83 and 87 %>), so despite being lower than 90%>, a comparison between formulations could be established.

Therefore, a non-parametrical statistical test was conducted using SPSS software (Mann Whitney U test) to compare F3 and F5 formulations, finding that there are significant statistical differences between them in regard to the amount of absorbed Paclitaxel (p<0.05). The comparison between formulations allowed concluding that F3 enhances significantly paclitaxel absorption in comparison with F5. However, it was not possible to apply statistical tests to compare F3 and the control formulation, due to the fact that only 3 cells were assayed for the latter, and a high variability was found between replicates. Nevertheless, despite this high variability, it is remarkable that in the case of the three control cells, two of them did not present detectable or quantifiable paclitaxel values, whereas the six cells assayed with F3 presented well quantifiable paclitaxel values. For that reason, it can be considered that F3 presented a higher tendency to enhance paclitaxel skin permeation than the control formulation.

A new study was conducted in order to have an estimation of the effect of a massage after the application of both F3 and control formulations. In this case, only three cells were used: the control formulation was administered on one cell and F3 was administered on the remaining two cells. The study procedure of this study remained the same as previously described, but in this case, after the administration of each formulation, a massage was applied for 15 seconds on each cell with a large and slim stirrer. Finally, the amount of drug adhered to the stirrer was analyzed and subtracted from the initial amount of paclitaxel added onto the skin.

As it can be seen from Table III, the amount of paclitaxel recovered from the skin after the administration of the control formulation was quantifiable and higher than the average obtained when no massage was applied (excluding the cell considered as an outlier). However, the amount of paclitaxel found in the skin after the administration of F3 was clearly higher when a massage was applied to the skin.

F3 ND 316.5 1.17 ND 88.9 0.6 89.5

F3 ND 230.4 1.71 ND 84.8 1.1 85.9

Average + 273,5 ±

'„ 1.44 ± 0.38 86.8 ± 2.9 0.8 ± 0.4 87.7 ± 2.5

SU 60.9 Table III. Total amounts of paclitaxel detected in the different compartments (skin, wash fluid and receptor compartment) after the administration of paclitaxel formulations with a skin massage.

Therefore, a clear increase in paclitaxel absorption was found in both F3 and the control formulation when a massage was applied on the skin after the administration of the formulation, being this effect higher in the case of F3. It is considered that F3 can enhance paclitaxel permeation in comparison with conventional paclitaxel formulations prepared in a cremophonethanol mixture. 6. Comparative examples of paclitaxel solutions for topical application and evaluation of their stability

Formulation preparation

In this study several paclitaxel-loaded topical formulations were prepared in order to evaluate their behavior when subjected to different stress conditions. These formulations contained approximately 1.0% (w/w) paclitaxel but different excipients.

For that purpose, different formulations were prepared:

• F3, Paclitaxel 1.0 % (w/w) solution having the composition as disclosed previously

· F3G, Paclitaxel 1.0 % (w/w) solution with 5 % polymer (Gantrez® ES-425)

• F3WTP, Paclitaxel 1.1 % (w/w) solution without triacetine and polymer (Gantrez® ES-425)

• FR-1, Paclitaxel 1.1 % (w/w) reference solution as described in Khandavilli S and Panchagnula R. 2007. Journal of Investigative Dermatology 127 (1): 154-162 having Labrasol, Labrafil M 1944CS and vitamin E-TPGS .

• FR-1GT, Paclitaxel 1.0 % (w/w) reference solution with polymer (Gantrez® ES- 425) and triacetine having also Labrasol, Labrafil M1944CS and vitamin E- TPGS. The composition of each of the formultations prepared for this study can be found in Table IV below. Component % F3WTP FR-1 FR-1GT F3 F3G

(w/w)

Paclitaxel 1.1 1.1 1.0 1.0 1.0

MCT 4.8 3.9 3.9

Triacetine 16.6 16.6 16.6

Propylene Glycol 26.7 21.9 14.3

GES 425 1.3 1.2 5.0

Ethanol 1.5 1.3 1.2 5.0

Tween 80 66.0 54.2 54.2

Labrasol 71.3 57.4

Vitamin E-TPGS 23.7 19.2

Labrafil 3.9 3.2

Table IV. Composition of the formulations subjected to the stability study

The formulations were prepared dissolving the appropriate amount of paclitaxel in triacetine, if applicable, and adding the rest of the excipients to the mixture. The preparation process was performed at room temperature.

For formulation F3WTP (without polymer and triacetine), the active ingredient was dissolved in the mixture of all the excipients while in formulation FR-1, paclitaxel was dispersed in Labrafil and the mixture Labrasohvitamin E-TPGS was added afterwards.

For the preparation of formulations FR-1 and FR-1GT it was necessary to heat the mixture Labrasohvitamin E-TPGS up to 60 °C in order to obtain a homogeneous mixture as vitamin E-TPGS was a wax-like product.

Each batch was analyzed for paclitaxel total content and macroscopical appearance. The formulations were subsequently aliquoted in glass vials (approximately 5 g of formulation per vial) that were sealed with a crimper and submitted to the different storage conditions.

Test conditions

One batch of each formulation was prepared and divided in three aliquots to be subjected to different stability tests. • Stress conditions: The formulations were subjected to 4 autoclave cycles (15 minutes at 121 °C each).

• Freeze-thaw stability: The formulations were subjected to three freeze- thaw cycles consisting of 24 hours at -20 °C followed by 1 hour heating at 70 °C.

• Accelerated storage conditions: The formulations were stored at 40 ±

2°C / 75 ± 5% RH for six weeks.

The stability after each treatment was evaluated by analyzing the drug content as well as evaluating the macroscopical appearance of the formulations.

After each stability condition, each batch was processed in triplicate.

For each analytical run, 9 freshly prepared paclitaxel quality control standards (3 Qmin, 3 Qmed and 3 Qmax) were measured in order to ensure the quality of the analysis.

Processing, evaluation and data interpretation

Result processing

Total amount of paclitaxel: The average concentration of the three preparations for each formulation was calculated after each of the stability treatments. Paclitaxel quantification was conducted using the previously validated analytical method disclosed in section 3.

Accuracy: Paclitaxel concentration after each of the different stability treatments was compared with the concentration at the initial time. For the quality control standards, the concentration calculated from the calibration curve was compared with the nominal concentration. Acceptance criteria

The accuracy (expressed as %) between paclitaxel concentration at the initial time point and paclitaxel concentration at each stability point should be 100 ± 5 % in order for a formulation to be considered stable at certain conditions.

The macroscopical appearance of the formulations should be a colorless or slightly yellow transparent solution without any aggregates of precipitates. No major color changes should be observed between the initial time point and any of the stability ones, the formulations will not be considered stable otherwise.

The back calculated concentration of the quality control standards should be within 100 ± 5 % of their nominal value. Only analytical runs with at least 8 quality control standards with less than 5 % relative error from the nominal concentration were accepted.

Results

Table V shows the characterization results for all the formulations after being subjected to the previously described conditions.

ent ent ent ent Transparen

Turbidity/Precipi solution/ solution/ solution/ solution/ t solution/ tates No No No No No

precipitat precipitat precipitat precipitat precipitates

es es es es

Table V. Characterization results of the formulations subjected to this study As it can be seen in table V, formulation FR-1 showed a higher concentration than expected when stored at 40 °CI 75% RH. This result could not be associated to an evaporation process as this formulation did not contain any volatile excipients.

This variation in concentration observed for formulation FR-1 was not observed in the same formulation when triacetine and GES-425 were added, thus generating formulation FR-1 GT.

Conclusions

The macroscopical characteristics of all the formulations remained unaltered throughout the study. Regarding paclitaxel content, formulations F3, F3G and FR-1GT showed a consistent paclitaxel content throughout the stability study. Formulation F3WTP (prepared without triacetine and polymer) suffered a dramatic decrease in paclitaxel content after being subjected to all the stability treatments. It was therefore considered unstable, while its counterparts with triacetine and polymer remained stable after every treatment.

Formulation FR-1 showed a slight increase in paclitaxel concentration. This result makes the formulation as not compliant with the stability requirements. Nevertheless, this variation in concentration observed for formulation FR-1 was not observed in the same formulation when triacetine and Gantrez® ES-425 were added (formulation FR-1GT).

As a final conclusion of the present study, F3 and F3G are good candidates for paclitaxel topical administration.

Claims

1. A topical pharmaceutical microemulsion comprising a plurality of core-shell nanocapsules, wherein said microemulsion comprises:

(i) paclitaxel,

(iv) triacetin,

wherein said microemulsion does not comprise more than 2% w/w of water and wherein each core-shell nanocapsule comprises a core and a shell, said shell comprising component (ii) and said core comprising components (i), (iv) and (vi).

2. The microemulsion according to claim 1 wherein said microemulsion does not comprise water.

3. The microemulsion according to any of claims 1 or 2, wherein component (i) is present in a concentration of 1% weight relative to the total weight of the microemulsion.

4. The microemulsion according to any of claims 1 to 3, wherein component (ii) is n- butyl ester of a PVM/MA copolymer.

5. The microemulsion according to any of claims 1 to 4, wherein component (iii) is propylene glycol.

6. The microemulsion according to any of claims 1 to 5 wherein component (vi) is caprylic/capric acid triglyceride.

7. The microemulsion according to any of claims 1 to 5 wherein component (vi) is medium-chain triglycerides.

8. The microemulsion according to any of claims 1 to 7 wherein component (v) is polysorbate 80.

9. The microemulsion according to any of claims 1 to 8, wherein said microemulsion does not contain additional preservatives.

10. The microemulsion according to any of claims 1 to 9 comprising:

(i) from 0.01 to 2% w/w of paclitaxel,

(ii) from 0.1 to 10% w/w of a half (d-C₄) alkyl ester of a PVM/MA copolymer,

(iii) from 10 to 30 % w/w of a non- volatile organic solvent capable of solubilizing component (ii) selected from the group consisting of propylene glycol and polyethylene glycol,

(iv) from 10 to 20% w/w of triacetin

(v) from 40 to 60%> w/w of a surfactant selected from the group consisting of polysorbates, anionic surfactants, block copolymers based on ethylene oxide and propylene oxide, polyvinylic alcohol, and a mixture thereof and

(vi) from 1 to 10%> w/w of an oil selected from the group consisting of medium- chain triglycerides, oleic acid, thyme oil, clove oil and a mixture thereof wherein w/w is the weight of each component relative to the total weight of the microemulsion.

11. The microemulsion according to claim 10 comprising:

(i) from 0.8 to 1.2% w/w of paclitaxel,

(ii) from 1.0 to 5.2% w/w of n-butyl ester of a PVM/MA copolymer,

(iii) from 13 to 22.1 % w/w of propylene glycol, (iv) from 16.4 to 16.8 % w/w of triacetin,

(v) from 3.7 to 4.2 % w/w of medium-chain triglycerides,

(vi) from 54.0 to 54.4% w/w of polysorbate 80

12. A process for producing a topical pharmaceutical microemulsion according to any of claims 1 to 11 comprising:

b) adding to the solution in a) a surfactant selected from the group consisting of polysorbates, anionic surfactants, block copolymers based on ethylene oxide and propylene oxide, polyvinylic alcohol, and a mixture thereof and an oil selected from the group consisting of medium-chain triglycerides, oleic acid, thyme oil and clove oil and stir until a homogeneous solution is obtained, and

13. A topical pharmaceutical microemulsion according to any of claims 1 to 11 for use in medicine.

14. A topical pharmaceutical microemulsion according to any of claims 1 to 11 for use in the prevention and/or treatment of a disease selected from the group consisting of actinic keratosis, squamous cell carcinoma, Kaposi's sarcoma, and psoriasis.

15. The microemulsion for use according to claim 14 wherein the disease is actinic keratosis.