IT201900011436A1 - MICROMETRIC PARTICLES, THEIR METHOD OF PREPARATION AND THEIR USES - Google Patents

Description

MICROMETRIC PARTICLES, THEIR METHOD OF PREPARATION E

THEIR USES

SECTOR OF THE INVENTION

The present invention relates to micrometric particles comprising cetyl decanoate, their method of preparation and their uses. In particular, the particles of the invention are useful for promoting transepithelial absorption of poorly soluble and / or permeable active ingredients.

FRONT TECHNIQUE

The clinical efficacy of many topically administered therapeutic agents is often suboptimal due to their poor penetration into the epithelia, and particularly into the skin.

In general, these are poorly soluble drugs belonging to class II and IV and / or poorly permeable belonging to class IV of the Biopharmaceutics Classification System (BCS).

Solubility and partition coefficient are important parameters that influence the transepithelial permeation of the drug. According to Fick's law, the flow of a drug is proportional to the concentration of the drug in the vehicle: a high solubility allows a high concentration of drug in the donor (vehicle) phase which produces a high drug concentration gradient and therefore a high permeation flow. However, the high solubility in aqueous liquids usually causes a decrease in the partition coefficient between the aqueous vehicle and the keratinized layers of the epithelia. Therefore, drugs with high water solubility are poorly distributed in the epithelia and skin, while drugs with low water solubility often show good partitioning properties in the stratum corneum and good transdermal transport, but their absorption is severely limited by the concentration in solution present in the vehicle, from which a limited permeation flow ensues.

The technological strategies used to improve the permeability of epithelial tissues generally focus on improving the solubilities of drugs in the vehicle, on diffusion through the vehicle, on promoting the breakdown of the permeant in the lipid phase and on the use of chemical absorption promoters. Methods developed to increase the solubility of drugs that are not water soluble are complexation, for example with cyclodextrins [1], and novel vehicle systems, such as liposome-based delivery systems, over-saturated formulations and microemulsions [2] .

One approach that can provide a means for efficient controlled release into epithelial tissues is the use of solid lipid microparticles (SLMs) [3]. SLMs are similar to conventional oil-in-water emulsions. However, compared to conventional emulsions, high molecular weight hydrophobic "oils" or saturated non-glyceride esters that are solid at room temperature are used. In the scientific literature, a distinction is made between micro and nano-lipid particles, which generally refer to dimensions measured in the range of micrometers and nanometers, however, there is currently no unambiguous worldwide accepted definition of nanoparticles [4]. While lipid nanoparticles (SLNs) are currently attracting great attention in the scientific community due to their interesting size-dependent properties, lipid microparticles (SLMs) have been less studied [5]. The literature reports that the components for obtaining SLM are lipids and surfactants. The main lipids reported in the literature are solid lipids at room and body temperature (melting point> 45 ° C) and include different classes of compounds, such as triacylglycerides (e.g. tristearin, tripalmitin), partial glycerides and their mixtures (e.g. di- and tri-esters of glycerol and behenic acid), fatty acids (e.g. stearic acid), fatty alcohols (e.g. cetyl alcohol), steroids (e.g. cholesterol), non-glyceride esters of saturated fatty acids with fat saturated alcohols (e.g. cetyl palmitate) and waxes (complex mixtures containing mainly esters of fatty acids and hydroxy acids with monohydric fatty alcohols, free fatty acids and hydrocarbons). Among the different types of surfactants that can be used (mainly anionic, non-ionic and amphoteric surfactants but also cationic surfactants) physiologically compatible substances are preferred, such as phosphatidylcholine and bile salts (sodium cholate, sodium taurocholate, sodium glycocolate) [6] . Documents US 5,227,165 and WO 91/07171 describe lipid particles having micrometric dimensions.

Among the various lipid-based delivery systems, SLMs have numerous advantages:

● possibility of obtaining controlled release and / or site-targeting of drugs;

● provide the active compounds incorporated with protection from degradation;

● the solid matrix is composed of physiological and well tolerated lipids;

● allow the incorporation of hydrophilic and / or hydrophobic drugs;

● high physical and chemical stability;

● feasibility of large-scale production;

● total minimum costs [5].

Cetyl Decanoate (CAS 29710-34-7, Synonyms: Cetyl Caprato; Hexadecyl decanoate; hexadecyl ester of decanoic acid; hexadecyl ester of capric acid; Hexadecyl decanoate), is an ester between capric acid and cetyl alcohol. It is an aliphatic ester without unsaturation and therefore very stable. Cetyl decanoate is a lipid.

Cetyl decanoate has a melting point of 26 ° C; showed a high solubilizing power of lipophilic active ingredients. Cetyl decanoate has remarkable "skin feel" properties, probably due to its structural similarity with the lipid portions present in the epidermis and mucosal epithelia.

Its synthesis is reported in the literature to: prove that some hydrates of multivalent metal salts of Fe <3>, Al <3>, Ga <3>, etc. they are effective catalysts for the esterification of fatty acids and alcohols [7]; to describe a liquid-liquid biphasic esterification of long-chain carboxylic acids with long-chain alcohols in a Lewis acid ionic liquid, using 2ZnCl2 choline chloride as catalyst [8]; to demonstrate that ZrOCl2 * 8H2O is an efficient catalyst for the esterification of long-chain carboxylic acids with long-chain primary and secondary alcohols [9]; to describe that a crude lipase preparation from Pseudomonas Fragi exhibited esterifying activity [10]. Nothing is reported on its obtainment by synthesis starting from the chloride of decanoic acid in the presence of cetyl alcohol.

Cetyl decanoate is a material already used in the pharmaceutical and cosmetic fields as a component of mixtures of esters obtained from the processing of vegetable oils (see Cocoyl Caprylocaprate European Pharmacopoeia (EP) monograph, Ph. Eur.) Or as a cosmetic excipient (INCI name: Cetyl Caprate, or in Italian: Cetil Caprato). Nothing is reported on its use in commercial products as a standardized pure raw material, capable of conferring unique characteristics to the finished product.

Cetyl Decanoate is classified as: Emollient, Skin conditioner.

Its use in the pharmaceutical / cosmetic field is described in some patent documents, see for example the references [11–14]. However, these documents do not describe microparticles comprising cetyl decanoate.

In a 2012 study [15] the thermal properties of long-chain fatty acid esters with higher alcohols as novel organic phase change (PCM) materials for thermal energy storage are investigated. This study highlights how thermal reliability is a very important property of materials for use in thermal applications as it defines the duration times of the materials used. Long-chain fatty acid esters with cetyl alcohol, including cetyl decanoate, show less than 1% variation in both their phase change temperatures and latent heat values after 1000 thermal cycles. Furthermore, it is highlighted that these materials are thermally stable up to about 150 ° C, which makes them suitable for exothermic coacervation to obtain micro- or macro-capsules [15].

G. Campisi et al., British Journal of Dermatology, 2004, 150, 984-990 discloses microparticles comprising isopropyl myristate and hexadecanol.

Therefore, there is still a need to develop pharmaceutical formulations for the administration of poorly soluble / permeable or insoluble / impermeable agents, which increase their bioavailability.

DESCRIPTION OF THE INVENTION

The authors surprisingly found that, when combined with other lipid substances with a higher melting point, cetyl decanoate forms homogeneous low-melting mixtures capable of partitioning, at body temperature, in the epithelial lipophilic domains.

Therefore, in the present invention, the authors have developed microparticles comprising cetyl decanoate, which are able to increase the bioavailability of agents, in particular active principles, poorly soluble / permeable or insoluble / impermeable. In fact, these microparticles allow the topical-zonal administration of drugs which, due to their very low solubility, are not found on the market in the form of topical preparations. In particular, the microparticles can be loaded with lipophilic active ingredients. Furthermore, they may include one or more long-chain aliphatic alcohols, in particular from C14 to C18, such as 1-hexadecanol for example. They can also support the addition of monoterpenes such as limonene, to improve their organoleptic properties and performance.

The particles of the invention, thanks in particular to their composition and their micrometric dimensions (at least 10 micrometers, preferably from 20 to 300 micrometers), appear in the solid state as powders, but by simple pressure with the fingers or at the body temperature of 37 ° C are able to fuse with epithelial lipids, dragging the active ingredient with it and promoting its breakdown in the tissues.

The microparticles of the invention are particularly advantageous because, for example:

- it is possible to encapsulate within them the desired quantity of agent with a load that can reach 20-30% (w / w) of the total weight of the preparation, in particular for poorly soluble compounds,

- they have a softening point from 30 ° C to 40 ° C ± 0.5 ° C, preferably from 35 ° C to 37 ° C ± 0.5 ° C and therefore they melt in contact with epithelial and / or mucosal tissues thus releasing the agent that contain,

- they are able to release high quantities of the agent they contain, higher than those released by commercial formulations,

- promote the penetration and permeation of the agent they contain in the epithelial / mucosal tissues, carrying out a topical-zonal action,

- come in the form of powder with spherical particles that have a high sliding capacity. Flowability can be defined as the ability of split solids (e.g. powders and granules) to flow vertically under defined conditions. It is the expression of the frictions between the particles or of the resistance to movement linked to the interaction of the particles. - have dimensions and dimensional distribution suitable for epithelial / mucosal administration,

- they can be incorporated into semi-solid / solid formulations, such as gels and films, allowing for easy application,

- they can be prepared with a quick, simple, economical, reproducible, environmentally friendly process, with very little waste and without using organic solvents.

Furthermore, the particles of the invention are skin-friendly and non-toxic. Advantageously, their composition is highly biocompatible and biodegradable. The addition of surfactants, in particular surfactants with a HLB (hydrophilic-lipophilic balance, Griffin scale) value between 8.7 and 14 is not necessary, but it may be appropriate to make the particles, for example: sorbitan laurate, polyoxyethylene sorbitan tristearate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan monolaurate.

The particles of the invention, thanks in particular to their composition and their micrometric dimensions, appear in the solid state as powders, but by simple pressure with the fingers or at a body temperature of 37 ° C they are able to fuse with the epithelial lipids dragging the active ingredient with it and promoting its breakdown in the tissues.

The particles of the invention are also able to keep the agent inside them stable for long periods of time (years).

Furthermore, the particles of the invention are suitable for use in medical and non-medical applications. In fact, the particles of the invention loaded with agent have a great versatility of use: they can be used as such (powders), be dispersed in gel for skin or mucosal application (oral, buccal, vaginal, etc.), be incorporated in a lattice hydrophilic dehydrated (patches and scaffolds applicable on injured mucous membranes and wounds), they can also be administered orally resulting in gastro-resistant and prolonged release in the intestinal environment.

The preparation of the particles of the invention with the melt dispersion method does not involve the use of organic solvents or surfactants. The method, which involves a hot emulsification, using water as a dispersing phase, followed by a sudden cooling that transforms the lipid droplets into solid spherical particles, is simple, economical and the preparation water can be reused for subsequent productions. Both the microspheres and the residual water are biodegradable.

The yields of the production process of the particles of the invention are high, since more than 95% of the starting lipid mixture is recovered in the form of particles and the encapsulation efficiency of the agents reaches up to 100% for highly lipophilic molecules.

To obtain particles with a softening temperature close to that of the body and loaded with high melting point agents, it is possible to add one or more suitable excipient (s) and / or diluent (s), for example a C14 long chain aliphatic alcohol. to C18, such as 1-hexadecanol, and vary the ratio between it / s and Cetyl decanoate.

An object of the present invention therefore forms a particle comprising cetyl decanoate and a C14 to C18 aliphatic chain alcohol, in which said particle has a diameter of at least 10 micrometers. The aliphatic chain from C14 to C18 can be linear, branched or cyclic. Preferably the diameter is from 20 to 400 micrometers.

Preferably the cetyl decanoate and the aliphatic chain alcohol from C14 to C18 are in a weight ratio from 20: 80 to 90: 10.

Still preferably said C14 to C18 aliphatic chain alcohol is selected from the group consisting of: hexadecanol, myristic alcohol, stearyl alcohol and a mixture thereof.

Preferably the particle further comprising at least one agent selected from the group consisting of: a therapeutic agent, a monoterpene, a cosmetic agent, a surfactant, an insecticide, a repellent, a fertilizer, a fungicide, a pesticide and a herbicide, preferably said monoterpene is limonene, preferably said surfactant is HLB 11.

Preferably said therapeutic agent belongs to class II or IV of the Biopharmaceutical Classification System.

More preferably, said therapeutic agent is selected from the group consisting of: an antifungal, a corticosteroid, an antibacterial and an anti-inflammatory, an antioxidant, a vitamin, an anti-aging agent, preferably said antifungal is selected from the group consisting of: miconazole, econazole, econazole nitrate, clotrimazole, bifonazole, ketoconazole and itraconazole, Fluconazole, Isavuconazole, Voriconazole, Amphotericin B, preferably called corticosteroid is clobetasol or clobetasol propionate, Beclometasone dipropionate, Dexamutoninone, Acetinone, Flucolone, Acetonide, Flucolone, Acetonide, Flucolone preferably said antibacterial is curcumin, Metronidazole, Colimycin, tobramycin, gentamicin, Sulfadiazine, preferably said anti-inflammatory is curcumin Diclofenac, Piroxicam,

Preferably said antioxidant is curcumin, quercetin, resveratrol, ferulic acid, preferably said anti-aging agent is the enzyme Q10, melatonin, small peptides, Sodium DNA, preferably the vitamin is a fat-soluble vitamin (vitamin A, D, E, K ).

By cosmetic agent we mean any substance or mixture intended to be applied on the external surfaces of the human body (epidermis, hair system and hair, nails, lips, external genital organs) or on the teeth and mucous membranes of the mouth for the sole or predominant purpose of clean them, perfume them, modify their appearance, protect them, keep them in good condition or correct body odors.

Cosmetic products may include creams, emulsions, lotions, gels and oils for the skin, beauty masks, foundations (liquids, pastes, powders), face powders, talcum powder for after bath and body care, beauty soaps, deodorant soaps , perfumes, toilet waters and cologne, preparations for baths and showers (salts, foams, oils, gels), products for hair removal, deodorants and antiperspirants, hair dyes, products for waving, straightening and fixing , styling products, hair cleaning products (lotions, powders, shampoos), products to keep hair in shape (lotions, creams, oils), hair styling products (lotions, sprays, brillantines) , products for shaving (creams, foams, lotions), products for make-up and make-up, products intended to be applied to the lips, products for the hygiene of the teeth and the mouth, products for the care of the nails and varnishes for the same, external intimate hygiene products a, sunscreen products, self-tanning products, skin lightening products and anti-wrinkle products.

In a preferred form the particle is characterized by a softening point from 30 ° C to 40 ° C, preferably from 34.5 ° C to 37.5 ° C.

The present invention further provides a process for obtaining at least one particle having a diameter of at least 10 micrometers comprising the steps of:

- heating cetyl decanoate and a C14 to C18 aliphatic chain alcohol and optionally an agent as defined above until melting to obtain a melted mixture;

- adding water heated to a temperature from 80 ° C to 100 ° C to the melted mixture to obtain a dilute mixture, said water being optionally added with at least one of: an acid, a base, a surfactant, glycerol or a mixture thereof;

- stir said diluted mixture at a speed from 500 rpm to 1000 rpm, preferably around 800 rpm, for a period of from 30 seconds to 5 minutes to obtain an emulsion;

- lower the temperature of said emulsion down to a temperature from 5 ° C to 15 ° C in a period of 60 seconds to 240 seconds while maintaining agitation to obtain the solidification of the emulsion; thus obtaining the at least one particle.

The present invention further provides a particle obtainable from the process described above.

The present invention further provides the particle as defined above obtainable from the process of the invention.

The present invention further provides the particle as defined above for use as a medicament, preferably for use in a method of prevention and / or treatment of: an inflammation, a fungal infection, a bacterial infection, an injury, a condition of the aging, a neurodegenerative disease, ischemia.

Preferably, the particle according to the invention is used in a method of prevention and / or treatment of: candidiasis, thrush, vulvovaginitis, intertrigo, onychomycosis, B. dermatitis infection, H. capsulatum infection, P. brasiliensis infection, infection with C. immitis, cutaneous sporotrichosis, extracutaneous sporotrichosis, tinea corporis, extensive tinea versicolor, lichen planus, oral lichen planus, dermatitis, eczema, atopic dermatitis, seborrheic dermatitis, psoriasis, alopecia, vitiligo, pseudallescheriasis, mycetoma or paracoccidioidomycosis.

In a preferred form, said particle is administered topically, preferably to the oral mucosa, vaginal mucosa, skin, nasal mucosa, or orally, preferably to the gastrointestinal mucosa.

The present invention also provides the use of the particle as defined above in the cosmetic field, preferably against aging, for the preservation of fragrances and / or in the conveyance of substances poorly soluble in water and / or unstable to light and / or unstable to the environment. 'oxygen.

Preferably, the particle of the invention is used to mask an odor and / or a taste or for the preservation of fragrances or in the agricultural field, preferably as an insecticide, repellent, fertilizer, fungicide, pesticide and / or herbicide.

The present invention provides a powder comprising at least two particles, said particles being as defined above.

Preferably in the powder, at least 50% by weight of said particles has a diameter from 75 micrometers to 250 micrometers.

The present invention provides a pharmaceutical formulation comprising:

- the particle as above and / or the dust as defined above; And

- a pharmaceutically acceptable vehicle.

Preferably, said formulation is characterized in that it is in solid or semi-solid form, preferably in the form of gel, film, cream, gauze, plaster or stick.

The present invention provides a formulation in the form of a gel or film in which the pharmaceutically acceptable carrier is selected from the group consisting of: water, trehalose, polyvinylpyrrolidone, hydroxyethylcellulose and / or a hydrophilic polymer, preferably said hydrophilic polymer is hyaluronic acid or chitosan.

The present invention provides a process for the production of cetyl decanoate comprising a reaction between decanoic acid or its salt and hexadecanol, said reaction being carried out adopting one or more of the following conditions:

- use of methylene chloride as a solvent;

- reaction temperature of about 60 ° C;

- reaction time of about 24 hours;

- absence of a catalyst.

In the present invention, the terms "micrometric particles", "microparticles" "microspheres" and "lipospheres" are used interchangeably.

The components of the particles can be present in any form, for example: in the form of salts (in particular pharmaceutically acceptable salts), in the solvated form, in the unsolved form, in the hydrated form, in the non-hydrated form, in the polymorphic form, in the zwitterionic, in isotopic form, etc.

The particles of the invention are solid, lipid and approximately spherical. They comprise a matrix, which in turn can comprise: lipids, surfactants, polymers, waxes, protective materials, proteins and / or fats. Furthermore, they are physiologically compatible and stable from a physico-chemical point of view. Advantageously, they can be produced on a large scale at a relatively low cost.

In general, a fundamental property of powders is the size and dimensional range of the particles (particle size distribution). If the particle is a sphere, the size of that particle can be uniquely defined by the diameter measurement. The dimensional analysis of the particles that make up a powder can be carried out with different methods depending on the size range of the powder. The simplest way to represent the data of a dimensional analysis of a powder is to fill in a table showing the dimensional classes (the dimensional range or average value of the range) and the number (or weight) of particles that belong to each class.

For example, methods for determining the size of a particle or dust include:

SIEVING. The sieve technique is the most popular method for measuring the diameter of particles. The sieve is a cylindrical container, delimited at a base by a metal mesh. The space between the meshes of the net or the diameter of the holes constitute the sieve openings (nominal opening). This value represents the minimum section through which the particle can pass. To carry out the particle size analysis, the sieves are stacked in order of decreasing opening from top to bottom and placed on a mechanical shaker (vibrating screen). One proceeds by placing a defined quantity of powder on the first sieve, the sieves are subjected to vibration for a defined period of time, after which the fractions of material retained by the various sieves are measured by weighing. The average diameter of the particles retained on each sieve is expressed in µm and is calculated as the average between the nominal opening of the sieve mesh on which the powder has stopped and that of the immediately preceding sieve in the pile;

SEDIMENTATION. Particles smaller than 5 microns can be analyzed by the sedimentation technique;

LASER DIFFRACTOMETRY. The laser diffraction technique is currently the most widely used for the determination of particle distribution in the pharmaceutical field. Instruments are used that are capable of emitting a low-intensity laser beam. The technique is based on the fact that light is diffracted by a particle at an angle that is inversely proportional to the volume of the same particle. A laser light detector analyzes the intensity of the radiation diffracted by the particles. These are passed through the laser beam by free fall or pushed by a jet of compressed air;

COULTER COUNTER. Its operation is based on the measurement of the variation of an electric field generated by a pair of electrodes immersed in a conducting liquid when a particle suspended in the liquid is interposed between them. The instrument is able to count up to 4000 particles per second having a diameter between 0.5 and 1000 µm and to quickly convert the volume distributions generated by the data into weight distributions;

MICROSCOPIC TECHNIQUES. Microscopic analysis techniques are very versatile because they allow the observation and measurement of particle sizes over a wide dimensional range. They include for example:

Optical microscopy: sample preparation takes place by suspending the powder in a liquid that is not solvent for the material to be observed. The suspension is placed on a slide and the particles are observed under the lens of a microscope, in reference to a calibrated micrometric grid;

Electron microscopy: includes two techniques that refer to the use of an electron beam which, deflected from the surface of the sample, is recorded and transformed into images. The transmission electron microscope, TEM, is used for direct determination of particle sizes in the range 0.001-5 µm. The scanning electron microscope, SEM, can, in some cases, be able to observe particles of 200 Å in diameter.

The particles of the invention, in particular when they are prepared starting from an O / W emulsion, have a spherical or substantially spherical shape. The shape can be confirmed by optical microscope analysis. Therefore, when defining the size of the particles, reference is made to their diameter. It should be understood that the diameter can vary according to the sphericity of the particles. If a particle of the invention is not strictly spherical, but is substantially spherical, the diameter can be calculated from the average of two or more diameters measured for the same particle. Preferably, the diameter of the particles of the invention is measured by sieving or laser diffractometry. The softening point can be defined as the temperature at which some substances reach fluidity (liquid state). We conventionally speak of softening point since some substances (fats, bitumens, etc.) pass from the solid to the liquid state in a more or less wide range of temperature, not having a precise melting point.

The melting point of a substance is defined as the temperature at which, at normal atmospheric pressure (101.3 kPa), the solid-liquid equilibrium is established.

The melting temperature of a crystalline substance depends on the stability of its lattice: the greater the forces of attraction between the particles within the lattice, the higher the melting temperature. Pure substances (molecular compounds, salts, etc.) have a net melting point, i.e. within a very small temperature range of 0.5 - 1 ° C. Amorphous solid substances do not have a net melting point, they melt in a wider temperature range, within which the amorphous body becomes soft and later liquid. Even in impure substances, the phase change from solid to liquid occurs over a temperature range that can be several degrees.

The microparticles are amorphous solids, which do not melt at a specific temperature (such as crystalline solids) but soften, presenting a melting or softening range (such as amorphous glasses and polymers).

The methods for evaluating the softening point are the same as those used to determine the melting point.

The rules for carrying out the determination of the melting point are set out in the pharmacopoeias (eg: paragraph 2.2.14. Of the Official Pharmacopoeia of the Italian Republic XII ed.). The texts agree in indicating the "capillary tube" method as the basic method for determining the melting point and the pharmacopoeias also explain the minimum requirements necessary for the equipment and for the determination procedure.

The particles of the invention can be administered for example topically, orally, ocularly, rectally, vaginally, and / or by inhalation. Furthermore, according to the treatment needs, the particles of the invention can be formulated as suitable dosage forms.

In particular, the particles of the invention can be administered topically as such or after formulation in an appropriate dosage form. Suitable topical formulations include, for example, gels (in particular hydrophilic gels), films, plasters, gauze, ointments, creams, oils, lotions, solutions, pastes, ointments.

Gels can consist of dispersed systems of colloidal nature, semisolids, in which a dispersed phase (polymer or colloid) forms a three-dimensional reticular macromolecular structure in whose meshes the liquid phase (dispersant) traps. Crosslinking of the polymer or colloid is responsible for the viscosity and structure of the gel. The hydrophilic gels (hydrogels) which usually contain water, glycerol or propylene glycol, gelled with suitable colloids such as starch, cellulose derivatives, caboxyvinyl polymers and magnesium-aluminum silicates, can be alcoholic or non-alcoholic.

Films can be defined as solid dosage forms in the form of thin flexible sheets, mainly consisting of water-soluble or water-dispersible polymers that allow a fast recall of water, determining important processes of hydration, adhesion and dissolution when placed in contact with the floor of the tongue, if administration is sublingual, or with the mucous membrane of the oral cavity, if administration is buccal, or with the epidermis, if administration is on the skin. Various categories of excipients are found in the composition of a film, including: mucoadhesive polymers such as polyvinylpyrrolidone, cellulose derivatives, Carbopol, Polyacrylates, Chitosan; plasticizers such as propylene glycol, glycerol, triacetin, triethyl citrate; absorption promoters, such as fatty acids, surfactants, terpenes, menthol; flavor masks such as cyclodextrins; sweeteners, flavorings and dyes.

Creams, as known to formulation experts, are viscous liquids or semi-solid emulsions, and comprise an oily phase, an emulsifier and an aqueous phase. The oil phase generally comprises petrolatum and an alcohol such as cetyl or stearic alcohol. Examples of ointments include oil-based ointments such as vegetable oils, animal fats, semisolid hydrocarbons, emulsifiable ointments such as hydroxystearyl sulfate, anhydrous lanolin, hydrophilic petrolatum, cetyl alcohol, glycerol monostearate, stearic acid, water-soluble ointments containing polyethylene glycols of various molecular weights.

Formulations suitable for ocular administration include in particular eye drops, in which the particles are dissolved or suspended in a suitable carrier, preferably an aqueous solvent.

It is possible to administer the particles orally as such or by dispersing them in water or transforming them into dosage forms, such as tablets, capsules, tablets, tablets, oral preparations, powders, granules, pills, etc. Tablets and capsules for oral administration are normally presented in unit doses and contain conventional excipients such as binders, fillers (including cellulose, mannitol, lactose), diluents, tableting agents, lubricants (including magnesium stearate), detergents, disintegrants (e.g. polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate), coloring agents, flavoring agents, and wetting agents (e.g. sodium lauryl sulfate). Oral liquid preparations can be in the form of, for example, aqueous suspensions, solutions, emulsions, syrups or elixirs, or they can be presented as a dry product by reconstitution with water or with a suitable vehicle prior to use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, emulsifying agents, such as lecithin, sorbitan monooleate, or acacia; cosolvents such as propylene glycol, or ethyl alcohol; preservatives, such as methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired, conventional flavoring or coloring agents. The oral formulations also include conventional slow-release formulations such as gastro-resistant tablets or granules. The solid oral compositions can be prepared by conventional mixing, filling or tableting methods.

The particles of the invention can be formulated for administration via inhalation, for example by inserting them in an insufflator or in a pressurized nebulizer. The particles can be pharmaceutically formulated in suppositories or retention enemas, for example suppositories containing conventional bases such as polyethylene glycols, for rectal administration.

A reference for the formulations is Remington's book (Remington "The Science and Practice of Pharmacy", Lippincott Williams & Wilkins, 2000).

The particles of the present invention can be used to deliver drugs useful for the treatment and / or prevention of various pathologies or conditions as reported above, and can deliver a single drug or even the combination of two or more drugs in the same microparticle or by mixing microparticles. each containing a type of drug.

Particles containing different therapeutic agents can be administered simultaneously or sequentially.

The particles of the invention can be administered to a patient in a total daily dose of, for example, from 0.001 to 1000 mg / kg of body weight daily. The particles can also be administered weekly or once a day. Preferably, a dose of 5 to 500 mg particles is administered. Determining optimum dosages for a particular patient is well known to one skilled in the art. As is common practice, the compositions are normally accompanied by written or printed instructions for use in the treatment in question.

The particles of the invention are versatile vehicles for multiple applications.

The particles of the invention can be used in the human and veterinary pharmaceutical field, for example in the preparation of solid pharmaceutical forms, in particular in powder form, for application on skin and mucous membranes and in the preparation of semi-solid pharmaceutical forms for application on skin and mucous membranes.

The particles of the invention can also be used for the masking of odor or taste.

The particles of the invention can also be used in the cosmetic field, for example in the preparation of anti-aging cosmetics, in the preservation of fragrances, in the maintenance of the fragrance of cosmetics and in the conveyance of active ingredients unstable to light or oxygen. Examples of active ingredients unstable to light or oxygen include vitamin E, Retinol, Coenzyme Q10, ferulic acid, ceramides, Curcumin, anti-aging peptides, Sodium DNA, essential oils of a terpenic nature.

The particles of the invention can also be used in the agricultural field, for example to convey insect control agents (such as insecticides and repellents, for example pyrethrins and artificial pyrethroids), fertilizers, fungicides, pesticides, herbicides. For example, the particles of the invention can be used to convey clothianidin, imidacloprid, thiamethoxam, glyphosate, Sulfonylureas, Aryloxyphenoxy-propionates.

The present invention will be described in non-limiting examples, with reference to the following figures:

Fig. 1. <1> H NMR of Cetyl Decanoate.

Fig. 2. <13> C NMR of Cetyl Decanoate.

Fig. 3. FT-IR of Cetyl Decanoate.

Fig. 4. Photograph with a Leica optical microscope, with a Photometrics digital camera and visualized by Image-Pro Plus of miconazole lipid microspheres (MCZ) at 10% w / w.

Fig. 5. Photograph with a Zeiss optical microscope, with Azio-Cam camera and Axio-Vision acquisition program of miconazole lipid microspheres (MCZ) at 10% w / w.

Fig. 6. Size distribution of miconazole lipospheres (MCZ) at 10% w / w, in terms of percentage of particles having a given diameter.

Fig. 7. Thermograms of: A = miconazole (MCZ); B = cetyl decanoate; C = Hexadecanol (ESA); D mixture of MCZ, ESA and cetyl decanoate obtained by melting the components in a mixture; E = 10% w / w miconazole lipid microspheres (MCZ).

Fig. 8. Amount of miconazole (MCZ) (mg): □ total (sum of MCZ penetrated into the mucosa and MCZ permeated into the acceptor compartment), ■ penetrated into the mucosa, after administration of: [1] Daktarin <®> gel 80 mg (1.6 mg MCZ), [2] lipospheres 15 mg (1.2 mg MCZ).

Fig. 9. Percentage of miconazole (MCZ) (%) with respect to the administered dose: □ total (sum of MCZ penetrated into the mucosa and MCZ permeated into the acceptor compartment); ■ penetrated into the mucosa, after administration of: [1] Daktarin <®> gel 80 mg (1.6 mg MCZ), [2] lipospheres 15 mg (1.2 mg MCZ).

Fig. 10. Trend over time (h) of the quantity of miconazole (MCZ) (mg): □ total (sum of MCZ penetrated into the mucosa and MCZ permeated into the acceptor compartment); ■ penetrated into the mucosa after administration of 15 mg lipospheres (1.2 mg MCZ).

Fig. 11. Amount of miconazole (MCZ) (mg): □ total (sum of MCZ penetrated into the mucosa and MCZ permeated to the acceptor compartment); ■ penetrated into the mucosa, after administration of: [1] Daktarin <®> gel 80 mg (1.6 mg MCZ), [2] lipospheres 15 mg (1.2 mg MCZ), [3] lipospheres 2.5 mg (0.2 mg MCZ).

Fig. 12. Percentage quantity of miconazole (MCZ) (%) with respect to the administered dose: □ total (sum of MCZ penetrated into the mucosa and MCZ permeated into the acceptor compartment); ■ penetrated into the mucosa, after administration of: [1] Daktarin <®> gel 80 mg (1.6 mg MCZ), [2] lipospheres 15 mg (1.2 mg MCZ), [3] lipospheres 2.5 mg (0.2 mg MCZ).

Fig. 13. Amount of miconazole (MCZ) (mg): □ total (sum of MCZ penetrated into the mucosa and MCZ permeated to the acceptor compartment); ■ penetrated into the mucosa, after administration of: [1] Daktarin <®> gel 80 mg (1.6 mg MCZ), [2] lipospheres 15 mg (1.2 mg MCZ), [3] lipospheres 2.5 mg (0.2 mg MCZ), [ 4] 22.5 mg liposphere gel (0.2 mg MCZ).

Fig. 14. Percentage quantity of miconazole (MCZ) (%) with respect to the administered dose: □ total (sum of MCZ penetrated into the mucosa and MCZ permeated into the acceptor compartment); ■ penetrated into the mucosa, after administration of: [1] Daktarin <®> gel 80 mg (1.6 mg MCZ), [2] lipospheres 15 mg (1.2 mg MCZ), [3] lipospheres 2.5 mg (0.2 mg MCZ), [ 4] 22.5 mg liposphere gel (0.2 mg MCZ).

Fig. 15. Leica DFC 420C scanning optical microscope photograph, connected to a FireWire 1394a color digital camera, of Itraconazole (ITZ) microspheres.

Fig. 16. Thermogram of pure Itraconazole (ITZ), with the enthalpy values of all melting peaks.

Fig. 17. Thermogram of the lipid microspheres containing 10% w / w of Itraconazole (ITZ), with the enthalpy values of all melting peaks.

Fig. 18. Comparison between thermograms of pure Itraconazole (ITZ) (solid line) and lipid microspheres (dashed line) containing 10% w / w ITZ.

Fig. 19. Size distribution of clobetasol propionate (CLB) microspheres. x = collected fraction (% w / w).

Fig. 20. Appearance of Liposfer-gels A, B and C.

Fig. 21. Amount of clobetasol propionate (CLB) trapped in porcine membrane as a function of time after administration of: Liposfer-gel A � (left histogram), Liposfer-gel B � (central histogram) and Liposfer-gel C� (histogram right).

Fig. 22. Percentage of clobetasol propionate (CLB) trapped in porcine membrane as a function of time after administration of: Liposfer-gel A � (left histogram), Liposfer-gel B � (central histogram) and Liposfer-gel C� (histogram right).

Fig. 23. Filling of the molds for the realization of clobetasol propionate (CLB) Lipospheric films, starting from the suspension of gel microspheres.

Fig. 24. Liposfer-Films of clobetasol propionate (CLB): on the left Liposfer-film �; on the right Liposfer-film β.

Fig. 25. Patch cut out of the lipospheric film α.

Fig. 26. Amount of clobetasol propionate (CLB) trapped in porcine membrane as a function of time after administration of patch � (dark gray) and patch� (light gray).

Fig. 27. Percentage of the dose of clobetasol propionate (CLB) trapped in porcine membrane as a function of time after administration of patch� (dark gray) and patch β (light gray).

Fig. 28. Packaging of clobetasol propionate patches (CLB) 1x2 cm <2> cut from lipospheric film α.

Fig. 29. Application of the patch obtained from the lipospheric film α.

Fig. 30.% of the dose of Curcumin extracted from the single epithelial layers (from S1 to S10) and cumulative (TOT).

DETAILED DESCRIPTION OF THE INVENTION

Materials and equipment

The cetyl decanoate was synthesized in the laboratory. Miconazole (MCZ, catalog number, cat., 005167) was purchased from Farmalabor (Bari, Italy), decanoic acid chloride (cat. 140295), 1-hexadecanol (cat. W255408) and the (R) - (+) - Limonene (cat. 183164) at Sigma-Aldrich Chemie (Steinheim, Germany), the Polyvinylpyrrolidone, P.M. ∼ 360,000 (K90) (cat. A2260), sodium bicarbonate and the components used for the preparation of buffer solutions at VWR International (Leuven, Belgium), citric acid monohydrate at A.C.E.F. (Fiorenzuola D'Arda, PC, Italy), trehalose at Hayashibara Shoij (Hayashibara Shoij Inc., Okayama, Japan), hydroxyethylcellulose (Natrosol <®> 250 MR) at the Galeno company (Florence, Italy).

Econazole Nitrate (ECZ, cat. E4632), Clotrimazole (CLZ, cat.C6019) and Bifonazole (BFZ, cat. B3563) were purchased from Sigma-Aldrich Chemie (Steinheim, Germany), Ketoconazole (KTZ, cat. 2108) and itraconazole (ITZ, cat. 4962) at the company Galeno s.r.l. (Carmignano PO, Italy); Curcumin (CUR, cat. B21573) at Alfa Aesar (Lancashire, UK), Clobetasol propionate at SICOR-Società Italiana Corticosteroidi (Milan).

A Polimix mechanical stirrer mod. RW 20, equipped with a digital speed indicator KCH-TRON (Kinematica, Switzerland), supported by a stainless steel propeller shaft with four blades, with a diameter of about 4.5 cm. Permeation studies were conducted using vertical Franz-type cells (hole diameter 9 mm, acceptor compartment volume 15 ml; SES GmbH -Analysesysteme, Bechenheim, Germany).

The isotonic solution used for conditioning the mucous membranes was prepared by solubilizing 9.0 g / L of NaCl in distilled water.

The solution used as acceptor medium was prepared by solubilizing 5g / L of citric acid in distilled water (pH 3).

The buccal mucosa and the porcini auricle were kindly made available by the Municipal Slaughterhouse of Villabate, Palermo, Italy.

The temperature and stirring, in the ex vivo permeation tests, were adjusted using a stirring and heating plate (Heidolph MR 3001 K, Germany), equipped with a thermostatic probe (Heidolph EKT 3001, Germany).

The UV-Vis spectra were recorded with a Shimadzu UV-VIS spectrophotometer, mod. 1700, Scans from 600 to 190 nm, analysis interval 0.2 nm.

The chemicals and solvents were used without undergoing further purification.

Method of synthesis of Cetyl Decanoate

A solution in anhydrous methylene chloride, corresponding to 0.5 moles (95.36 g) of decanoic acid chloride (MW 190.71) was gently added to a lukewarm solution (60 ° C), kept under stirring, of 0.5 moles ( 121.22 g) of 1-hexadecanol in anhydrous methylene chloride. The reaction mixture was heated under reflux to 60 ° C and kept under stirring for 24 hours, during which time the HCl formed evaporated and a product separated. The mixture was then evaporated under reduced pressure, repeatedly washed with an aqueous solution of sodium bicarbonate, and subsequently with water at pH 7. The resulting waxy solid mass was collected by filtration, dried under vacuum, and repeatedly crystallized from acetone and alcohol. ethyl until obtaining a gas chromatographic purity of the cetyl decanoate> 98%. Four recrystallizations were sufficient to obtain samples of analytical purity. The obtained and purified product was analyzed to confirm its structure and to assess that there were no residues of the starting products.

NMR analysis

<1> H NMR (300 MHz, CDCl3): δ = 0.88 (br t, 6 H), 1.26 (br s, 38 H), 1.58–1.64 (br m, 4 H), 2.27–2.35 (t, J = 7.5 Hz, 2 H), 4.04–4.12 (t, J = 6.7 Hz, 2 H).

<13> C NMR (75 MHz, CDCl3): δ = 14.15, 22.68, 25.03, 25.94, 28.66, 29.16, 29.28, 29.42, 29.55, 29.69, 29.84, 31.91, 34.40, 64.38, 173.99.

Table 1. <1> H NMR of Cetyl Decanoate

Table 2. <13> C NMR of Cetyl Decanoate

The yield of the process was over 80%. The melting point of the product obtained, measured with a Büchi B-540 instrument, is 32 ° C. The analytical and spectroscopic data (Fig. 1-3) are in agreement with the expected structure. The FT-IR spectrum (Fig. 3) of the synthesized ester shows that the final product does not contain impurities of alcohol and unreacted fatty acid. Crystallization with acetone and ethyl alcohol is effective for removing unreacted impurities.

General method of preparation of lipid microspheres

For the preparation of microspheres containing the drug, the “melt dispersion” method was used. The mixture consisting of Pharmaco, 1-hexadecanol and Cetyl Decanoate, placed in a beaker, was melted on a plate or in a bain-marie in a silicone bath. After the melt was well mixed, limonene was optionally added (10 µL to 20 µL, eg 10 µL, 15 µL, 20 µL). Then 100 ml of distilled water or 120 ml of a 2: 1 (v / v) water / glycerol mixture were added to the melt, which was stirred, previously heated to a temperature from 90 ° C to 100 ° C and optionally add NaHCO3 (for example 0.5 g) if the drug is basic, HCl 1 N (for example 0.5 ml) if the drug is acidic, limonene (for example 1 mL) and / or HLB 11 surfactant (e.g. 0.05 g). The agitation (700-900 rpm, for example 700 rpm, 800 rpm or 900 rpm) carried out with a whisk with a 4-blade steel rod, determines the formation of an emulsion (O / W). The system was kept in these conditions for about 1-2 minutes, at the end of which the temperature was suddenly lowered, by means of an ice and water bath placed outside the beaker or by adding a mixture of water, ice and NaCl (for example 200-300 ml), maintaining constant stirring. When the system reached a temperature of about 10 ° C, the stirring was stopped. Due to the cooling effect, the melted mass, which constitutes the internal phase of the emulsion, solidifies. The microspheres formed were recovered by flotation and subsequent filtration, and dried at room temperature. The yield of the process was calculated using the following formula,

where Pmc and Pc respectively represent the weight of the microspheres and that of the components used for their preparation. It is on average around 90%. The validation of the composition and method of preparation was carried out by obtaining the following formulas:

Lipid microspheres at 10% w / w of Miconazole (MCZ),

Lipid microspheres at 10% w / w of econazole nitrate (ECZ),

10% w / w clotrimazole lipid microspheres (CLZ),

Bifonazole (BFZ) 10% w / w lipid microspheres,

Lipid microspheres at 10% w / w of ketoconazole (KTZ),

Lipid microspheres at 10% w / w of Itraconazole (ITZ),

Lipid microspheres at 0.1% w / w of clobetasol propionate (CLB),

Lipid microspheres with 2.5% w / w curcumin (CUR).

Example 1: miconazole lipid microspheres (MCZ) at 10% w / w

Miconazole (MCZ, m.p. 84 ° C) is a broad spectrum antifungal agent of the imidazole group. It is a widely used antifungal agent, but its use in topical formulations is not effective because deep fungal infections are difficult to treat with conventional topical formulations. Candida, in particular, represents one of the most frequently observed opportunistic pathogens, especially in lesions located at the mucosal level (thrush, vulvovaginitis), and more rarely at the cutaneous (intertrigo) and nail (onychomycosis) level. The progressive increase in the incidence of candidiasis in the oral cavity would seem to be attributable to an increasing, and often incongruous, consumption of antibiotics and immunosuppressants, with a significant role also attributable to antiblastics and anticancer radiotherapy [16]. Candida infections are also frequently observed vaginally; in fact, it is estimated that 75% of women have encountered this infection at least once in their life [17].

Methods

Method of preparation of miconazole 10% w / w lipid microspheres

The mixture consisting of MCZ (0.2 g), 1-hexadecanol (1.44 g) and Cetyl decanoate (0.36 g) was melted on a plate. 100 ml of distilled water, previously heated to 90 ° C and added with 0.5 g of NaHCO3 and 1 ml of Limonene, were added to the obtained melt, placed under stirring. The agitation (800 rpm) resulted in the formation of an O / W emulsion. The system was maintained in such conditions for about 2 minutes, at the end of which the temperature was suddenly lowered, by means of an ice and water bath placed outside the beaker, maintaining constant agitation. When the system reached a temperature of about 10 ° C, the stirring was stopped. Due to the cooling effect, the melted mass, which constitutes the internal phase of the emulsion, solidifies. The microspheres formed were recovered by flotation and subsequent filtration, and dried at room temperature. The process yield was 97 ± 0.5%.

Morphological analysis of microspheres under the microscope

The morphological analysis was performed using a Leitz DMRB optical microscope (Leica Microsystems, Germany), connected to a Photometrics digital camera and viewed using the Image-Pro Plus program. The analysis was also repeated with a Zeiss optical microscope. , an Azio-Cam camera and an Axio-Vision acquisition program. We then proceeded to observe and evaluate the geometric and morphological characteristics of the surface, photographing some representative samples of microspheres.

Determination of the drug content of the microspheres

5 mg of microspheres were transferred to a 5 ml flask and solubilized in methanol. The solution obtained was brought to volume with the same solvent and analyzed by UV-Vis spectrophotometry. Due to the interference of the excipients constituting the microspheres in the quantitative analysis, the absorption patterns were processed using the second derivative technique. Consistently with this, a calibration straight line was preliminarily carried out by analyzing 6 standard solutions in the concentration range 0.05-0.5 mg / ml and reprocessing the relative UV spectra with the second derivative technique.

The percentage content of MCZ actually present in the microspheres (Drug Loading, DL) was calculated as:

where "MCZ quantity determined" corresponds to the quantity of MCZ evaluated by UV spectrophotometry in an exactly weighted sample of microspheres ("quantity of microspheres") The DL was 8.0 ± 0.50% w / w.

Furthermore, the percentage of encapsulated MCZ compared to the theoretical one (Drug Recovery, DR or encapsulation efficiency), determined as:

where "quantity of MCZ in the formulation" corresponds to the actual quantity found within the formulation and "quantity of MCZ added" is the quantity of MCZ used during the preparation process of the microspheres. DR% was 80%, therefore the loss of active ingredient is 10%

Evaluation of the size distribution of the microspheres

The size distribution was determined by laser diffractometry using a Mastersizer 3000 (Malvern) laser light scattering instrument.

Before analysis, the samples were dispersed in an aqueous medium and a surfactant was added to facilitate the formation of the suspension.

Evaluation of the softening point of the microspheres

The softening point was evaluated, in capillary, on each batch of microspheres using a Büchi 530 instrument and setting the temperature increase at 0.5 ° C / min.

Differential Scanning Calorimetric Analysis (DSC) of the microspheres

DSC analyzes (Perkin-Elmer Diamond, Cryofill liquid nitrogen) were carried out on samples weighing between 8 and 10 mg, hermetically sealed in Perkin-Elmer aluminum capsules with a punching machine. The determinations were carried out at a heating rate of 10 ° C min <–1>, in a temperature range from - 30 ° C to 130 ° C; indium was used as the calibration standard. The difference in heat flow between the test sample and the reference sample is measured when these are subjected to controlled heating or cooling.

Preparation of the gel containing microspheres

To 50 ml of distilled water, previously heated (60 ° C), the following were added in order:

- trehalose, 0.15g,

- PVP K90, 0.05g,

- Natrosol 0.80g,

The system thus composed was gently mixed and then left to rest at room temperature for 24 h, until complete gelation.

We therefore proceeded to incorporate the microspheres by manually dispersing them in the gel until a homogeneous dispersion (microspheres ratio: gel 1: 8 w / w) was obtained, trying to avoid the incorporation of air.

Ex vivo permeation studies through porcine buccal mucosa

1) Preparation and conditioning of the porcine buccal mucosa

Samples of porcine buccal mucosa, taken immediately after the sacrifice of domestic pigs of an average age of 12 months, were obtained from the tissue removed from the vestibular area of the retro molar trigone. Subsequently, they were refrigerated and transferred within 1 h of the animal's sacrifice to the laboratories of the Department (STEBICEF), where they were appropriately stored at a temperature of about -20 ° C. Before use, the mucosal samples were subjected to the separation of the mucosal epithelium from the adipose tissue and the submucosal connective tissue by means of thermal shock. The separation of the epithelium was achieved by placing the sample in an isotonic solution, at a temperature of 60 ° C, for about 2 minutes; then, the detachment from the submucosal tissue was carried out, thus obtaining mucosal portions having a thickness of 250 ± 25µm. The heat treatment does not involve any modification of the permeability and integrity of the components of the oral mucosa [18]. The mucosal epithelium was then subjected to conditioning in isotonic solution, at room temperature, for 24 h.

2) Evaluation of permeation

For the comparative evaluation of the permeation of MCZ released by Daktarin <®> 2% oral gel, lipid microspheres and microspheres gel, a vertical Franz-type cell was used, representing a two-compartment open model. The dosage form to be tested was placed in the donor compartment; the acceptor compartment was filled with a solution of citric acid at pH 3, in which the drug is considered soluble enough to allow the maintenance of the "sink" conditions without the occurrence of premature saturation of the acceptor medium. The porcine buccal mucosal epithelium was interposed between the two compartments, as described in point 1). Comparative permeation studies were carried out using the different formulations from time to time. In particular, the following were loaded into the donor compartment:

● 80 mg of Daktarin <®> gel at 2% w / w (corresponding to 1.6 mg of MCZ), roughly corresponding to the conventionally used dose of Daktarin <®>

● lipid microspheres 15 mg (corresponding to 1.2 mg of MCZ)

● lipid microspheres 2.5 mg (corresponding to 0.2 mg of MCZ)

● 22.5 mg microspheres gel (corresponding to 0.2 mg of MCZ)

The permeation experiments were conducted at a temperature of 37 ± 0.5 ° C. At regular intervals of time (60 min) 0.5 ml of solution were withdrawn from the acceptor compartment, immediately replaced with an identical volume of fresh acceptor fluid. The amount of drug permeated through the porcine mucosa was evaluated by UV-Vis spectrophotometric analysis using the appropriate blank (citric acid solution). Due to the reduced concentration of drug in the acceptor solution after 5 hours, as well as the interference of the excipients constituting the microspheres in the quantitative analysis, the traces were processed using the second derivative technique. Therefore the calibration curve was performed using standard solutions of MCZ in citric acid (concentration range 0.005-0.05 mg / ml).

Each experiment was conducted for 5 hours and repeated 6 times. At the end of each permeation experiment, the cell was disassembled and the porcine membrane removed.

3) Extraction of the MCZ from the porcine buccal mucosa

At the end of each permeation experiment, the drug was extracted from the mucosal tissue sample. The mucosa was rapidly washed with isotonic solution (5 ml), to eliminate any residual MCZ retained on the surface, divided into small pieces and subjected to cold extraction in methanol (5 ml) for 24 h. The methanolic solution was transferred to a 5 ml flask and made up to volume with the same solvent. The quantity of drug extracted was determined by UV-Vis spectrophotometric analysis using methanol as blank and the relative calibration curve.

Results

The aim of the research was to develop a new dosage form which, through the topical administration of the MCZ, allows a massive penetration of the drug into the mucosal tissue, overcoming the limits of the conventional pharmaceutical form. The creation of a therapeutic system, capable of incorporating MCZ and promoting its absorption at the level of the buccal mucosa, could be useful in the local therapy of oral candidiasis. This therapeutic system could also be administered on the vaginal mucosa, for the therapy of Candida vulvovaginitis. From a histological point of view, in fact, it is not risky to compare the structure of the vaginal mucosa to the structure of the oral mucosa: in both cases, in fact, they are tissues in continuous reworking, on which delicate defensive systems (saliva and its enzymes and the acidity of the vaginal milieu) in substantial balance with a resident microbial flora [19].

Lipid microspheres containing MCZ were made as a matrix system in which the drug is homogeneously distributed in molecular form. The release of the drug should occur following the fusion of the microspheres in contact with the mucosa. The authors hypothesized that the materials constituting the matrix, thanks to their peculiar characteristics, should "lipidize" the mucosa by promoting the penetration of the drug into the tissue: this would lead to an optimization of the bioavailability of the drug at the site of action, or in the buccal mucosa or vaginal.

The preparation of the lipospheres was carried out using the hot melt dispersion method [20,21]; this process involves a biphasic system, in which the formation of the lipospheres is induced by thermal shock. It is a quick, simple, inexpensive and reproducible method; moreover, by not requiring the use of organic solvents, it avoids the presence of toxic residues in the preparation.

In order to be able to administer the microspheres on the buccal mucosa, the materials used as support should have chemical-physical properties such as to make them similar to the administration site. Taking into account some ideal characteristics, such as lipophilicity, non-toxicity, biocompatibility, as well as flow capacity and softening point of the microparticles to be obtained, a higher fatty alcohol was chosen, hexadecanol, to be used in mixture with Cetyl Decanoate. The poor water solubility of MCZ, together with the lipophilicity of the matrix, allowed a good encapsulation efficiency. In order to obtain particles with suitable geometric (spherical) and softening properties (melting range 36-40 ° C), in consideration of the fact that MCZ contributes to the final softening point of the microparticles, mixtures of the above components were tested, all containing 10% w / w MCZ, in different proportions cetyl decanoate: 1-hexadecanol (from 90:10 to 10:90 by weight). The optimal weight ratio between Cetyl Decanoate and 1-hexadecanol was found to be 20:80. In fact, this ratio of components, combined with 10% of MCZ, leads to obtaining a lipid mixture with a melting range close to that of body temperature, therefore compatible with administration. At the same time, the spherical shape of the microparticles allows for high sliding capacity.

In consideration of the fact that the long-chain alcohol used in the preparation shows surfactant characteristics, during the process of encapsulating the drug in the lipid support it was not necessary to use any emulsifier.

The main factor affecting the size range of the microspheres is the stirring speed used during preparation. By operating at a speed of 800 rpm, particles below 300 µm are obtained, suitable for buccal administration. Lower speeds, on the other hand, lead to the formation of larger particles or non-spherical aggregates.

To obtain spherical and sufficiently small particles (<300 µm), the cooling rate of the system was also decisive. The solidification of the lipid support is, in fact, promoted by a rapid lowering of the temperature. The use of an ice bath proved to be suitable for determining the rapid solidification of the material.

The yield of the microencapsulation process was 97 ± 0.5%.

All the batches obtained were analyzed under an optical microscope in order to confirm the morphological characteristics and evaluate their quality, size and homogeneity. Under the microscope, the particles appear homogeneous, spherical and translucent; furthermore, no irregular or crystalline material is observed between the particles, indicating that no crystallization phenomena of the drug have occurred outside the microparticles during the process of solidification of the same (Fig. 4 and 5). The average value of the softening point, performed on 9 batches, of the lipospheres containing MCZ was 35 - 37 ° C ± 0.5 ° C, perfectly compatible with the temperature present inside the oral cavity.

The particle size and dimensional distribution can affect the smoothness of the product, its handling and its appearance; they can also influence the speed with which the fusion takes place and, consequently, the release of the active principle.

The dimensional analysis of the lipospheres was carried out using laser light scattering. The results were expressed in terms of the percentage of particles having a given diameter. Fig. 6 shows, on a semi-logarithmic scale, the dimensional distribution of the analyzed microparticles.

The dimensional distribution is sufficiently homogeneous in a narrow range of diameter; in particular, 90% of the lipospheres fall within a dimensional range between 16.4 and 163 µm. These dimensions indicate that the powder obtained is sufficiently homogeneous for its handling (e.g. processing, mixing) and the particles are small enough to be able to distribute well on the tissues but not too small to be lost during application or even inhaled. during handling.

By means of UV-Vis spectrophotometric analysis, the average MCZ content in the lipospheres was evaluated. Since the lipid materials used in the formulation showed UV absorption in the wavelength range in which the MCZ absorbs, the absorption spectra plots were processed using the derivative II technique. Under these conditions, the MCZ content in the lipospheres was equal to 8% � 0.5% w / w (80% encapsulation efficiency) Therefore only a small amount of drug is lost during the preparation process.

In order to evaluate the physical state in which the MCZ is located within the microspheres (eg crystalline or amorphous) and any MCZ-excipients interactions, differential scanning calorimetry (DSC) analyzes were conducted. Fig. 7 shows the thermograms relating to MCZ, hexadecanol, Cetyl decanoate, lipospheres and the mixture obtained by fusion of MCZ-hexadecanol-Decanoate before the microencapsulation process.

The thermograms of the individual components (Fig. 7 A, B, C) are characterized by the presence of endothermic peaks relative to the melting point of the active ingredient and of the excipients. The thermogram of the lipospheres (E) keeps the endothermic peaks related to the excipients unaltered, but shows the disappearance of the endotherm related to the MCZ, suggesting a process of amorphization of the same. It should also be noted that the ratio between the areas of the two excipients reflects their weight ratio in the liposphere formulation. From the thermogram of the mixture (D) of all components, it is possible to highlight that already in the mixing phase, drug-excipients interactions take place such as to induce the amorphization of the active ingredient itself. The presence of MCZ in the amorphous and non-crystalline microspheres allows the drug to increase its solubility in the lipids of the epithelial tissues and thus allow a better distribution. In fact, this results in a better penetration into the biological tissue.

In order to evaluate the ability of the lipospheres to release the drug in the oral mucosa and favor its penetration, a comparative study was conducted with the commercial product Daktarin <®> oral gel containing 2% drug (MCZ). These studies were conducted through ex vivo experiments using porcine buccal mucosa. The latter, in fact, is considered the most representative of human tissue from the point of view of lipid content, composition and morphology, as the pig's epithelium is not keratinized. Furthermore, the aforementioned mucosa is extremely comparable to the human one also in terms of thickness.

The system chosen for the experimentation is represented by a Franz type diffusion cell which represents a model with two open compartments. In consideration of the poor solubility of the active ingredient in aqueous solution at pH 7.4, which leads to the cancellation of the concentration gradient, the actual driving force of the diffusion process, a solution other than artificial plasma was used as the accepting phase; in particular, an aqueous solution of citric acid 5 mg / ml, pH 3, was used, in which the MCZ, being a molecule with pKa� 6.5, is partially ionized due to the protonation of the imidazole ring, and therefore soluble.

A first series of comparative experiments was performed by administering Daktarin <®> (80 mg, containing 1.6 mg of active principle) and lipospheres (15 mg; containing an average quantity of active principle equal to 1.2 mg of MCZ). The results obtained (Fig. 8 and 9) showed that the overall quantity of active principle (sum of the drug penetrated into the mucosa and of the drug permeated in the acceptor compartment) was equal to 0.30 mg, corresponding to 25% of the administered dose; on the other hand, the administration of Daktarin <®> at the usual dose results in an overall quantity of MCZ equal to 0.06 mg, corresponding to just under 4% of the administered dose.

On the basis of the results presented, it is possible to state that the MCZ released by the lipospheres is significantly greater than that released by Daktarin <®>, both in terms of overall quantity and in terms of the percentage of drug with respect to the administered dose.

In order to establish the time necessary for the MCZ released by the lipospheres to be able to saturate the mucosa, a battery of experiments was conducted, in which the amount of MCZ penetrated into the mucosa at different "end time points" was evaluated, including 1 and 6 hours, after administration of 15 mg lipospheres (1.2 mg MCZ). At the same time, the cumulative quantity of drug present in the acceptor compartment was also monitored. As can be seen from the graph (Fig. 10), after 2 h from the administration of the lipospheres, the membrane saturates, while the quantity that permeates in the acceptor compartment continues to progressively increase. This indicates that the microspheres promote the penetration of the MCZ into the mucosal tissue by also promoting the breakdown of the MCZ also in the underlying hydrophilic tissues such as the blood (possible systemic effect).

The effects of a drastic reduction in the dose of liposphere administered on the penetration of MCZ into the porcine mucosal membrane were then evaluated. A third series of experiments was therefore undertaken, reducing the quantity of lipospheres administered by 90% (equal to 1/8 of the dose of MCZ in Daktarin <®>); in particular, a quantity of lipospheres equal to 2.5 mg, corresponding to 0.2 mg of MCZ, was placed in the donor compartment.

The results obtained (Fig. 11 and 12) showed that the quantity of MCZ penetrated into the mucosa and the total amount (sum of the drug penetrated into the mucosa and of the drug permeated to the acceptor compartment) was equal to 0.096 and 0.124 mg respectively (corresponding to the 48 and 62% of the administered dose). It can therefore be stated that, by reducing the dose of drug administered by 90%, the quantity of MCZ released by the lipospheres to the mucosa is double compared to that released by a dose of 80 mg of Daktarin <®> gel.

Since the direct application of such a small amount of lipospheres (2.5 mg) on the buccal mucosa could be difficult and imprecise, a semi-solid dosage form containing MCZ lipospheres was formulated.

For this purpose, a hydrophilic gel was prepared, in which the lipospheres were incorporated.

A pharmaceutical form easily applicable on the buccal mucosa and suitably dosable was made using a 1: 8 liposphere-gel weight ratio.

To evaluate the ability of the liposphere gel to release MCZ, ensuring adequate penetration of the same at the level of the buccal mucosa, another series of ex vivo experiments was undertaken, placing a quantity of liposphere gel equal to 22.5 in the donor compartment. mg (corresponding to 0.2 mg of MCZ).

The results obtained by ex vivo experiments (Fig. 13 and 14), 5 hours after administration, show that the quantity of MCZ penetrated and the total amount (sum of the drug penetrated into the mucosa and of the drug permeated in the acceptor compartment) was equal to 0.063 and 0.086 mg respectively (corresponding to 31.6 and 43.0% of the administered dose).

It is therefore possible to observe that by administering MCZ in liposphere gel at a dose that is 1/8 compared to that conventionally used for the commercial product, the quantity of drug penetrated into the buccal mucosa is always greater than that released by the Daktarin <®> gel ( Fig. 13).

By observing the same data as a function of the dose fraction administered (expressed as a percentage), it can be deduced that the percentage of bioavailable dose from the liposphere gel is approximately 43% of the administered dose (Fig. 14), against the 4% of the Daktarin <®> gel.

Therefore, the application of this new dosage form allows to exploit the administered dose to a greater extent. This evidently results in an increase in the therapeutic potential of the same dosage form.

Conclusions

The study undertaken in order to create a topical formulation of MCZ capable of improving its bioavailability at the site of action and consequently optimizing the topical therapy of oral candidiasis, showed that the liposphere gel is suitable for achieving this goal.

The lipospheres, preferably obtained in the form of free flowing powder, represent a new dosage form, but their further dispersion in a hydrophilic gel allows a more manageable and pleasant form of administration.

The new formulation proved to be easily applicable on the buccal mucosa and suitably dosable. Furthermore, the low-melting lipid materials, which make up the lipospheres, allow a rapid release of the drug and a "lipidization" of the membrane, with a consequent increase in the penetration of the drug into it. The MCZ, in fact, acquires a greater affinity towards the mucosa thanks to the penetration in it of the lipidic materials of the matrix, which determine an increase in the lipophilicity of the tissue itself.

The administration of a dose of MCZ in liposphere gel equal to 1/8 of the usual dose of MCZ in the Daktarin <®> gel allows to obtain quantities of MCZ in the membrane useful for a therapeutic response.

In fact, the new dosage form allows, in addition to a reduction in the dose of active ingredient to be administered, also to a reduction in the number of daily applications required.

Furthermore, Limonene, by improving the smell and taste of the dosage form, helps to increase patient compliance.

Example 2: Lipid microspheres containing Econazole nitrate (ECZ), Clotrimazole (CLZ), Bifonazole (BFZ) and Ketoconazole (KTZ)

This example shows that several imidazole anti-infectives, whether they are found in salt or free form, Econazole nitrate (ECZ, mp 162 ° C), Clotrimazole (CLZ, 147 ° C), Bifonazole (BFZ, mp 142 ° C) ), Ketoconazole (KTZ, m.p. 146 ° C), lend themselves favorably to technological manipulation for the preparation of an administration system that can be used for both topical and systemic administration [22]. Also for these drugs the incorporation in microspheres, consisting of suitable mixtures of Cetyl Decanoate and hexadecanol, was the best expedient in order to optimize the absorption of the active ingredient whose chemical-physical characteristics did not allow, with the traditional forms pharmaceuticals, a complete and uniform absorption. Numerous reproducible batches of microspheres containing the following antifungals have been prepared: Econazole Nitrate (ECZ), Clotrimazole (CLZ), Bifonazole (BFZ) and Ketoconazole (KTZ).

Methods

Method of preparation of lipid microspheres containing Econazole nitrate (ECZ), Clotrimazole (CLZ), Bifonazole (BFZ) and Ketoconazole (KTZ)

Each batch of 2 grams of microspheres has the following compositions:

Table 3. Composition of lipid microspheres containing antifungals

Bifonazole 0.2 0.36 1.44 20:80 Ketoconazole 0.2 0.63 1.17 35:65

Each lipid mixture composed of drug, cetyl decanoate, 1-hexadecanol in the quantities indicated in Table 3 was melted on a plate. To the melt obtained, placed under stirring, 100 ml of distilled water were added, previously heated to 90 ° C and added with 0.5 g of NaHCO3. The agitation (800 rpm) resulted in the formation of an emulsion (O / W). The system was kept in these conditions for about 2 minutes, at the end of which the temperature was suddenly lowered, by means of an ice and water bath placed outside the beaker, maintaining constant agitation. When the system reached a temperature of about 10 ° C, the stirring was stopped. The microspheres formed were recovered by flotation and subsequent filtration, and dried at room temperature.

The obtained lipid microspheres were evaluated in terms of morphology (by optical microscopy), content (by UV-Vis spectrophotometry), size distribution by sieving, softening point (by melting point).

Morphological analysis of microspheres under the microscope

It was carried out in analogy to example 1.

Determination of the drug content of the microspheres

The quantitative determination of each drug trapped inside the microspheres was carried out by solubilizing the microspheres in Methanol.

For the microspheres containing ECZ and CLZ, a quantity equal to 5 mg of microspheres was placed in a 5 ml volumetric flask with an aliquot of solvent and placed in an ultrasonic bath until the sample was completely dissolved. with the volume flow with the same solvent. For the microspheres containing BFZ and KTZ a quantity equal to 5 mg of microspheres was placed in a 25 ml volumetric flask and solubilized in the same way as described for the other drugs.

Each solution was analyzed by UV-Vis spectrophotometer. The quantification was performed on the maximum absorption peak of each drug (λmax) using the relative calibration curve and methanol as blank. The calibration curves were carried out using 6 standard solutions for each drug in the concentration range 0.05-0.5 mg / ml. The percentage content of drug and the percentage of encapsulated drug were calculated as described in Example 1.

Measurement of the size distribution of lipid microparticles

The separation of the microspheres into fractions of various sizes was performed using an Endecotts Octagon 200 vibrating screen (Endecotts Ltd., U.K.) and standard mesh sieves (Endecotts). A series of eight standard stainless steel sieves in the range of 75-500µm were arranged in the order of decreasing aperture size. The microspheres (5 g) were placed on the upper sieve of the series. The sieves were mounted on the mechanical stirrer running for an adequate period of time for complete separation (approximately 15 minutes). The average sphere size of each fraction was determined as the arithmetic mean of the size of the sieve opening they were held over and the size of the sieve opening they passed. The weight of the separated material was measured and the size distribution determined (Table 4).

Evaluation of the softening point of the microspheres

It was determined in analogy to Example 1.

Results

The quantity of drug initially used for each batch was 10% by weight of the entire preparation; this percentage, although below the maximum encapsulation capacity of the system, was thus pre-established in order to obtain sufficiently charged lipid microspheres, to be able to be further dispersed in a semisolid, (e.g. gel) and to obtain drug administrations that can be correlated to the doses on average contained in the topical pharmaceutical forms available on the market. The microspheres were prepared using the "melt dispersion" method. The drug and the coating material (i.e. cetyl decanoate and 1-hexadecanol in the present example) - are emulsified in an aqueous external phase, without the use of emulsifying agents. The dispersion is kept for 2-4 minutes at a temperature of 90-95 ° C and mechanically stirred at a speed of 800 rpm.

The main factor affecting the size distribution of the microspheres is the stirring speed used during preparation. At a stirring speed of 800 rpm, small variations in the average diameter of the microspheres were observed and the recovered material was approximately 92-98% of the initial one. By increasing the stirring speed in a range between 900 and 1200 rpm, the average diameter of the particles decreases, but the quantity of material recovered also decreases; this appears to be due to the loss of smaller microspheres during filtration. An agitation speed lower than 800 rpm determines the formation of large particles and a fair amount of molten material that adheres to the walls of the vessel during the cooling process. The emulsified lipid droplets solidify on cooling, with the formation of drug-laden microspheres. Cooling is done through an ice-water bain-marie and is maintained until the system reaches room temperature. The particles obtained are then separated by flotation and filtration and left to dry at room temperature. The overall yield of the process is, for all drugs, greater than 90% compared to the material initially used. In order to obtain microspheres with an appropriate softening point (and therefore melting on the epithelia, 35-38 ° C), mixtures of hexadecanol and cetyl decanoate were used in various ratios, making small changes in the ratio between the constituents due to the evident chemical-physical differences of the drugs used. The microspheres were analyzed under a microscope and their actual sphericity was verified, then they were mechanically sieved to determine their size distribution. The results obtained are shown in Table 4.

Table 4. Size distribution of lipid microspheres of antifungals

For all the prepared microspheres, the particle size obtained is below 500 micrometers, however it can be observed that the lipid microspheres containing Bifonazole and Ketoconazole have a smaller average size since more than 80% of the particles have a diameter less than 300 µm.

The drug content was determined for each batch of microspheres and for each size class. The analysis was performed spectrophotometrically using methanol as blank and the related calibration curve. Table 5 shows the instrumental values used and the most important experimental data for each formulation of lipid microspheres.

Table 5. Parameters for the quantitative analysis of drugs using UV-Vis and results on the efficiency of the microspheres preparation process

� λmax = maximum absorption peak for each drug; E1% = Absorbance of a 0.01 mg / ml solution of drug in Methanol.

Also in terms of the actual amount of drug trapped in the microspheres, the best results were obtained with Bifonazole and Ketoconazole. In particular, for Ketoconazole it is observed that 99% of the drug used in the preparation is trapped inside the microspheres. This means that there is no drug prediction during the preparation process.

The method is simple, fast, low cost, easy to apply for a large number of molecules with an appropriate partition coefficient and does not involve the use of organic solvents which could be toxic. In the case of drugs that are not soluble in water, the quantity that can be encapsulated is close to 100% of the active ingredient used.

The coating supports used in this example have therefore shown broad compatibility with antifungals with an imidazole structure, excellent yields of the finished product, reproducibility of the method, simplicity of analysis and quality control.

Example 3: Lipid microspheres containing Itraconazole (ITZ) at 10% w / w

Itraconazole (ITZ) is a synthetic triazole whose structure is closely related to ketoconazole. ITZ is the drug of choice for patients with indolent, non-fatal infections due to B. dermatitidis, H. capsulatum, P. brasiliensis, and C. immitis. ITZ is often the best choice for the treatment of pseudallescheria, an infection that does not respond to amphotericin B therapy, as well as cutaneous or extracutaneous sporotrichosis, tinea corporis and extensive tinea versicolor. However ITZ is poorly soluble in aqueous medium (less than 1 µg / ml in aqueous solutions at pH 1-12.7) with a partition coefficient> 5 in octanol / water at pH 6. Therefore, despite the high antifungal activity, the crystalline ITZ bioavailability is extremely low [23]. To overcome its poor solubility, hydroxypropyl-β-cyclodextrin (cyclodextrin) is often used [24]. To date ITZ is used only orally and injected, in order to obtain a systemic effect and this administration is not free from consistent side effects [25].

The IST was formulated in 1% cream, for example Roticor cream, http://pharmamart.in/jabs-biotech/dermatology-products-for-franchise-punjab-corium.php and Itrasen cream, https: / /www.trademyntra.com/product/cream/itrasen/6273. However, both within the European Community and in the United States there are no pharmaceutical forms for topical application of the ZZ.

Methods

Method of preparing lipid microspheres containing Itraconazole (ITZ)

The mixture consisting of ITZ (0.2 g), Cetyl decanoate (1.08 g) and 1-hexadecanol (0.72 g) (DeCet / Exa ratio 60:40) was melted on a plate. After the melt was well mixed, 120 ml of a 2: 1 v / v water / glycerol mixture were added, previously heated to 100 ° C and added with 0.05 g of HLB 11 surfactant (TWEEN <®> 85, Merck, cat. P4634). The stirring (900 rpm) was carried out with a whisk with a 4-blade steel rod. The system was kept under these conditions for about 2 minutes, at the end of which the temperature was suddenly lowered by adding about 300 ml of a mixture of ice water and NaCl to the preparation. At the same time, an ice and water bath was placed outside the beaker.

When the system reached a temperature of about 10 ° C, the stirring was stopped. The microspheres formed were recovered by flotation and subsequent filtration, washed with distilled water and dried at room temperature.

The obtained lipospheres were evaluated in terms of morphology (by optical microscopy), content (by UV spectrophotometry), size distribution by sieving, DSC analysis, softening point (by melting point apparatus).

Morphological analysis of the microspheres under an optical microscope

The morphological analysis was performed using a Leica DFC 420C scanning optical microscope, connected to a FireWire 1394a color digital camera. We proceeded with the observation and evaluation of the geometric and morphological characteristics of the surface, photographing some representative samples of microspheres.

Determination of the drug content of the microspheres

A quantity equal to 5 mg of microspheres was placed in a 5 ml volumetric flask with an aliquot of methanol and placed in an ultrasonic bath until the sample was completely dissolved, then the volume flow was carried out with the same solvent . The solution was analyzed by UV-Vis spectrophotometry. The quantification was performed on the maximum absorption peak λmax 261 nm, using the relative calibration curve and methanol as blank. The calibration curve was carried out using 6 standard solutions of ITZ in the concentration range 0.05-0.5 mg / ml. E1% 0.4786. The percentage content of drug and the percentage of encapsulated drug were calculated as described in Example 1.

Measurement of the size distribution of lipid microparticles

It was determined as in Example 2.

Differential Scanning Calorimetric Analysis (DSC) of the microspheres

The DSC analyzes were carried out with a Setaram DSC 131 EVO differential scanning calorimeter, on samples weighing 8 mg placed in aluminum capsules. The determinations were carried out by means of a gradient equal to 10 ° C x min <-1>, in a temperature range between - 10 ° C and 300 ° C. Indium was used as the calibration standard. The difference in heat flow between the test sample and the reference sample is measured when these are subjected to controlled heating or cooling.

Evaluation of the softening point of the microspheres

It was determined as in Example 1.

Results

The present example shows that the behavior of Itraconazole in the microencapsulation process is strictly correlated to that of the other antifungals with an imidazole structure.

In particular, for the preparation of the 10% ITZ lipid microparticles, it was appropriate to modify some parameters, in consideration of the chemical-physical characteristics of the active ingredient, such as the high melting point (mp 166 ° C, from the literature). In particular:

a) The weight ratio between Cetyl Decanoate and Hexadecanol; to obtain lipospheres with a softening point close to that of the body (about 35 ° C), the two excipients were placed in a 60:40 ratio (DecCet: Exa),

b) the addition of a small amount of surfactant,

c) the use, as a dispersing phase, of a water-glycerin mixture, to increase the viscosity of the medium,

d) the stirring speed of 900 rpm,

e) cooling, by pouring a mixture of ice-water-NaCl directly into the preparation in order to make the thermal drop as rapid as possible. The saline solution also facilitates the breaking of the emulsion, resulting in a rapid formation of easily filterable and separable sliding microspheres.

Under the optical microscope (Fig. 15) the particles appear spherical, homogeneous and translucent; furthermore, no presence of irregular or crystalline shaped material is observed between the particles, indicating that no ITZ crystallization phenomena have occurred outside the microparticles during the solidification process of the same. Therefore, there is no drug left outside the solid microparticles.

The yield of the process was 95% with respect to the initial material; the actual ITZ load inside the microspheres is 8.8% w / w, measured by UV-Vis spectrophotometry. This leads to obtaining microspheres with a high content of ITZ which allows, in the formulation phase of a topical product, to be able to insert the particles in a semisolid (eg gel) obtaining an optimal drug concentration to obtain a therapeutic effect.

The dimensional distribution (Table 6), by sieving, of the solid particles obtained indicates that more than 95% by weight of them have an average diameter below 300 µm, optimal dimensions for subsequent manipulations that can foresee their use both as a powder as such that to be dispersed in a gel or film as the final formulation.

Table 6. Size distribution of ITZ lipid microspheres

A DSC analysis of a sample of lipid microspheres obtained was performed (Fig. 17) and was compared with the thermogram of the pure IST (Fig. 16).

As can be seen from Figure 16, the pure ITZ thermogram is characterized by the presence of a main endothermic peak relative to the melting point of the active ingredient at 168 ° C and by two secondary peaks probably related to impurities in the material. As can be seen from Figure 17, the thermogram of the lipid microspheres charged at 9% (w / w) (approximately) is characterized by the presence of two endothermic peaks relative to the melting point of the two excipients (cetyl decanoate and hexadecanol, 31 ° C and 41 ° C, respectively) whose areas correspond to their weight ratio. A third widened peak (150 ° C), of very low intensity, could be attributed to the presence of traces of the active principle in crystalline form.

By comparing the two thermograms normalized to the weight of the samples (Fig. 18), the disappearance of the endothermic peak of the drug is immediately evident, an indication of its amorphization. ITZ, being in the amorphous, and non-crystalline state, increases its solubility in the lipids of the epithelial tissues and can be distributed there in greater quantities. In fact, this results in better ITZ penetration into biological tissue.

Example 4: Clobetasol (CLB) microspheres dispersed in a hydrophilic polymeric matrix and application in the oral cavity of gel vs film formulations

Oral lichen planus (OLP) is a chronic autoimmune inflammatory mucocutaneous disease that can cause bilateral white streaks, papules or plaques with or without erythema and ulcerations involving the buccal mucosa [26]. The main symptom is painful oral lesions that affect the quality of life. The current standard clinical treatment is topical administration of high potency topical corticosteroids, such as clobetasol propionate (CLB, m.p. 196 ° C) to control symptoms [27]. Since the buccal dosage forms of CLB are not commercially available, administration is carried out using semi-solid preparations for skin application mixed with an adhesive paste, namely Orabase ™ [28]. This approach has several drawbacks, including difficulties in applying the drug to various oral sites, altered taste, limited contact times and possible swallowing of a formulation not designed for the buccal route. Therefore, to improve patient compliance and reduce the risk of side effects, the development of a solid mucoadhesive dosage form could be of interest. In a previous study, the efficacy and compliance of a new dosage form consisting of lipid microspheres containing hexadecanol and isopropyl myristate and loaded with CLB at 0.025% w / w was evaluated, compared to a standard formulation of CLB in Orabase (adhesive medium). The results indicate that the administration of the drug delivered in liposphere determines an improvement of the lesion up to the remission of symptoms. Furthermore, it seems that this formulation significantly reduces the painful symptoms associated with the disease [20]. However, their application, in the form of a lipid powder with no mucoadhesive at all, represented the main disadvantage.

Materials and methods

Materials

The cetyl decanoate was synthesized in the laboratory. Clobetasol-17-propionate (CLB) was purchased from SICOR-Società Italiana Corticosteroidi (Milan), 1-hexadecanol, polyvinylpyrrolidone (PVP K90) and limonene from Sigma-Aldrich Chemie (Steinheim, Germany ), trehalose at Hayashibara Shoij (Hayashibara Shoij Inc., Okayama, Japan), hydroxyethylcellulose (Natrosol <®> 250 MR) at Galeno (Florence, Italy). The salts and components used for the preparation of the buffer solutions were purchased from VWR International (Leuven, Belgium).

The experiments were conducted using the following solutions:

● Isotonic 0.9% NaCl solution (9.0 g of NaCl in 1 L of distilled water);

● Non-enzymatic artificial plasma pH 7.4 (20.5 g of Na2HPO4�2H20 and 2.8 g of KH2PO4 in 1 L of distilled water);

● Non-enzymatic artificial saliva pH 6.8 (1.5 g of KCl; 1.75 g of NaHCO3; 0.5 g of KSCN; 0.5 g of NaH2PO4�H20 and 1 g of lactic acid in 1 L of distilled water).

The chemicals and solvents were used without undergoing further purification. The porcine buccal mucosa taken from the retromandibular trigone of domestic pigs of 10-12 months of age at the time of slaughter, was kindly provided by the Municipal Slaughterhouse of Villabate, Palermo.

Instruments

A stirring and heating plate (Heidolph MR 3001 K, Germany) equipped with a thermostating probe (Heidolph EKT 3001, Germany) and an ultrasonic sonicator (Branson Model 1200) were used for the preparation of the gels. Thermo-Shaker Grant Instruments Ltd (Shepreth, Cambridgeshire, England) was used for the preparation of the films. Permeation studies were conducted using vertical Franz-type cells with a hole diameter of 9 mm and an acceptor compartment volume of 15 mL (SES GmbH-Analysesysteme, Bechenheim, Germany).

The determination of the film thickness was carried out with an external analog micrometer (Mitutoyo Italiana SRL, Milan, Italy) which allows a measurement range of 0-25 mm (sensitivity: 0.01 mm).

Quantitative determination of CLB by HPLC

HPLC analyzes were conducted with an Agilent instrument equipped with an Agilent 1260 Infinity quaternary pump (G1311B), a 1260 VL Infinity UV detector and a 20 µL injector, properly connected to the ChemStation Workstation (equipped with OpenLAB CDS processing software). The chromatographic separation was carried out using a Merck Chromolith <®> Performance reverse phase column (RP-18, 100-4.6 mm) and a mobile phase composed of the mixture water: methanol = 35:65 (v / v) under isocratic conditions . The flow was set at 1 mL / min and the wavelength of the UV lamp at 239 nm. Under these conditions, CLB is visible in a single peak having a retention time of 4.7 minutes.

For the quantitative determination of the drug, the areas underlying the chromatographic peaks were integrated and compared with those obtained by injecting standard solutions of known concentration.

The calibration curve used for the determination of CLB in microspheres, as well as in porcine mucosal membrane, was made with standard solutions of CLB in methanol. For the determination of CLB in films, a calibration curve made in aqueous methanol was used (ratio MeOH: H2O = 4: 1 v / v) in order to guarantee the complete dissolution of the formulation, given the poor solubility of the constituents of the polymer matrix in methanol only.

The calibration curves were performed in a concentration range between 0.0008 mg / mL and 0.05 mg / mL and were respectively:

y = -37.912 36634x (R = 0.99834) for CLB standards in methanol and

y = 0.95794 33470x (R = 0.99981) in aqueous methanol.

For the analysis of the data relating to the integrated areas of the peaks, the aid of the Curve Expert 1.4 and Kaleidagraph 3.5 softwares was used.

Preparation and characterization of lipospheres

For the preparation of the microspheres containing 1% (w / w) of CLB, the melt dispersion method was used, using a Polimix mechanical stirrer, mod. RW 20, equipped with a digital speed indicator KCH-TRON (Kinematica, Switzerland), supported by a stainless steel propeller shaft with four blades, with a diameter of 4.5 cm. The mixture consisting of CLB (0.020g), Cetyl decanoate (0.400g) and 1-hexadecanol (1.580g), (DeCet / Exa ratio 20:80) was melted on a plate and immediately after adding the appropriate quantity of Limonene (10, 15 or 20�L), (Table 7).

Table 7. Composition of lipospheres

The melted mass was stirred (800 rpm) by adding 100 mL of distilled water, previously brought to boiling.

The system was maintained in this condition for 1 minute. Subsequently, the temperature was abruptly reduced with a water / ice bath placed outside, maintaining the agitation. The microspheres thus obtained were separated by flotation, collected by filtration, and dried at room temperature. The yield of the process was then calculated.

Determination of the drug content of the microspheres

The quantitative determination of the CLB trapped inside the microspheres was carried out on all batches, by solubilizing 10 mg of microspheres in 10 mL methanol. An aliquot of this solution was centrifuged for 10 minutes at 14000 rpm (Centrifuge 5415 C Eppendorf, Germany), and the supernatant was analyzed by HPLC using the relative calibration curve. The result is expressed as the average of the tests relating to 5 lots for each type of formulation prepared. The percentage content of drug and the percentage of encapsulated drug were calculated as described in Example 1.

Morphological analysis under the microscope

The morphological analysis was conducted using a Leitz DMRB optical microscope (Leica Microsystems, Germany), on all batches of microspheres produced by observation under an optical microscope in order to evaluate the geometric and morphological characteristics of the surface.

Analysis of the size distribution of the microspheres

Each batch of microspheres was placed on the first of a series of standard sieves (Endecotts Ltd, England), with mesh openings in the range between 106 and 250 µm, mounted on a mechanical sieve stirrer (Endecotts, Octagon 200) and stirred to 15 min. The material on each sieve was carefully collected and weighed to determine the percentage size distribution of the microspheres. The result is expressed as an average of three lots, for each type of formulation prepared.

Evaluation of the softening point of the lipospheres

It was determined in analogy to Example 1.

Preparation of gels containing lipospheres (liposfer-gel)

Three types of formulations were made, corresponding to the three different liposphere compositions.

The semi-solid formulations, hereinafter called Liposfer-gel, were made by incorporating a known quantity of lipospheres into the gel described below.

For the preparation of the base gel, 15 mL of distilled water, previously heated (60 ° C), was added in the following order:

● Trehalose 0.15 g,

● PVP K900.05 g,

● Natrosol <®> 0.80 g,

Once the preparation was completed, the gel was placed in an ultrasonic bath for about 2 hours at 30 ° C. Following sonication, the gel appears compact, homogeneous and clear. The system thus obtained was left to stand at 4 ° C for one night.

To make 5.00g of lipospheric gel, 0.42g of lipospheres were suspended in 4.58g of base gel. The mixture was subjected to slow stirring in order to homogeneously disperse the lipospheres, minimizing the incorporation of air into the final preparation.

Table 8 shows the compositions of the respective semisolid preparations, corresponding to the lots of lipospheres used.

Table 8. Composition of lipospheric gels

The homogeneous preparation was left to rest again at 4 ° C until it was used for ex vivo permeation tests through porcine buccal mucosa.

In addition, liposfer-gels with a similar composition but with the addition of 0.1% (w / w) methyl p-oxybenzoate (Farmalabor, cat. 001594) as antimicrobial were prepared, in order to conduct physical stability tests in the time without microbial alteration phenomena intervening.

Evaluation of the stability of lipospheric gels over time

The lipospheric gels were stored refrigerated at 4 ° C for 16 weeks. Each week the gels were taken and visually observed to assess their homogeneity and the possible appearance of molds.

Preparation and characterization of Liposfer-films

For the preparation of the buccal films containing the lipospheres (hereinafter called "liposferfilm") only type B lipospheres were used. 2 types of liposferfilms were made, having a different quantity of lipospheres, hereinafter referred to as � α and β.

For the preparation of the lipospheric films, a gel was initially made having the same composition as the base gel previously described in the preparation of the lipospheric gels, but prepared by solubilizing the various components in 30 mL of hot distilled water at 60 ° C. To make the films loaded with microspheres, a portion of lipospheres was added to the gel created. The relationship between the amount of lipospheres added and the amount of gel is summarized in Table 9.

Table 9. Composition of Lipospheric films

The components, suitably weighed in a beaker, were slowly stirred manually in order to ensure complete mixing, minimizing the incorporation of air into the final preparation. Subsequently, the suspension was poured into a silicone mold with a surface of 20.25 cm <2>.

The mold was weighed before and after filling, in order to monitor the loss of water on drying.

The molds were placed inside the Thermo-Shaker at 30 ° C for 60 hours, at the end of which they were removed and left to rest for 2 hours at ambient temperature and humidity, thus obtaining the lipospheric films.

With the aid of a cutter, disks with a diameter of 0.6 cm and a surface of 0.28 cm <2> were obtained, used for subsequent analyzes.

All discs made from the same film, considered as belonging to the same batch, were placed in sealed polyethylene bags for storage and divided into batches.

Determination of the drug content in Liposfer-films

A disc (0.28 cm <2>), from each batch of preparation, was carefully weighed, placed in a 5 mL flask and solubilized in aqueous methanol (MeOH: H2O ratio = 4: 1 v / v). The solution obtained was placed in an ultrasonic bath for 20 min and, at the end, brought to volume with the same solvent. After complete solubilization of the disc, the solution was centrifuged for 10 minutes at 14000 rpm and the supernatant obtained was analyzed in HPLC, using the relative calibration curve.

Determination of the thickness of the Liposfer-films

The analysis of the thickness of the Liposfer-films was carried out with the aid of a micrometer. The analysis was carried out on 6 discs (0.28 cm <2>) taken from three different batches of preparation, for a total of 18 samples. The results were expressed as the average of all measurements made.

In vitro mucoadhesion test

The evaluation of the mucoadhesive strength, for each Liposfer-film formulation, was performed on 4 discs coming from different batches of preparation. The tests were performed using a modified analytical balance [29].

A portion of porcine buccal mucosa, previously conditioned for 30 minutes at a temperature of 37.0 ± 0.5 ° C in artificial saliva, was placed in a Petri dish containing artificial saliva, pre-heated to a temperature of 37.0 ± 0.5 ° C, sufficient to keep the surface of the mucosal tissue constantly wet for the duration of the experiment. The Liposfer-film disk was fixed to one arm of the scale, by means of a support, in order to ensure direct contact of the other surface with the mucosa. The disc was left to adhere to the mucosa for 3 minutes, and then weights were added on the other plate of the scale until the film was detached from the mucosa. The mucoadhesive force was expressed as the mass (g) necessary to obtain the detachment of the film from the surface of the buccal mucosa, and was calculated according to the equation [30]:

where 9.81 is the value of the acceleration of gravity.

By normalizing the adhesive force obtained (N) to the surface of the film (expressed in m <2>), the detachment force is obtained, calculated according to the equation:

Ex vivo permeation test through porcine buccal mucosa

Ex vivo permeation tests were conducted using Franz type vertical diffusion cells as a model with two open compartments [29], in analogy to what is described above in Example 1.

The acceptor compartment was filled with a phosphate buffer solution pH 7.4 (F.U. XII ed.). The donor compartment was loaded with the appropriate amount of lipospheric gel or lipospheric film. The porcine buccal mucosa is interposed between the two compartments. In particular, different experiments were carried out by loading in the donor compartment:

• 80 mg of Liposfer-gel A or Liposfer-gel B or Liposfer-gel C;

• A disc of Liposfer-film α or Liposfer-film β with an area of 0.28 cm <2>, added with 0.2 mL of artificial saliva at pH 6.8.

The experiments were carried out by keeping Franz's cells at a temperature of 40 ± 0.5 ° C, in order to guarantee a temperature in the donor compartment that mimics the in vivo conditions of the oral cavity (37 ° C). The experiments lasted a total of 2, 4 or 6 hours.

At regular intervals (60 minutes) 0.5 mL fluid samples were withdrawn from the acceptor compartment. The volumes withdrawn were immediately replaced with an identical volume of fresh acceptor fluid to maintain the "sink" conditions of the system. The collected samples were immediately analyzed by HPLC, using the relative calibration curve. The results obtained are expressed as the average of 6 experiments.

Extraction of CLB from the porcine buccal mucosa

At the end of each experiment, the extraction of the CLB trapped in the mucosal tissue was carried out. The mucosa, coming from the permeation test, was washed with fresh isotonic solution in order to eliminate any formulation residues present on the mucosal surface. Subsequently the tissue was placed in 2 mL of methanol at a temperature of 60 ° C for 5 minutes.

The extraction procedure was repeated twice. The collected fractions were pooled, transferred to a 5 mL flask and made up to volume with methanol. The quantity of drug extracted was determined by HPLC analysis, using the relative calibration curve.

Results

Using the microencapsulation technique, lipid microspheres containing CLB at 1% (w / w) homogeneously dispersed in a lipid matrix, the components of which are biocompatible with natural epithelial tissue, were created and characterized. The technique used for making the microspheres is the Hot Melt dispersion method [20,21]. The optimal ratio between Cetyl Decanoate and Hexadecanol was 20:80 in order to obtain smoothness of the particles obtained and softening capacity at suitable oral cavity temperatures.

For the realization of the lipospheres, limonene was also added. The latter was added in different quantities in order to evaluate its influence on the softening temperature of the microspheres, on the loading efficiency of the CLB in the same and on its ability to act as a promoter of absorption. Considering that hexadecanol has surfactant characteristics, it was not necessary to add emulsifying agents. The agitation speed of 800 rpm and the cooling speed of the system resulted in particles of suitable size for buccal administration. The rapid solidification of the lipid support was, in fact, promoted by a rapid lowering of the temperature, obtained through the use of a water and ice bath placed outside the preparation beaker. The microspheres produced were separated by flotation and subsequent filtration. After 24 hours at room temperature on a filter, the dry microspheres can be collected and analyzed.

Three formulations of 1% (w / w) CLB lipospheres were made, different in Limonene content (10, 15 and 20 µl).

The microspheres, observed under an optical microscope, appear translucent and spherical and regular in shape.

The process of preparation of the lipid microspheres allowed to obtain a yield of 93.5 � 0.3%, determined as the ratio between the weight of the collected microspheres and the weight of the lipid and drug components, used for their preparation. The CLB content in the lipospheres, determined by HPLC analysis, is reported in Table 10 as an average of 5 batches per formulation:

Table 10. CLB contained in the lipospheres.

The low water solubility of CLB and the lipophilicity of the matrix favored a good encapsulation efficiency, guaranteeing an average content of active principle in the lipospheres equal to 0.94% w / w. The different amount of limonene added to the formulations does not affect the encapsulation efficiency of the drug in the lipospheres. The optical microscope analysis of all batches of liposphere formulations made it possible to evaluate the morphological characteristics and evaluate their quality, size and homogeneity, confirming that the method used leads to the formation of spherical particles with a smooth surface.

Since the microspheres should release the drug by "fusion" of the same and partition of the active principle between the liquid matrix and the buccal epithelium, it is appropriate to determine the softening point. The latter was between 36 and 37 ° C for all formulations, confirming that the components of the matrix are in the right ratios so that the lipospheres melt at the temperature present in the oral cavity.

The size of the particles and their dimensional distribution can affect the smoothness of the product, its handling and its appearance.

The particle size analysis showed that 62% of the particles have an average diameter between 250 and 106 μm, as shown by the dimensional distribution trend shown in Fig. 19 and that all particles have a diameter of less than 300 µm; the data are reproducible for each batch prepared. The dimensions are therefore optimal for subsequent manipulations which may involve their use both as a powder as it is and to be dispersed in a gel or film as the final formulation.

Since the direct application of the lipospheres on the buccal mucosa could be difficult and imprecise, we proceeded with the creation of a semi-solid dosage form containing CLB lipospheres in the form of a gel.

Which components of the base gel were used:

● Natrosol <®>

● Trehalose

● Polyvinylpyrrolidone (PVP).

Three semi-solid formulations were produced, hereinafter referred to as Liposfer-gel with lipospheres A, B and C respectively (Fig. 20 and Table 11). The formulations were easy to produce, inexpensive, easy to handle and above all simple to administer.

Table 11. Amount of CLB in gel formulations.

To study the physical stability of the Liposfer-gels over time, they were prepared and stored in a refrigerated environment at 4 ° C. It was observed that after 5 weeks the suspensions appeared compact, homogeneous, without any flotation or caking of the lipospheres in the gel. However, after 7 weeks there was a clear separation of phases and the formation of hyphae and molds. For this reason, a 0.1% (w / w) mixture of methyl p-oxybenzoate was added to the gel formulation. The new formulations were then stored in a refrigerated environment at 4 ° C and observed for 16 weeks. By the end of this period, the Liposfer-gels had retained their initial homogeneous appearance.

To evaluate the ability of Liposfer-gels to release CLB and to allow adequate penetration into the buccal mucosa, a series of ex vivo permeation experiments were undertaken, using the porcine buccal mucosa.

For the experimentation, the use of vertical Franz-type diffusion cells was used, as a model with two open compartments.

The donor compartment of the Franz cell was loaded with 80 mg of Liposfer-gel A, B or C, while the acceptor compartment was filled with a plasma-simulating pH 7.4 phosphate buffer solution. At regular intervals of time, samples of acceptor fluid were taken and immediately replaced with fresh acceptor fluid to maintain the system's sink conditions. The thermostating jacket was kept at a temperature of 40.0 ± 0.5 ° C in order to guarantee a temperature in the donor compartment that mimics that of the oral cavity (37 ° C) and to allow the fusion of the lipospheres on the mucosa. The experiments were conducted for 2, 4, 6 hours.

In order to establish the amount of CLB that remains trapped in the porcine mucosal membrane, at the end of all the experiments the membrane was removed and extracted with methanol to recover the trapped drug.

The data obtained provided a measure of the ability of the lipospheres to act as a "promoter" of the penetration of the drug into the mucosa.

At the same time, the quantity of CLB present in the accepting compartment was assessed and monitored, however, it was found to be small and close to the minimum quantifiable quantity (LOQ). This result was attributed to the chemical-physical characteristics of the CLB, which made the permeation of the CLB in the acceptor compartment null, ensuring a locoregional action of the drug. The purpose of the study was in fact to clarify whether the lipid microparticles are able to increase the entrapment of the drug into the buccal mucosa, favoring its penetration rather than permeation, thus carrying out only the topical-zonal action.

The CLB extraction data from the membranes, relative to the experiments conducted at 2, 4 and 6 hours are reported in Table 12 and Fig. 21.

Table 12. Results of CLB extraction from membranes

From the graphs (Fig. 21 and 22) it is possible to deduce that already at 2 hours, quantities of CLB capable of saturating the membrane are reached. The slight reduction in the percentage of CLB found between 4 and 6 hours of experiment, could be attributed to the possible formation of metabolites of the drug.

In particular, it was observed that the application of Liposfer-gel B allows to obtain a greater quantity of CLB trapped in the membrane.

On the basis of the findings, a mucoadhesive film was made, containing the lipospheres. In fact, since even semisolid preparations can present drawbacks in terms of patient compliance, as well as loss of active ingredient due to leaching or swallowing, a further step of the research involved the formulation of solid and flexible films containing CLB lipospheres. Films, compared to semisolids, could have the additional advantage of behaving like "wound dressing" by covering the lesion and protecting it from external insults.

In particular, the preparation of mucoadhesive films containing the B lipospheres was perfected which gave the best results in the gel formulation. Due to their function of covering and protecting the application area, the films are able to adhere to the mucous membranes, to swell, to allow the microspheres to fuse and to release the active ingredient on the lesion.

The use of a mucoadhesive film containing lipospheres allows you to take advantage of the advantages offered by both technologies, namely the precise localization of the application / absorption site, greater adhesion and the prolongation of the drug's residence time on the target site. In addition, the entrapment within the lipid microspheres allows the delivery and easier absorption of lipophilic drugs.

The films are flexible and elastic but, at the same time, resistant and stable, with suitable mucus adhesive properties and swelling capacity so as to be able to properly release the drug. The method used to make the films was that of "solvent casting".

2 different formulations were made, hereinafter called Liposfer-film α and β, different in the amount of microspheres present per cm <2>. For the preparation of the Liposfer-films α and β we started from a gel composed as follows:

Trehalose 0.15g

PVP K90 0.05g

Natrosol <®> 0.80g

Water 30 ml.

The appropriate amount of microspheres was added to the base gel, as indicated in Table 13.

Table 13. Composition of the films before drying and final weight of the dried films as an average of 5 lots.

The system thus created was poured into silicone trays and dried for 60 hours (Fig. 23).

The Liposfer-films obtained (Fig. 24) showed good characteristics of homogeneity in the dispersion of the lipospheres.

The method used for the realization of the Liposfer-films was therefore found to be simple, cheap, reproducible and also did not involve the use of organic solvents. With the help of a cutter, film discs were obtained, hereinafter respectively called patch α (obtained from the liposfer-film α) and β (obtained from the liposfer-film β), with a diameter of 0.6 cm (Fig. 25) or of 0.28 cm <2> used for the subsequent analyzes.

The first tests carried out were aimed at evaluating the uniformity of the patches both in terms of drug content and overall weight and thickness.

To verify the uniformity of the content of the Liposfer-Films obtained and demonstrate the reproducibility of the preparation process, we proceeded with the quantitative determination of the CLB contained in each batch produced: 3 discs were taken from each film (2 along the edges and 1 central to the mold), all having an area 0.28 cm <2>, each placed in a 5 ml flask, solubilized in a mixture of MeOH: H2O = 4: 1 v / v, centrifuged and analyzed by HPLC. The results, reported in Table 14, are expressed as the average of three batches from which 3 film discs (n = 9) were taken for each. The analysis of the thickness of the patches was carried out on 6 discs with an area of 0.28 cm <2>, previously weighed and coming from each batch produced. The results (Table 14) are expressed as an overall average for both weight and thickness evaluation.

Table 14. Weight (mg), thickness (mm) and CLB content of the α and β patches cut from the Liposfer-Films α and β respectively.

The results obtained show that the average drug content in each patch has a maximum oscillation of ± 7.7%, a value within the limits dictated by the pharmacopoeia. of drug therefore it is homogeneously distributed throughout the Liposfer-Film both α and β, indicating that the preparation process is reproducible and that during the drying process of the gel with dispersed the microspheres no aggregation phenomena of the particles occurred. The thickness and weight of the patches cut from each Liposfer-Film are also homogeneous, highlighting the reproducibility of the method of preparation of the Liposfer-Film. The patches were subsequently subjected to a series of tests aimed at evaluating their swelling capacity, the strength of mucoadhesion, to release the drug on the mucosa and to promote its penetration.

In the realization of Adhesive Drug Delivery Systems an important parameter is the adhesion force that is established between the formulation and the mucosal surface. This parameter is important in evaluating the residence time of the formulation on the application site and therefore in the release of the drug in situ. The mucoadhesion force and the detachment force, expressed as an average of four tests, are reported in Table 15.

Table 15. Adhesive strength and Detachment force of patches.

It can be observed that the adhesive strength is significantly lower for the β formulation; it follows that as the quantity of lipospheres in the patch increases, adhesiveness increases.

The different detachment force value of the two patches could be explained as a function of the different thickness of the two patches: it is possible that the greater thickness of the patch α determines a greater ability to "dissipate" the force applied along its entire thickness. In fact, the α patch, despite having a slightly lower quantity of hydrophilic matrix and an almost double quantity of lipospheres compared to the β patch, shows a higher mucoadhesive value. Depending on the clinical aims of the design, the α patch would guarantee a longer residence time on the target site, allowing the release of therapeutically useful amounts of CLB at the loco-regional level.

In order to verify whether the quantity of CLB released by the patches over time was sufficient to guarantee an adequate concentration of drug at the target site level, ex vivo permeation tests were conducted, similar to what has already been described for liposfer-gels. . All experiments were conducted for 2, 4 and 6 hours and the results are reported as mean of 6 experiments.

Again, the low tendency of CLB to permeate into the acceptor compartment (systemic effect) and its tendency to accumulate in the buccal mucosa was observed. The amount of CLB reaching the acceptor compartment was found to be zero. Therefore, at the end of all the experiments, we proceeded to the extraction of the drug from the membranes (Table 16, Fig. 26 and 27) and to the quantitative determination.

Table 16. Values of CLB trapped in porcine membrane after patch administration.

Fig. 27 shows the data of the extractions carried out on the two patch formulations, expressed as a percentage of the administered dose.

From the data reported, comparing the outcome of the extractions at 2, 4 and 6 hours of the α and β patches, the amount of drug trapped in the membrane increases up to 6 hours, reaching membrane saturation values. It can be deduced that, with different loading of the formulation used, the entrapment in the mucosa is completely coincident. Therefore in terms of the percentage of the administered dose that is bioavailable, the β patch formulation is more advantageous since it contains half the dose of the α patch, but will exhaust its content in a shorter period of time.

In a second analysis, comparing the data of the extractions at 2, 4 and 6 hours of the lipospher-gel B and Patch α, having the same quantity of lipospheres, it is clear that the quantity and the percentage of CLB extracted from the membrane at the end of each experiment , is greater for the liposfergel.

Ultimately all formulations, both in the form of lipospheres in gel and lipospheres in films, have shown a remarkable ability to promote the penetration of CLB into the mucosa and therefore could be effectively used in therapy, leaving the opportunity to choose the type of formulation in based on the extent of the lesion and the patient's compliance.

Conclusions

Two new therapeutic systems were created, in the form of semi-solid and patches, containing lipid microspheres loaded with 1% CLB, in order to optimize the bioavailability of the active ingredient at the site of action.

The lipospheres, in the form of free-flowing powders, could already in themselves represent a new dosage form, but their dispersion in an appropriate hydrophilic vehicle has made it possible to obtain formulations with greater ease of administration and considerable patient compliance with respect to pharmaceutical products. currently employed. The gel containing the Lipospheres, compared to the formulations currently used in therapy, allows easier application of the active ingredient on the mucous membranes of the oral cavity. However, the washout due to salivary secretions, together with the potential swallowing of part of the dosage form following application, could be critical for achieving the effective therapeutic concentration of CLB.

As evidenced by the experimental data of entrapment in the mucosal membrane, the high amount of CLB found compared to the administered dose, and the simultaneous absence of significant drug concentrations in the plasma compartment, underline the effective loco-regional action of the proposed formulations. In fact, the low-melting lipid materials, with which the lipospheres are formulated, allow a rapid release of the drug while the gel dispersion guarantees a close and prolonged contact between the dosage form and the injured mucosal portion.

In this way, it is possible to obtain high concentrations of drug in situ for a prolonged period of time, with a consequent reduction in the frequency of administration. This results in an optimization of the bioavailable dose and in a decrease in any side effects related to the systemic distribution of the drug.

By exploiting the formulation of the gel, patches have also been created, containing the lipospheres, which constitute innovative dosage forms due to their uniformity, flexibility and ease of application.

The two formulated patches made it possible to obtain an effective quantity of drug in the membrane already two hours after application and are able to guarantee intimate contact with the injured mucosal surface for a sufficient period of time, without the disaggregative phenomena and of salivary turnover compromise the release of CLB.

The patches, by virtue of the mucoadhesive properties shown, and the ability to behave as "wound dressing" could constitute the formulations of choice for the treatment of PLO. The Patch formulation would also allow both the decrease in the amount of drug administered and the decrease in the number of pro die applications necessary for the resolution of the typical symptom picture of OLP.

In conclusion, all the formulations have shown a remarkable ability to promote the penetration of CLB into the mucosa and therefore could be effectively used in therapy, leaving the opportunity to choose the type of formulation based on the extent of the lesion, the therapeutic needs and compliance. of the patient.

Example 5: Clinical Trial of the patch of microspheres containing Clobetasol It was approved by the Ethics Committee of the O.U. of Oral Medicine of the A.O.U.P. "P. Giaccone ”of Palermo, a clinical trial protocol entitled“ MODIFIED RELEASE MUCOADHESIVE FORMULATIONS FOR THE TREATMENT OF SYMPTOMATIC ORAL LICHEN PLANUS ”. This is a phase IV prospective case-control longitudinal study.

The primary objective of the study is to propose and evaluate the therapeutic efficiency of the daily application (once / day) of a modified release mucoadhesive patch containing 0.2 mg / cm <2> of clobetasol propionate (Liposfer-film α), compared to standard therapy of about 0.15 g of Clobetasol Propionate cream at 0.05 w / w%, 2-3 times / day in subjects with symptomatic Oral Lichen Planus (OLP).

The secondary objectives were respectively the determination of the degree of compliance and adherence to the formulation.

For this purpose, lots of Liposfer-film α (with a higher content of lipospheres) were prepared and, to facilitate application in the oral cavity, patch rectangles measuring 1x2cm were cut out and packaged in polypropylene bags. for food, taking care to seal dose by dose with a heat sealer (Fig. 28).

For this clinical trial, 10 patients were enrolled at the O.U. of Oral Medicine of the A.O.U.P. "P. Giaccone "of Palermo, which fall within certain inclusion and exclusion criteria (Table 17).

Table 17. Criteria for inclusion and exclusion from the clinical study.

Patients not excluded were treated with modified release mucoadhesive patches containing 0.20 mg \ cm² of clobetasol propionate to be applied to the site of the symptomatic lesion 1 \ day for 15 days (Fig. 29). The patch remains adhered until it comes off on its own a few hours after application.

During the therapy the patients were subjected to rinses twice a day, with alcohol-free mouthwash, based on 0.12 w / v% Chlorhexidine Gluconate; and a selective diet was practiced in order to avoid foods irritating the mucous membranes.

Clinical data, photographic images of the lesion were acquired for each patient with the aid of an L-shaped landmark (Tenore G et al 2016 [32]) and the parameters useful for evaluating the objectives at the time of enrollment (T0), after 30 days from the beginning of therapy (T1) which included 1 application per day for 15 days followed by 15 days of wash-out, and after 2 months (T2).

Table 18 shows the results relating to the primary objective of the study protocol.

Table 18. Preliminary results of the primary objective

In particular, by analyzing the parameters taken into consideration, we can observe that:

Linear Numeric Analogue Scale (NAS), with range 0-10, for pain assessment.

Patients are asked to mark their perception of pain on a horizontal line from 0 to 10 (where 0 = no pain; 10 = strongest pain ever experienced). The average for pain assessment, out of 10 patients, was 5.2 at T0, or at the time of enrollment. At T1, after 30 days from the start of therapy, the average is 2.9, so there is a reduction in painful symptoms.

� Clinical grading: 0 = absence of lesion, 1 = presence of white streaks, 2 = presence of white streaks and erosion ≤ 1 cm <2>, 3 = presence of white streaks and erosion ≥ 1 cm <2>, 4 = presence of white streaks and ulceration ≤ 1 cm <2>, 5 = presence of white streaks and ulceration ≥ 1 cm <2> [31].

The measurement was carried out with the aid of photographic surveys and the L-shaped landmark described by Tenore G et al. 2016 [32].

The average of the clinical grading at T0 is 2.7, this indicates the presence of lesions, at T1 the average is reduced to 1.2, therefore the lesions treated with the mucoadhesive patch are reduced. � Modified clinical resolution index, measured at T2, according to which three nominal values are considered: complete, partial, absent. In this index the "Complete" value corresponds to a total remission of symptoms and lesions, to the "absent" value a persistence, or a worsening of these.

The cohort shows a partial resolution of 9/10 patients, and a complete resolution.

Table 19 shows the results relating to the questionnaire dedicated to the assessment of compliance and adherence to the experimental therapy, administered only to the Test group at the time of the control on the 30th day, after having performed 15 days of therapy (T1), which represents the secondary objective of the study protocol.

Table 19. Parameters of secondary objectives

In particular, by analyzing the parameters taken into consideration, we can observe that:

1. "Did you have difficulty applying the patch we sent you?" Always / Almost always / Occasionally / Almost never / Never

As reported in Table 19, 6/10 have never reported difficulties in using the patch, 2/10 almost never, 1/10 almost always and finally 1/10 always. Overall, out of 10 patients, the majority did not report difficulties in use.

2. "Did you notice any changes in taste?" Yes / No - "If yes, which ones?" Sweet / Bitter / Salty 4 \ 10 patients reported alterations in taste, in particular bitter, the remaining 6 \ 10 did not report any alterations.

3. "Did you experience any oral complaints related to the patch (burning, tingling)?" Yes No.

8 \ 10 patients did not report oral disorders, 2 reported tingling in the application area.

4. Supplementary result to the compliance / adherence questionnaire, for evaluating the patch's adhesiveness: "How long after did you observe the detachment of the patch?".

Patients have reported that the patch comes off in <1 day.

In conclusion, the new formulation has shown a remarkable ability to promote the penetration of CBL into the mucosa and therefore could effectively be used in therapy, instead of conventional galenic preparations.

Example 6: Curcumin microspheres (CUR) at 2.5% w / w

Curcumin (CUR), a molecule obtained from the rhizome of Curcuma Longa Linn, a plant belonging to the Zingiberaceae family, has been the focus of numerous studies in recent years that have recognized its interesting therapeutic properties. Curcumin, or 1,7-bis- (4-hydroxy-3-methoxyphenyl) -epta-1,6-dien-3,5-dione, occurs as a crystalline solid, with a molecular weight of 368.39 g / mol and melting temperature of 170 ° C; being a lipophilic substance it is insoluble in water and in ether, but is soluble in acetone, ethanol and methanol.

Curcumin is a polyphenolic compound whose antibacterial and anti-inflammatory activity has been widely demonstrated [33].

It is one of the most powerful inhibitors of NF-kB, a crucial molecule in all pathological conditions that are recognized in chronic inflammation. In fact, curcumin exerts anti-inflammatory effects by interrupting the NF-kB signaling pathway at various levels. Downregulates both the activity of cyclooxygenase-2 (COX-2) and the transcription for the iNOS (inducible nitric oxide synthase) gene in activated macrophages, demonstrating a powerful scavenger activity for free radicals [34]. It inhibits cell proliferation and the production of pro-inflammatory cytokines, such as interleukin 1β (IL-1β), interleukin 6 (IL-6), necrosis factor α (TNF-α) and cyclin E [35]

Although possessing various potentially beneficial activities, Curcumin is a photosensitive and unstable substance, it has poor absorption and rapid metabolization and elimination which results in reduced bioavailability, accentuated by poor solubility in water [36,37]

Given the countless beneficial properties of Curcumin, this example aims to develop lipid microspheres in which Curcumin is homogeneously distributed in molecular form.

Methods

Method of preparation of lipid microspheres containing curcumin (CUR)

The mixture consisting of CUR (0.05 g) Cetyl decanoate (1.755 g) and 1-hexadecanol (0.195 g) (DeCet / Exa ratio 90:10) was melted on a plate. After the melt was well mixed, 100 ml of water were added, previously heated to 100 ° C and added with 0.05 g of surfactant HLB 11 (TWEEN <®> 85, Merck, cat. P4634). The stirring (900 rpm) was carried out with a whisk with a 4-blade steel rod. The system was maintained in these conditions for about 2 minutes, at the end of which the temperature was suddenly lowered by adding about 200 ml of a mixture of ice water and NaCl to the preparation. At the same time, an ice and water bath was placed outside the beaker.

When the system reached a temperature of about 10 ° C, the stirring was stopped. The microspheres formed were recovered by flotation and subsequent filtration, washed with water and dried at room temperature. The obtained lipospheres were evaluated in terms of morphology (by optical microscopy), content (by UV spectrophotometry), size distribution (by sieving), softening point (by melting point apparatus).

Morphological analysis of the microspheres under an optical microscope

It was performed as in Example 3.

Determination of the drug content of the microspheres

A quantity equal to 5 mg of microspheres was placed in a 5 ml volumetric flask with an aliquot of methanol and placed in an ultrasonic bath until the sample was completely dissolved, then the volume flow was carried out with the same solvent . The solution was analyzed by UV-Vis spectrophotometry. The quantification was carried out on the maximum absorption peak λmax 420 nm, using the relative calibration curve and methanol as blank. The calibration straight line was carried out using 6 standard CUR solutions in the concentration range 0.0005-0.0075mg / ml. Equation of the line: Abs = 0.034 129.2 conc. (mg / ml), with correlation coefficient 0.999. The percentage content of drug and the percentage of encapsulated drug were calculated as described in Example 1.

Measurement of the size distribution of lipid microparticles

It was determined as in Example 2.

Evaluation of the softening point of the microspheres

It was determined as in Example 1.

Experimental technique of tape stripping

For the tape stripping technique, skin samples taken from the ear of domestic pigs with an average age of 12 months were used. Samples of porcine auricular epithelium, taken immediately after the sacrifice of domestic pigs of an average age of 12 months, were obtained from the tissue of the external auricle. Subsequently, they were refrigerated and transferred within 1 h of the animal's sacrifice to the laboratories of the Department (STEBICEF), where they were appropriately stored at a temperature of about -20 ° C. Before use, the epithelium was subjected to conditioning in isotonic solution, at room temperature, for 24 h and was then cut, with the aid of a scalpel, into rectangular sections of about 4 cm in height and 3 cm wide. The epithelium, in order to condition it, was placed for a few minutes on a system thermostated at 35 ° C containing a PBS solution and then removed and dried. In order to standardize a surface of 2 cm², a rectangular stencil with corresponding area was applied to the epithelium from time to time, through which 5 mg of lipid microspheres were applied to the porcine tissue.

After applying the sample and massaging the epithelium until the microspheres have completely disappeared (fusion) (about 1 minute), we proceeded with the separation of the stratum corneum by applying adhesive tapes (Scotch 810 Magic Tape 3M). It is important that the pressure exerted on the tape before tearing it off remains the same throughout the process, therefore it was preferred to use a roller to make the adhesive tape adhere to the epithelium. The adhesive was then removed and the latter was deposited in a vial. This process is repeated 10 times with 10 different adhesive tapes. The different adhesives are deposited in vials according to the following sequence: S1, S2, S3, S4, S5; S6; S7-S8; S9-S10. This process was replicated 6 times.

The adhesive tape samples obtained with the tape stripping method were subjected to extraction in methanol (2x2.5mL) at 60 ° C. The methanolic solutions were then transferred into 10mL flasks, brought to volume with methanol and the quantitative determination of the trapped CUR was performed by UV-Vis spectrophotometry using the relative calibration curve and methanol as blank.

Results

The study carried out showed that the behavior of Curcumin, in the microencapsulation process, is also similar to the drugs studied so far. Furthermore, also in this case, due to the high melting point (mp 170 ° C) of the active ingredient, it was appropriate to change the ratio between the excipients and some parameters of the preparation method. In particular:

a) The weight ratio between Cetyl Decanoate and Hexadecanol; to obtain lipospheres with a softening point close to that of the body (about 35 ° C), the two excipients were placed in a 90:10 ratio (DecCet: Exa),

b) the addition of a small amount of surfactant,

c) the stirring speed of 900 rpm

d) cooling, by pouring a mixture of ice water-NaCl directly into the preparation in order to make the thermal drop as rapid as possible. The saline solution also facilitates the breaking of the emulsion, resulting in a rapid filtration of the particles.

Under the optical microscope, the sphericity of the particles and the absence of CUR crystals between the particles are observed, indicating that there has been no dispersion of the CUR in the dispersing medium and that the CUR is homogeneously dispersed within the particles. The softening point of the microparticles was 35 ± 0.5 ° C, compatible with their application on the epithelia. In fact, once applied to the skin surface, by simple pressure and / or friction of the fingers they can melt and divide in the horny layer. The yield of the process was 95% with respect to the initial material; the percentage of effective CUR in the microspheres was 2.38% w / w, measured by UV-Vis spectrophotometry (� λmax 420 nm, E1% 1.392). This data confirms that in the case of water-insoluble drugs, such as CUR, the quantity that can be encapsulated is close to 100% of the active ingredient used.

The analysis of the size distribution (Table 20) was carried out by sieving and the results indicate that the particles are 99% below 300 µm and this allows both an easy handling of the same for the formulation of semisolid products or film, both their application as it is as a powder.

Table 20. Dimensional distribution of lipid microspheres of CUR

Studies to promote absorption through the skin

The tape-stripping technique is applied in vitro for the analysis of the penetration of the drug into the stratum corneum. The technique involves sequential removal of the cell layers of the stratum corneum by applying pieces of adhesive tape. The residues of the formulation are previously removed from the surface of the skin and the tape is applied by pressure to ensure its adhesion to the skin. When the tape is removed, part of the stratum corneum is also removed [38]. The results expressed as a percentage of the dose applied on 2 cm <2> of skin trapped in each cell layer collected as an average of 6 tests are reported in Fig. 30. It is possible to highlight that a total of 39% of the administered dose of CUR managed to penetrate the epithelium and that this was also divided into deeper layers. Thanks to the vehiculation in lipid microparticles, the CUR has been massively absorbed by the epithelial layers where it can perform antioxidant and anti-inflammatory effects.

Conclusions

Curcumin, a molecule that is extremely poorly soluble in an aqueous environment and, for this reason, poorly absorbable through the epithelia, is also well suited to be conveyed by solid lipid microparticles with high affinity to biological tissues. The microparticles in contact with the tissue surface, at a temperature of 36-37 ° C, and with the pressure exerted with the massage, melt, promoting the penetration of the solubilized CUR in the lipids of the stratum corneum. Furthermore, given the photosensitivity of the CUR, the lipid microspheres, being in solid form, can represent a useful means of keeping the CUR stable within a semi-solid formulation for topical use.

Claims (24)

CLAIMS 1. A particle comprising cetyl decanoate and a C14 to C18 aliphatic chain alcohol, wherein said particle has a diameter of at least 10 micrometers. The particle according to claim 1, wherein the diameter is 20 to 400 micrometers. 3. The particle according to claim 1 or 2 wherein the cetyl decanoate and the aliphatic chain alcohol from C14 to C18 are in a weight ratio from 20: 80 to 90: 10. The particle according to any one of the preceding claims, wherein said C14 to C18 aliphatic chain alcohol is selected from the group consisting of: hexadecanol, myristic alcohol, stearyl alcohol and a mixture thereof. The particle according to any one of the preceding claims further comprising at least one agent selected from the group consisting of: a therapeutic agent, a monoterpene, a cosmetic agent, a surfactant, an insecticide, a repellent, a fertilizer, a fungicide, a pesticide and a herbicide, preferably said monoterpene is limonene, preferably said surfactant is HLB 11. The particle according to claim 5, wherein said therapeutic agent belongs to class II or IV of the Biopharmaceutical Classification System. The particle according to claim 5 or 6, wherein said therapeutic agent is selected from the group consisting of: an antifungal, a corticosteroid, an antibacterial, an anti-inflammatory, an antioxidant, a vitamin, an anti-aging agent, preferably said antifungal is selected from the group consisting of: miconazole, econazole, econazole nitrate, clotrimazole, bifonazole, ketoconazole, itraconazole, Fluconazole, Isavuconazole, Voriconazole, Amphotericin B, preferably called corticosteroid is clobetasol or clobetasolone propionate, Fluasometasol propionate, Prednisolone, Triamcinolone acetonide, Amphotericin B, preferably called antibacterial is curcumin, Metronidazole, Colimycin, tobramycin, gentamicin, Sulfadiazine, preferably called anti-inflammatory is curcumin Diclofenac, Piroxicam. The particle according to any one of the preceding claims, characterized by a softening point from 30 ° C to 40 ° C, preferably from 34.5 ° C to 37.5 ° C. 9. A process for obtaining at least one particle having a diameter of at least 10 micrometers comprising the steps of: - heating cetyl decanoate and a C14 to C18 aliphatic chain alcohol and optionally an agent as defined in claim 4, 5 or 6 until melting to obtain a melted mixture; - adding water heated to a temperature from 80 ° C to 100 ° C to the melted mixture to obtain a dilute mixture, said water being optionally added with at least one of: an acid, a base, a surfactant, glycerol or a mixture thereof; - stir said diluted mixture at a speed from 500 rpm to 1000 rpm, preferably around 800 rpm, for a period of from 30 seconds to 5 minutes to obtain an emulsion; - lower the temperature of said emulsion down to a temperature from 5 ° C to 15 ° C in a period of 60 seconds to 240 seconds while maintaining agitation to obtain the solidification of the emulsion; thus obtaining the at least one particle. 10. A particle obtainable from the process according to claim 9. The particle as defined in any one of claims 1 to 8 obtainable from the process according to claim 9. The particle as defined in any one of claims 1 to 8, 10 or 11 for use as a medicament. 13. The particle according to claim 12 for use in a method of prevention and / or treatment of: an inflammation, a fungal infection, a bacterial infection, an injury, an aging condition, a neurodegenerative disease, a ischemia. The particle according to claim 12 or 13 for use in a method of prevention and / or treatment of: candidiasis, thrush, vulvovaginitis, intertrigo, onychomycosis, B. dermatitis infection, H. capsulatum infection, P. brasiliensis infection , C. immitis infection, cutaneous sporotrichosis, extracutaneous sporotrichosis, tinea corporis, extensive tinea versicolor, lichen planus, oral lichen planus, dermatitis, eczema, atopic dermatitis, seborrheic dermatitis, psoriasis, alopecia, vitiligo, pseudallescheriasis, mycetomycidium. The particle for use according to any one of claims 12 to 14 wherein said particle is administered topically, preferably to the oral mucosa, vaginal mucosa, skin, nasal mucosa, or orally, preferably to the gastrointestinal mucosa . 16. Use of the particle as defined in any one of claims 1 to 8, 10 or 11 in the cosmetic field, preferably against aging, for the preservation of fragrances and / or in the conveyance of substances that are poorly soluble in water and / or unstable. to light and / or oxygen. Use of a particle as defined in any one of claims 1 to 8, 10 or 11 to mask an odor and / or taste or for the preservation of fragrances. 18. Use of a particle as defined in any one of claims 1 to 8, 10 or 11 in the agricultural field, preferably as an insecticide, repellent, fertilizer, fungicide, pesticide and / or herbicide. 19. A powder comprising at least two particles, said particles being as defined in any one of claims 1 to 8, 10 or 11. The powder according to claim 19, wherein at least 50% by weight of said particles has a diameter of from 75 micrometers to 250 micrometers. 21. A pharmaceutical formulation comprising: - the particle as defined in any one of claims 1 to 8, 10 or 11 and / or the powder as defined in claim 19 or 20; And - a pharmaceutically acceptable vehicle. The formulation according to claim 21, characterized in that it is in solid or semi-solid form, preferably in the form of gel, film, cream, gauze, plaster or stick. 23. The gel or film formulation according to claim 21 or 22 wherein the pharmaceutically acceptable carrier is selected from the group consisting of: water, trehalose, polyvinylpyrrolidone, hydroxyethylcellulose and / or a hydrophilic polymer, preferably said hydrophilic polymer is hyaluronic acid or chitosan. 24. A process for the production of cetyl decanoate comprising a reaction between decanoic acid or salt thereof and hexadecanol, said reaction being carried out adopting one or more of the following conditions: - use of methylene chloride as a solvent; - reaction temperature of about 60 ° C; - reaction time of about 24 hours; - absence of a catalyst.