KR20010040726A - Pharmaceutical compositions in form of nanoparticles comprising lipidic substances and amphiphilic substances and related preparation process - Google Patents

Abstract

A pharmaceutical composition in the form of nanoparticles comprising a composite of at least one lipid material and at least one amphipathic material, and a pharmaceutically active ingredient. Due to the surface and central properties of the composite, the composition improves the incorporation efficiency of the active ingredient and improves the biocompatibility of the active ingredient which is hardly absorbed.

Description

Pharmaceutical compositions in form of nanoparticles comprising lipidic substances and amphiphilic substances and related preparation process

Among the polymers commonly used, polyalkylcyanoacrylate, poly-lactic acid (PLA) and poly-lactic glycolic acid (PLA-PLGA) derivatives are exemplified. However, such a system presents some problems.

For example, polyalkylcyanoacrylates are metabolized every 24 hours by the organism, releasing formaldehyde, a potentially toxic derivative; PLA and PLA-PLGA do not provide toxic metabolites, but they can present dangerous accumulations, with long degradation times from weeks to months.

In addition, the method of making these systems requires the use of potentially toxic organic solvents that may remain in trace amounts in the final form.

As a result, foreign bodies having a size of 5 μm or more injected into blood vessels can cause embolism, so most of the systems are difficult to use in the form of intravenous injection.

These problems have led to greater interest in biocompatible and low toxicity dosing systems: of all these, the preferred systems are oil / water emulsions, liposomes, lipid microparticles and nanoparticles. Lipid lipid colloid system.

Oil-in-water emulsions consisting of lipid droplets with a size in the nanometer range and dispersed in an external aqueous phase have been used as carriers for parenteral administration (Japanese Patent No. 55,476, 1979, Okamota, Tsuda and Yokoama).

Oil-in-water emulsions comprising the active ingredient are disclosed in WO 91/02517 (1991, Davis and Washington). Such a system has a high capacity to include the active ingredient in the internal lipid phase, but the active ingredient easily diffuses from this phase to the external phase, causing limitations and stability problems for the selective development of sustained release. .

Liposomes are colloidal structures with an internal water phase surrounded by one or more phospholipid layers. The use of liposomes as carriers for the administration of drugs is disclosed, for example, in US Pat. No. 3,993,754 (Raaman and Cherni, 1976).

Typically, however, such systems present problems with stability in storage, poorly reproducible manufacturing methods, and difficulties with incorporation and maintenance of active ingredients.

Fountain et al. Invented spherical lipid microparticles having a size in the range of 0.5-100 μm as a carrier for the administration of the active ingredient. This invention is disclosed in US Pat. No. 4,610,868 (1986).

Dome et al. (US Pat. No. 435,546) are enclosed in an aqueous environment, in a composition consisting of an internal lipophilic phase that is added to the composition and surrounded by an external phospholipid layer adsorbed on the surface of the particles themselves Invented Liposheres ^TM , an insoluble particle having a size of about 40 μm added and adsorbed onto the surface of the particle itself. These systems have been developed to control the release of active ingredients with pesticidal effects (Dome et al., US Pat. No. 5,227,535) and anesthetics (Dome et al., US Pat. No. 5,227,165). However, the techniques for the preparation of such systems require the help of solvents remaining in trace form in the final form.

It has already been found to be difficult to orally administer to active ingredients that are poorly soluble and not well absorbed in the gastrointestinal tract or sensitive to pH or the activity of proteases (proteins and peptides). By incorporating such materials into lipidic nanoparticles, these difficulties can be overcome because these nanoparticle systems can be absorbed along the gastrointestinal tract. Their reduced size may utilize passive transmucosal absorption mechanisms, pass intercellular binding or ion channels, use endocytosis mechanisms, or allow lymphatic fluxes to enter.

Lipid solid systems consisting of nanopellets have been developed by Spacer et al. (US Pat. No. 4,880,634, 1989) and have been used for oral administration of drugs that are poorly absorbed. After emulsifying the lipid material in an aqueous environment with a high energy mixer to obtain the lipid pellet, the emulsion is cooled at room temperature and the pellet is obtained by sonication.

Gasco (European Patent No. 0526666A1, 05/08/1991) invented a technique for preparing lipid nanoparticles. Lipids dissolved in the presence of a surfactant and a cosurfactant are added to the aqueous phase to obtain a microemulsion, which is then dispersed in an aqueous environment maintained at a temperature of about 10 ° C. Solid nanoparticles are obtained in an aqueous suspension, but immediately afterwards the surfactant remaining in the ultrafilter can be removed and recovered by filtration or lyophilization.

This technique has proven to be advantageous in terms of energy savings compared to homogenizing treatments that require a lot of energy, with an average diameter of 90 to 900 nm, smaller with more uniform size distribution and a low polydispersion index. Nanoparticles can be obtained. However, the preparation of microemulsions requires melting of the lipidic material, and about 70 ° C. for the most used lipidic materials, thus limiting the use of this technique for heat-sensitive materials.

In the field of researching new carriers suitable for administering active principles, great attention is paid to polymer systems having sizes in the micrometer range and polymer systems having sizes in the nanometer range.

The present invention relates to a pharmaceutical composition in the form of nanoparticles having a diameter of 1000 nm or less, preferably 50 to 500 nm, comprising a composite consisting of at least one lipid material and at least one amphiphilic material and a pharmaceutically active ingredient. will be.

Unexpectedly, the inventors have found that the mixtures and related particles according to the invention have characteristics which cannot be achieved by routine mixing of lipid and amphiphilic materials or by adsorbing amphiphilic materials to lipid particles.

Preferably the amphiphilic material may be dispersed on the surface of the nanoparticles, preferably inside the nanoparticles, or uniformly dispersed on and inside the nanoparticles.

The formation of the complex is:

1. having surface properties that aid in oral administration absorption and half-life in the circulatory system;

2. having mass properties that enable incorporation of thermally labile drugs, such as low melting points;

3. Suitable for transporting both water soluble and fat soluble drugs due to the presence of lipophilic and partially hydrophilic regions in the composite;

4. Inherently homogeneous incorporation of hydrophilic agents (eg peptides) within the lipophilic matrix;

Nanoparticles can be provided.

The present invention relates to the preparation of a particle form composition for pharmaceutical use having a size of less than 1 micron (nanometer) comprising a composite consisting of an amphiphilic material, which is a lipidic or polymeric species, and a composite of lipidic material.

In general, nanoparticles according to the present invention are prepared starting from composites obtained by eutectic or covalent dissolution of lipid and amphiphilic materials. The eutectic mixture, in the subsequent cooling step, exhibits new properties that indicate more hydrophilic and more lipophilic regions due to separation of the amphiphilic material or reverse alignment of the constituents towards the nanoparticle surface or into the core for the two starting materials. Composites are created. These features are substantially different from the adsorption of amphiphilic material on the lipophilic surface. This property will be described in more detail in the characteristic experimental examples described below.

Drugs can be dissolved or suspended in the eutectic mixture in the manufacturing process and, due to the new nature of the composite, can be classified according to their properties, preferably within the more hydrophilic or more lipophilic regions. . Moreover, hydrophilic agents (e.g. peptides) filled on the nanoparticles can unexpectedly be uniformly dispersed within the nanoparticles themselves, while the proportion of peptides adsorbed on the surface is very low, and the examples of the prior art (lipid core and adsorption) Lower particles compared to the amphiphilic material).

Nanoparticles obtained from eutectic mixtures retain the same characteristics as the starting composites.

Nanoparticles can be obtained using other manufacturing techniques, such as:

1. Dispersion of oil-in-water microemulsions (comprising lipid and amphiphilic material as an oil phase, left at temperatures above the melting point of the composite and one or more surfactants and cosurfactants) in an aqueous medium using a temperature gradient. Providing technology.

2. A technique provided for high pressure homogenization of a pure emulsion of a composite at a temperature above the melting point of the material forming the composite, or below the melting point of the composite, in the presence of a surfactant.

The process of the present invention according to the microemulsion-dispersion process (Technology 1) is provided for the initial eutectic or covalent dissolution of two or more lipid and amphiphilic components, where the melting point of the component itself, or the latter, is dissolved in the former Raises the melting point of one of the two components; An appropriate amount of an aqueous solution comprising at least one surfactant and cosurfactant, heated at the same temperature as the molten composite, is added to the molten composite as described above with gentle stirring.

It is also possible to simultaneously form the microemulsion by raising the lipid and amphipathic components to the melting point in the presence of water, surfactants and cosurfactants necessary for the formation of the microemulsion itself. The pharmaceutically active ingredient can be dissolved or dispersed in the molten composite that is the starting material or added directly to the microemulsion in the preparation of the microemulsion itself, depending on the nature of the active ingredient itself. Dispersion of the active ingredient occurs in the complex, causing a surprising reduction in the amount of chemicals adsorbed on the surface and subject to degradation of enzymes and the external environment.

The oil-in-water microemulsion thus formed is then dispersed in water or an aqueous medium under controlled amounts and stirring conditions, usually in the temperature range of +1 to + 10 ° C., but using a non-aqueous solvent which can be mixed with water. It is possible in the temperature range of 30 ° C. and thus obtains composite nanoparticles in solid form in an aqueous suspension. The nanoparticles have a diameter of 1000 nm or less. Nanoparticles have been found to be different compared to techniques obtained by adsorption of amphiphiles on the surface of lipidic particles (domes) or systems using amphiphilic particles (CASCO) as surfactants for forming lipidic nanoparticles. It became.

The nanoparticle suspension can then be washed with water or an aqueous solution using an ultrafiltration system (or dialysis) capable of removing surfactants, cosurfactants and excess free agent. This process can thus eliminate unwanted effects due to the surfactants present in pharmaceutical form. In addition to this process it is possible to quantitatively determine the percentage of active ingredients that are not incorporated or adsorbed to the nanoparticles.

The compositions described above can be administered as aqueous suspensions and recovered as a solid by lyophilization, filtration, distillation of an aqueous solvent and spray-drying techniques.

Nanoparticles according to the invention have the following quantitative weight composition.

0.5-99.5%, preferably 10-90% lipid material;

0.5-99.5%, preferably 10-90% amphiphilic material;

Pharmacologically active ingredient of 0.001 to 99%, preferably 0.01 to 50%, compared to the sum of lipid and amphipathic substances.

In the method for producing nanoparticles according to technique (1), the component materials are used in the following weight ratios:

In microemulsion

0.1-50% by weight, preferably 10-25% by weight of lipidic material;

0.1-50% by weight, preferably 0.5-25% by weight of amphipathic material;

5-30% by weight of surfactant, preferably 10-20% by weight;

40 to 75% by weight, preferably 50 to 70% by weight of water or an aqueous solution;

Based on incorporation effects and preferred dosages and incorporated directly into the complex or dissolved in microemulsions at a concentration of 0.001 to 99% by weight, preferably 0.001 to 50% by weight compared to the sum of the lipidic and amphiphilic components Pharmaceutically active ingredients.

In the dispersion:

The microemulsions prepared as described above are dispersed in an aqueous environment (water or aqueous solution) diluted in an amount of 1: 2 to 1: 200, preferably 1: 5 to 1: 50.

In dispersion

0.05 to 5% by weight of dispersing aid

-0.05 to 5% by weight of polymer type viscosity agent

Can be added.

There is provided a production process according to high pressure homogenization techniques for the dispersion of complexes in which one or more auxiliary substances are added in an aqueous environment. The composite is homogenized to form nanoparticles that hold the system at the melting point of the material itself or just below that temperature (“softening”) or at a temperature at which the composite remains solid.

The complex is initially melted or covalently dissolved, similar to the method reported in Technique 1, in which eutectic lipid and amphiphilic substances are carried out to the melting point of the component, or to the melting point of one of the two components when the latter melts in the former. Can be provided.

The composite can be widely dispersed in an aqueous solution containing surfactants, stabilizers and / or viscosifiers by dispersion or low energy homogenization techniques (eg using Silverson L2R or Ultra-Turrax type of equipment). After such treatment, which may be unnecessary if the composite exhibits sufficient surface properties to aid dispersion in water, the system is then subjected to a high pressure homogenizer (e.g. APV Gaulin) for repeated homogeneous circulation processes that can lead to nanoparticle dispersion. , APV Rannie Mini-Lab, Microfluidizer Class). High pressure homogenization can occur at the melting point of the composite, at a "softening" temperature, or at a temperature where the material is in a solid state in a refined form.

The active ingredient is incorporated into the composite during the eutectic process of the system, or is eutectic, covalently or dispersed in each of the components, or by itself or in a subsequent process in the presence of a surfactant that aids incorporation into or adsorbing to the nanoparticles. Can be added.

The nanoparticle suspension can then be washed with water or an aqueous solution using an ultrafiltration system similarly as described in technique (1).

The composition can likewise be administered as an aqueous suspension or recovered as a solid using lyophilization, filtration, aqueous solvent distillation under vacuum or spray-drying techniques.

In the method for producing nanospheres according to the technique (2), the materials forming the present invention are used in the following weight ratios.

0.1-50% by weight, preferably 0.5-15% by weight of the lipidic component;

0.1-50% by weight, preferably 0.5-15% by weight of amphipathic component;

0.05 to 10% by weight of surfactant, preferably 0.5 to 5% by weight;

45-99.5% by weight, preferably 50-80% by weight of water or an aqueous solution of a water soluble component;

0.05-5% by weight of dispersing aid;

0.05 to 1% by weight of viscous agent

Based on incorporation effects and preferred dosages and incorporated directly into the complex or dissolved in microemulsions at a concentration of 0.001 to 99% by weight, preferably 0.001 to 50% by weight compared to the sum of the lipidic and amphiphilic components Pharmaceutically active ingredients.

Among the lipidic substances usable in accordance with the present invention are both natural and synthetic or semisynthetic, which can be defined as "fats" in that they can only be mixed with water or only partially.

1) saturated or unsaturated natural fats, and partially or fully hydrogenated vegetable oils such as hydrogenated cottonseed oil (Lubritab ^™ ), hydrogenated palm oil (Dynasan ^™ P60) and hydrogenated soymilk (Sterotex ^™ HM);

2) semi-synthetic and synthetic mono-, di- and triglycerides comprising saturated and / or unsaturated fatty acids (with aliphatic chains of C ₁₀ to C ₂₂ ) and their polyhydroxyethylated derivatives, for example tristearin, Monoglycerides such as capriccocapryl triglycerides (Mygliol ^™ , Captex ^™ , Labrafac ^™ Lipo), behenic triglycerides (Compritol ^™ ), glyceryl monostearate (Myvaplex ^™ 600) or glyceryl palmitostearate (Precirol ^™ ) Rides, and saturated or unsaturated polyhydroxy triglycerides (Labarfil ^™ , Labrafac ^™ Hydro, Gelucire ^™ series);

3) "liquid waxes" such as isopropyl myristate, isopropyl-caprinate, -caprylate, -laurate, -palmitate, -stearate, and fatty acids such as ethyl oleate and oleyl oleate ester;

4) "solid waxes" such as carnauba wax and bees-wax;

Aliphatic alcohols such as cetyl alcohol, stearyl alcohol, lauryl alcohol, cetylstearyl alcohol and polyhydroxyethylated derivatives thereof;

6) Preferably, aliphatic carboxylic acids having medium and long chains (C ₁₀ to C ₂₂ ), saturated (decanoic acid, lauric acid, palmitic acid, stearic acid, docoic acid, etc.), unsaturated (oleic acid, linoleic acid, etc.) ) And polyhydroxyethylated derivatives thereof.

Among the amphipathic substances of the lipidic class, lipids having some hydrophilic components in the structure, for example, can be used:

1) Phospholipids belonging to the following series: phosphatidyl glycerol, phosphatidylcholine and phosphatidic acid (eg dimyristoyl phosphatidyl glycerol);

2) mono- and diglycerides such as glyceryl monostearate (Myvaplex ^™ 600) or glyceryl palmitostearate (Precirol ^™ );

3) triglycerides, and saturated or unsaturated polyhydroxy triglycerides (eg, Labrafil ^™ , Labrafac ^™ Hydro, Gelucire ^™ );

4) decyl esters of oleic acid: fatty acid esters such as Cetiol ^™ V and isopropyl myristate;

5) medium chain fatty acids (capric acid, capronic acid and lauric acid).

Among the amphipathic materials of the polymer type, the following polymers may be used:

1) both polyethylene glycol (PEG), liquids (PEG 200 to PEG 1000) and solids (PEG 1500 to PEG 20,000)

2) poly- (propyleneoxide) poly- (ethyleneoxide) copolymer, poloxamer (Lutrol ^™ 188, Lutrol ^™ 407);

3) polyvinyl alcohol;

4) polyacrylates (Carbopol ^™ , Pemulen ^™ , Noveon ^™ );

5) poly- (methylvinyl ether) -maleic anhydride (Gantrez ^™ ) copolymer;

6) chitosan and derivatives, ialuronic acid and derivatives, xanthan, scleroglucan, gellan, guar gum, locust bean gum, alginate ( alginate) and dextran;

7) polyesters, for example poly-ε-caprolactone.

As described above, the composites of the present invention can be obtained by mixing, eutectic or covalently mixing a component selected between a lipidic material and an amphiphilic material of the lipidic or polymer type. For example, the complex of the present invention may be prepared by combining fatty acid mixtures (stearic acid-decanoic acid), mixtures of fatty acids and phospholipids (stearic acid-dimyristoyl phosphatidyl glycerol or dimyristoyl phosphatidylcholine), fatty acids and triglycerides or polyhydroxides. Silylated triglycerides (stearic acid and Labrafil ^TM 2130), fatty acids and mono- and di-glycerides (stearic acid and glyceryl palmitostearate), polyethylene glycols and fatty acids (stearic acid-PEG), polyethylene oxide-polypropylene oxide Copolymers and mono and diglycerides (poloxamer and glyceryl palmitostearate).

As aqueous phases of the invention (aqueous phases of microemulsions according to technique 1 and / or dispersed phases according to techniques 1 and 2), the following may be mentioned:

1) water buffered by itself or other pH and ionic strength

2) polyethylene glycol, polyvinyl pyrrolidone, polyacrylic acid and derivatives (for example Carbopol , Pemulen Etc.), polymethacrylic acid and derivatives (eg Eudragit ), Polyoxyethylene-polyoxypropylene copolymers (eg poloxamers, Lutrol ), For example, hydrophilic, water soluble or water dispersible polymers, such as various natural polysaccharides such as dextran, xanthan, scleroglucan, arabic gum, guar gum, chitosan, cellulose and starch derivatives. Aqueous solution;

3) aqueous solutions of saccharides (eg sorbitol, mannitol, xylitol);

3) mono or polyhydroxyaliphatic alcohols, preferably having short chains (C ₂ to C ₄ );

4) polyethylene glycol (eg PEG 200, PEG 400, PEG 600, PEG 1000)

5) polyglycol glycerides (eg Labrasol ^™ );

6) Polyglycols (Transcutol ^™ ) such as propylene glycol, tetraglycol, ethoxydiglycol.

Among the surfactants for use in Techniques 1 and 2, all nonionic surfactants having a typical HLB value but not necessarily having a value of 7 or more are not completely exemplified, but for example: Non-ester (e.g. Span , Arlacel , Brij ), Polyoxyethylene sorbitan esters of fatty acids (eg Tween , Capmul , Liposorb ), Copolymers of polypropylene oxide-polyethylene oxide (poloxamer), esters of polyethylene glycol (PEG) -glycerol (Labrasol with HLB 6-7) , Labrafil ), Polyglyceride esters (Plurol ), Saccharides and esters of fatty acids (sucrose-esters). If necessary, surfactants with low HLB values, lecithin itself (Lipoid S75) and hydrogenated lecithin (eg Lipoid S75, S75-3), phospholipids and their semisynthetic or synthetic derivatives, as well as even anionic surfactants ( For example sodium lauryl sulfate, sodium stearate, sodium oleate), bile salts (e.g. sodium glycocholate, taurodeoxycholate, taurocholate, ursodeoxycholate) or cationic interfaces Active agents (eg tricetol) can be used.

Among the cosurfactants required for the formation of the microemulsion, for example, short-chain alcohols such as ethanol, 2-propanol, n-butanol, isopropanol; Short or medium chain fatty acids (eg butyric acid, valeric acid and capronic acid), aromatic alcohols (eg benzyl alcohol); Mention may be made of heavy chain alcohols and fatty acids (C ₈ to C ₁₂ ), such as decanoic acid, lauric acid, caprylyl alcohol and lauryl alcohol. It is also possible to use esters or ethers of acids with mono- or polyhydroxylated alcohols, or aliphatic alcohols of medium chain length as cosurfactants. Some of the components mentioned among the cosurfactants may simultaneously form the oil phase of the microemulsion.

Pharmaceutically active ingredients that can be used in the present invention may not only be insoluble in both carriers (eg acyclovir), but also soluble in water (eg peptides or proteins) and in lipids (eg steroidal) Hormones) can melt. The surface and mass properties of the nanoparticles according to the invention have the following advantages:

1) the possibility of administering orally or transmucosal methods of molecules (eg polypeptides and proteins) which are not normally absorbed by such methods;

2) the possibility of oral and / or parenteral administration of lipophilic molecules that are hardly soluble and insoluble;

3) enhancement of the biopharmaceutical properties of the active ingredient (eg, an increase in plasma half-life and controlled or prolonged release);

4) the possibility of topical administration of active molecules (eg antiviral, antifungal, anti-psoriasis) at the mucosal or dermal stage;

5) Possibility of encapsulating active ingredients with an unpleasant odor that can be administered in an immediate release formulation.

Active ingredient groups that can be used in the present invention include nonsteroidal (NSAID) and steroidal (SAID) anti-inflammatory agents, estrogens or progesterone, cardiovascular agents, antiviral agents, antifungal agents, malignant tumor inhibitors, peptides and proteins with other activities. Include.

Examples of the above-mentioned active ingredients include, but are not exhaustive.

Ergot alkaloids and derivatives: dihydroergotamine, dihydroergotoxine and bromocriptine.

Analgesics and nonsteroidal anti-inflammatory agents and salts thereof: diclofenac sodium, diclofenac hydroxyethyl pyrrolidine, diclofenac diethylamine, ibuprofen, flubiprofen, ketoprofen, indomethacin, mefenamic acid, naproxen, nimesulide (nimesulide) and pyroxicam.

Antiarrhythmics: Amiodarone, diisopyramid, propranolol and verapamil.

Antibacterial agents: amoxicillin, flucoxacillin, gentamicin, rifampicin, erythromycin and cephalosporins.

Antimicrobial and anti-psoriasis agents: amphotericin, butoconazole nitrate, ketoconazole, econazol, etretinate, fluconazole, flucitocin, griseofulvin, itraconazole, myconazole, nystatin, sulfonazole and thiocazole.

Antiviral agents: Acyclovir, ganciclovir, AZT and protease inhibitors.

Antihypertensives: amlodipine, clonidine, diltiazem, pelodipine, guanabenz acetate, isradipine, minoxidil, nicardipine hydrochloride, nimodipine, nifedipine, prazosin hydrochloride and papaverine.

Antidepressant: Carbamazepine.

Antihistamines: diphenylhydramine, chlorophenylamine, pyrylamine, chlorocycline, promethazine, acribastin, cinnarizine, loratadine and terpenadine.

Malignant Tumor Suppression and Immune Inhibitors: Cyclosporin, Dacarbazine, Etretinate, Etoposide, Romustine, Melparan, Mitomycin, Mitoxantrone, Paclitaxel, Procarbazine, Tamoxifen, Taxol and Derivatives and Taxotere.

Anti-anxiety, sedative, hypnotics: alprazolam, bromazepam, diazepam, lorazepam, oxazepam, temazepam, sulfide and triazolam.

β-blockers: alprenolol, atenolol, oxprenolol, pindolol and propanolol.

β-antagonists: salbutamol, salmeterol.

Muscle contractors of the heart and cardiovascular: amlinone, digitoxin, digoxin, lanatoside C, medigoxin and ubidecarenone.

Corticosteroids: beclomethasone, betamethasone, budesonide, cortisone acetate, desoxymethasone, dexamethasone, fludrocortisone acetate, flunisolidide, hydrocortisone, methylprednisolone, methylprednisone and triamcinolone.

Gastrointestinal and anti-H2-histamines: cimetidine, cisapride, domperidone, famotidine, loperamide, mesalazine, omeprazole, ondansetron hydrochloride and ranitidine.

Hypolipidemic agents: bezafibrate, clofibrate, gemfibrozil, probucol and lovastatin.

Antianginases: amyl nitrate, glyceryl trinitrate, isosorbide dinitrate and mononitrate and pentaerythritol tetranitrate.

Central nervous agent: for example nicotine.

Vitamins and Nutritional Supplements: Beta-carotene, vitamin A, vitamin B2, vitamin D and derivatives, vitamin E and derivatives and vitamin K.

Opioid analgesics: codeine, dextrosepropoxyphene, dihydrocodeine, morphine, pentazosin and methadone.

Sex hormones: danazol, ethynyl estradiol, methoxyprogesterone acetate, methyltestosterone, testosterone, noredistrone, norgestrel, estradiol, estriol, progesterone, stilbestrol and diethylstilbestrol.

Peptide, protein or polysaccharide molecules with other activities: leuprolide and LH-RH analogues, calcitonin, glutathione, somatotropin (GH), somatostatin, desmopressin (DDAVP), interferon, morphramostine, Epidermal growth factor (EGF), nerve growth factor (NGF), insulin, glucagon, toxin or toxoid (e.g. tetanus toxin), antigen factor of the protein or polysaccharide class, heparin, heparin with low molecular weight and Heparinoids.

Molecules with specific local activity: for example sun protectants (UV absorbers); Skin nutrients, ceramides and glycolic acid.

The characteristics of the components according to the invention can be measured by several physicochemical methods, for example, in the following way.

Thermal analysis (DSC, TGA and “hot stage” microscopy) to identify changes in core properties,

-Surface analysis (contact angle method) for measuring changes in surface properties

Laser light scattering (LLS) technology for measuring the size distribution of nanoparticles,

Zeta potential measurement techniques for measuring surface charge properties of nanoparticles,

-Active ingredient incorporation efficiency measurement technology (marking, separation and analysis with fluorescent molecules),

Structure observation technique (electron transfer microscope: TEM).

This technique demonstrates that the composites forming the nanoparticles according to the invention and the nanoparticles themselves exhibit innovative and beneficial properties. When appropriate combinations of lipid and amphipathic materials are obtained, the following materials and composite nanoparticles can be obtained.

Increases the incorporation efficiency of hydrophilic agents (eg peptides) in the lipidic matrix, which aids in the dissolution / dispersion of the active ingredient in the largest hydrophilic region of the nanoparticles,

Modification in a way that does not anticipate localization of molecules mixed into the nanoparticles: enabling uniform dispersion of peptide molecules, typically absorbed at the surface, for example inside lipophilic nanoparticles,

Lower surface energy compared to the starting component, resulting in better biocompatibility,

Exhibits a more lipophilic surface than a single component, resulting in better absorption by the oral method,

When applied, it shows a surface more hydrophilic than a single component and increases the plasma half-life of the particles injected by parenteral methods,

At specific proportions of amphiphilic and lipidic substances, causing two phases with distinctive characteristics:

1) Amphiphilic compounds are preferably located on the surface of the nanoparticles ("separated from the surface")

2) the amphiphilic compound is preferably located inside the nanoparticle ("isolated within")

3) the amphiphilic compound is uniformly located on the surface and inside of the nanosphere itself,

At certain ratios, nanoparticles consisting of complexes with unique and different properties to a single original material can melt at physiological temperatures to release the active ingredient in a fast way (eg topical / dermal therapy and flavors). Shielding),

At other ratios, the nanoparticles can remain at physiological temperature, ensuring release of the active ingredient by diffusion and / or degradation of the nanoparticles themselves,

In the manufacturing technique (1), at certain proportions of lipid and amphiphilic substances, the thermal intervals at which the microemulsions themselves are based on oil-water microemulsions of the complexes can be extended to lower temperatures. Incorporation of thermolabile molecules can be enabled.

Independently of the method of making the nanoparticles, an excellent feature underlying the present invention is the unexpected formation of composites with new and different surface and central properties compared to a single component. Based on the qualitative characteristics of the selected starting material (e.g. chemical structure, melting point, hydrophobicity and wettability), it is possible to obtain a large amount of complexes at a relative percentage of the substance in the determined mixture, resulting in a large amount of different characteristics Nanoparticles can be obtained.

An advantage of the present invention is that it can increase the incorporation of hydrophilic active ingredients inside lipophilic nanoparticles and modify their distribution.

As a result, an important advantage of the present invention is the possibility of having a vector system (composite nanoparticles) consisting of new materials based on exclusive physical changes in the component materials, thus eliminating the need for long toxicological experimental test procedures.

Examples of the preparation method of the composition according to the present invention for the detailed description and the comparative example of the preparation method according to the technique 1 (Examples 1 to 36), Technique 2 (Examples 37 to 45) and the prior art (A to E) Together. The properties of the product obtained in comparison with the products of the prior art and the advantageous behavior in vivo are shown in Examples (F-Z1).

Example 1

4 g of stearic acid and 19.9 mg of di-myristoyl-phosphatidyl glycerol (% DMPG in the mixture: 0.5%) are mixed and heated to about 72 ° C. to form a molten complex, to which 4 ml of n-butanol are added. 2 g of sodium taurodioxycholate and 3.85 mg of salmon calcitonin were dissolved in 20 ml of water acidified to pH 3, and the solution was heated to 72 ° C. and added to the molten composite mixture. 3 ml of Tween 20 was added while stirring the emulsion thus formed to obtain a transparent, anisotropic microemulsion. Complex solid lipid composition consisting of stearic acid-dimyristoyl-phosphatidyl glycerol containing calcitonin by diffusion in microemulsion at about 60 ° C. (250 rpm) and 5 volumes of water at pH 3 cooled to 3-5 ° C. Nanoparticles were formed. The suspension was washed by ultrafiltration to remove excess surfactant. The calcitonin incorporation efficiency in the nanoparticles, measured by fluorescence and chromatographic techniques, is about 10.5%. The average diameter of the nanoparticles is 226 nm and the polydispersity index is 0.200.

Example 2

The preparation of Example 1 was repeated with a DMPG percentage of 1.0% in the composite mixture. The calcitonin incorporation efficiency was 10.2%, the average diameter of the nanoparticles was 220 nm, and the polydispersity index was 0.268.

Example 3

The preparation of Example 1 was repeated with a DMPG percentage of 4.5% in the composite mixture. The calcitonin incorporation efficiency was 13.2%, the average diameter of the nanoparticles was 199 nm, and the polydispersity index was 0.212.

Example 4

The preparation of Example 1 was repeated with a DMPG percentage of 5.2% in the composite mixture. The calcitonin incorporation efficiency was 13.4%, the average diameter of the nanoparticles was 195 nm, and the polydispersity index was 0.200.

Example 5

The preparation of Example 1 was repeated with a DMPG percentage of 9.5% in the composite mixture. The calcitonin incorporation efficiency was 9.19%, the average diameter of the nanoparticles was 231 nm, and the polydispersity index was 0.186.

Example 6

The preparation of Example 1 was repeated with a DMPG percentage of 25% in the composite mixture. The calcitonin incorporation efficiency was 9.51%, the average diameter of the nanoparticles was 205 nm, and the polydispersity index was 0.275.

Example 7

The preparation of Example 1 was repeated with the distearoyl phosphatidic acid (DSPA) phospholipid percentage as 1.2% as the amphipathic material in the composite mixture. The calcitonin incorporation efficiency was 9.9%, the average diameter of the nanoparticles was 245 nm, and the polydispersity index was 0.261.

Example 8

The preparation of Example 7 was repeated with a DSPA percentage of 5.2% as the amphipathic material in the composite mixture. The calcitonin incorporation efficiency was 14.4%, the average diameter of the nanoparticles was 324 nm, and the polydispersity index was 0.341.

Example 9

The preparation of Example 1 was repeated with low molecular weight heparin (average molecular weight 4000 Da) as the active ingredient in the composite mixture and dimyristoyl phosphatidylcholine (DMPC) phospholipid percentage as 4.0% as amphiphilic material. The heparin incorporation efficiency was about 1.0%, the average diameter of the nanoparticles was 213 nm and the polydispersity index was 0.186.

Example 10

The preparation of Example 9 was repeated with a dimyristoyl phosphatidylcholine (DMPC) phospholipid percentage in the complex mixture of 8.0%. The average diameter of the nanoparticles is 179 nm, and the polydispersity index is 0.249.

Example 11

The preparation of Example 9 was repeated with a dimyristoyl phosphatidylcholine (DMPC) phospholipid percentage in the complex mixture as 10%. The average diameter of the nanoparticles is 290 nm, and the polydispersity index is 0.270.

Example 12

The preparation of Example 9 was repeated with a dimyristoyl phosphatidylcholine (DMPC) phospholipid percentage in the complex mixture as 14%. The average diameter of the nanoparticles is 369 nm, and the polydispersity index is 0.360.

Example 13

The preparation method of Example 1 was repeated with a Labradac Hydro ^™ polyhydroxylated triglyceride percentage of 1.0% as amphiphilic material in the composite mixture. The average diameter of the nanoparticles is 307 nm, and the polydispersity index is 0.234.

Example 14

The preparation of Example 13 was repeated with a Labradac Hydro ^™ polyhydroxylated triglyceride percentage of 2.5% in the composite mixture. The average diameter of the nanoparticles is 307 nm, and the polydispersity index is 0.234.

Example 15

The preparation of Example 13 was repeated with a Labradac Hydro ^™ polyhydroxylated triglyceride percentage of 50% in the composite mixture. The average diameter of the nanoparticles is 310 nm, and the polydispersity index is 0.332.

Example 16

The preparation of Example 13 was repeated with the Labradac Hydro ^™ polyhydroxylated triglyceride percentage in the complex mixture as 10.0%. The average diameter of the nanoparticles is 326 nm, and the polydispersity index is 0.325.

Example 17

The preparation of Example 13 was repeated by mixing Labrafac Hydro ^™ polyhydroxylated triglyceride percentage in the complex mixture to 10.0% and mixing 3.87 mg of calcitonin in the complex mixture against 3.8 g of the mixture. The average diameter of the nanoparticles is 326 nm, and the polydispersity index is 0.325.

Example 18

2.18 g of stearic acid and 67 mg of Labarafil ^™ M 2130CS polyhydroxylated triglyceride (percent of Labrafil in the composite mixture: 3.1%) are mixed and heated to about 75 ° C. to form a molten composite. The mixture is cooled to solidify. Dissolve 1.34 g of sodium taurodioxycholate and 3.85 mg of salmon calcitonin in 10 ml of water acidified to pH 3, heat the solution to 72 ° C., and add to the molten composite mixture at about 72 ° C., here To 1 ml of n-butanol was added. 4 ml of Tween 20 was added while stirring the emulsion thus formed to obtain a transparent and anisotropic microemulsion. The microemulsion heated up to about 50 ° C. was diffused in 50 volumes of water at pH 3 cooled to about 3-5 ° C. with constant stirring (250 rpm) to form solid nanoparticles comprising calcitonin. The suspension was washed by ultrafiltration to remove excess surfactant. The calcitonin incorporation efficiency in the nanoparticles was about 11.5%. The average diameter of the nanoparticles is 185 nm, and the polydispersity index is 0.300.

Example 19

The preparation of Example 18 is repeated without mixing calcitonin. The average diameter of the composite nanoparticles is 182 nm and the polydispersity index is 0.295.

Example 20

The preparation of Example 18 is repeated with Labrafil 2130CS polyhydroxylated triglyceride in 8.9% in the complex mixture. The composite mixture is melted at about 70 ° C. The mean diameter of the nanoparticles is 173 nm and the polydispersity index is 0.268.

Example 21

The preparation of Example 18 is repeated with 10% of Labrafil 2130CS polyhydroxylated triglyceride in the composite mixture. The composite mixture is melted at about 66 ° C. The average diameter of the nanoparticles is 216 nm and the polydispersity index is 0.287.

Example 22

The preparation of Example 18 is repeated with 15% of Labrafil 2130CS polyhydroxylated triglyceride in the composite mixture. The mean diameter of the nanoparticles is 188 nm and the polydispersity index is 0.247.

Example 23

The preparation method of Example 18 is repeated with 50% of Labrafil 2130CS polyhydroxylated triglyceride in the composite mixture, and the composite mixture is melted at 60 ° C. The average diameter of the nanoparticles is 312 nm and the polydispersity index is 0.424.

Example 24

The preparation of Example 18 is repeated with 75% Labrafil 2130CS polyhydroxylated triglyceride in the composite mixture and the composite mixture is melted at 54 ° C. The average diameter of the nanoparticles is 162 nm and the polydispersity index is 0.315.

Example 25

The preparation of Example 18 is repeated with 95% Labrafil 2130CS polyhydroxylated triglyceride in the composite mixture and the composite mixture is melted at 35 ° C. The average diameter of the nanoparticles is 205 nm and the polydispersity index is 0.281.

Example 26

1.5 g stearic acid and 0.5 g decanoic acid (percent decanoic acid in the mixture: 25%) are mixed and heated to about 75 ° C. to form a composite mixture. The mixture is cooled to solidify. 1.30 g of sodium taurodioxycholate was dissolved in 10 ml of acidified to pH 3, and the solution heated to 55 ° C. was added to the molten composite mixture at about 55 ° C., and 1 ml of n-butanol was added thereto. While stirring the emulsion thus formed, 2.6 ml of Tween 20 was added to obtain a transparent and anisotropic microemulsion. The microemulsion maintained at about 45 [deg.] C. was stirred (250 rpm) with diffusion in 50 volumes of water at pH 3 cooled to about 3-5 [deg.] C. to form solid nanoparticles that melt at low temperatures. The suspension was washed by ultrafiltration to remove excess surfactant. The average diameter of the nanoparticles is 280 nm.

Example 27

The preparation method of Example 26 was repeated with 50% decanoic acid in the composite mixture. The composite mixture is melted at 50 ° C. The average diameter of the nanoparticles is 310 nm and the polydispersity index is 0.280.

Example 28

The preparation method of Example 26 is repeated with 75% decanoic acid in the composite mixture. The composite mixture is melted at 35 ° C. The average diameter of the nanoparticles is 300 nm and the polydispersity index is 0.250.

Example 29

1.8 g of stearic acid and 0.2 g of polyethylene glycol PEG 4000 (percent PEG 4000 in the mixture: 10.0%) are mixed and heated to about 75 ° C. to form a complex mixture. 1.30 g of sodium taurodioxycholate was dissolved in 10 ml of acidified to pH 3, and the solution heated to 50 ° C. was added to the molten composite mixture at about 50 ° C., and 1 ml of n-butanol was added thereto. 2.4 ml of Tween 20 was added, stirring the emulsion thus formed to obtain a transparent, anisotropic microemulsion. The microemulsion maintained at about 45 ° C. was diffused in 50 volumes of water at pH 3 cooled to about 3-5 ° C. with constant stirring (250 rpm) to form composite lipidic solid nanoparticles. The suspension was washed by ultrafiltration to remove excess surfactant. The average diameter of the nanoparticles is 184 nm and the polydispersity index is 0.302.

Example 30

The preparation method of Example 29 is repeated with 20% of polyethylene glycol PEG 4000 in the composite mixture. The composite mixture is melted at 50 ° C. The mean diameter of the nanoparticles is 263 nm and the polydispersity index is 0.334.

Example 31

The preparation of Example 29 is repeated with 10% of Lutrol ^™ 188, a poly- (propyleneoxide) poly- (ethyleneoxide) copolymer, as the amphipathic material in the composite mixture. The composite mixture is melted at 50 ° C. The mean diameter of the nanoparticles is 353 nm and the polydispersity index is 0.314.

Example 32

The root roll (Lutrol ^TM) 188 in the composite mixture and repeats the method of Example 29 to 20%. The composite mixture is melted at 50 ° C. The average diameter of the nanoparticles is 375 nm and the polydispersity index is 0.300.

Example 33

1.4 g of stearic acid and 607 mg of Labrafil ^™ M2130CS polyhydroxylated triglyceride (percent of Labrafil in the mixture: 30.1%) are mixed and heated to about 70 ° C. to form a complex mixture. To this mixture is added partially hydrogenated soy lecithin (Lipoid S75-35). 10 ml of an aqueous solution of pH 3 was added to the molten composite mixture at about 70 ° C., and 1 ml of n-butanol was added thereto. While stirring the emulsion thus formed, 6 ml of Tween 20 was added to obtain a transparent and anisotropic microemulsion. Add about 200 mg of cyclosporin to the microemulsion. The microemulsion heated to about 50 ° C. was diffused in 50 volumes of water at pH 3 cooled to about 3 to 5 ° C. with constant stirring (250 rpm) to form solid nanoparticles containing cyclosporin. The suspension was washed by ultrafiltration to remove excess surfactant. The average diameter of the nanoparticles comprising cyclosporin is 304 nm and the polydispersity index is 0.365.

Example 34

100 mg of etoposide was mixed into the microemulsion and the preparation method of Example 33 was repeated. The composite mixture is melted at 50 ° C. The average diameter of the composite nanoparticles is 288 nm and the polydispersity index is 0.211.

Example 35

The root roll (Lutrol ^TM) 188 in the composite mixture and repeats the method of Example 29 to 20%. The composite mixture is melted at 50 ° C. and acyclovir is added in an amount of 100 mg per gram of the composite mixture. The average diameter of the nanoparticles containing acyclovir is 360 nm and the polydispersity index is 0.302.

Example 36

980 mg of stearic acid and 510 mg of Labrafil ^™ M2130CS polyhydroxylated triglyceride (percent of Labrafil in the mixture: 30%) are mixed and heated to about 70 ° C. to form a complex mixture. To this mixture is added 300 mg of Q10 coenzyme (Ubidecarenone) and 1.06 g of partially hydrogenated soy lecithin (Lipoid S75-35). 10 ml of an aqueous solution of pH 3 was added to the molten composite mixture at about 70 ° C., and 1 ml of n-butanol was added thereto. While stirring the emulsion thus formed, 6 ml of Tween 20 was added to obtain a transparent and anisotropic microemulsion. The microemulsion heated to about 50 ° C. was diffused in 50 volumes of water at pH 3 cooled to about 3 to 5 ° C. with constant stirring (250 rpm) to form solid nanoparticles containing ubidecarenone. The suspension was washed by ultrafiltration to remove excess surfactant. The percentage of ubidecarenone incorporation in the nanoparticles is 99%, the average diameter of the nanoparticles is 195 nm, and the polydispersity index is 0.214.

Example 37

6 g of stearic acid and 0.9 g of Labrafil ^™ M2130CS polyhydroxylated triglyceride (15% of the mixture) are mixed and melted at a temperature of about 70 ° C. 1.5 g of soy lecithin (Lipoid S75-35) is added to the molten composite mixture, and 300 ml of an aqueous solution of pH 5.5 containing 3 g of Tween 20 is added. The emulsion thus formed is passed through a high pressure Rannie-MiniLab 8.30 homogenizer at a temperature of 70 ° C. and a pressure of 750 bar over 0-15 minutes. The dispersion was recovered and cooled directly to 4 ° C. by continuously stirring at 250 rpm in a thermostatically controlled bath, to obtain composite lipid solid nanoparticles. The average diameter is as follows:

Process time (minutes) Diameter (nm) Polydispersity Index 5 257 0.36 10 264 0.392 15 282 0.400

Example 38

When the composite is obtained by eutectic, the production method of Example 37 is repeated below the melting point of the composite itself (T < 50 ° C.) while maintaining the temperature of the system. The resulting dispersion is pre-uniformed for 5 minutes in a low energy homogenizer-mixer (Silverson mod. L2R) and then homogenized at constant temperature (45 ° C.) and high pressure (750 bar).

Example 39

0.39 g of stearic acid and 5.5 g of Labrafil ^™ M2130CS polyhydroxylated triglyceride (65% of the mixture) are mixed and melted at a temperature up to about 75 ° C. To the molten composite mixture 3 g soy lecithin (Lipoid S75-35) are added and 300 ml of an aqueous solution of pH 5.5 containing 12 g Tween 20 is added. The emulsion thus formed is passed through a high pressure Ranimini Lab 8.30 homogenizer at a temperature of 70 ° C. and a pressure of 750 bar over 0-15 minutes. The dispersion is cooled directly to 4 ° C. by continuously stirring at 250 rpm in a thermostatic bath to obtain composite lipid solid nanoparticles. The average diameter is as follows:

Process time (minutes) Diameter (nm) Polydispersity Index 3 106 0.205 5 72 0.161 10 91 0.176

Example 40

When the composite is obtained by eutectic, the production method of Example 39 is repeated below the melting point of the composite itself (T < 35 ° C) while maintaining the temperature of the system. The dispersion obtained is pre-uniformed for 5 minutes in a low energy homogenizer-mixer (Silverson mod. L2R) and then homogenized at constant temperature (30 ° C.) and high pressure (750 bar).

Example 41

3.75 g of stearic acid and 3.75 g of polyethylene glycol 20000 (PEG 20000) (50% of the mixture) are mixed and melted at a temperature up to 75 ° C. 3 g of soy lecithin (Lipoid S75-35) is added to the molten composite mixture, and 300 ml of an aqueous solution of pH 5.5 containing 6 g of Tween 20 is added. The emulsion thus formed is passed through a high pressure Rannie-MiniLab 8.30 homogenizer at a temperature of 70 ° C. and a pressure of 750 bar over 1-10 minutes. The dispersion is cooled to 4 ° C. directly by continuous stirring at 250 rpm in a thermostatically controlled bath to give solid nanoparticles. The average diameter is as follows:

Process time (minutes) Diameter (nm) Polydispersity Index 5 156 0.274 10 188 0.308

Example 42

When the composite is obtained by eutectic, the production method of Example 41 is repeated below the melting point of the composite itself (T < 45 ° C.) while maintaining the temperature of the system. The resulting dispersion is pre-uniformed in a low energy homogenizer-mixer (Silverson mod. L2R) and then homogenized at constant temperature (45 ° C.) and high pressure (750 bar).

Example 43

With 15% PEG percentage in the composite mixture, 300 mg of Salmon Calcitonin is mixed into the composite mixture itself and the preparation of Example 41 is repeated. The calcitonin incorporation efficiency in the PEG-stearic acid mixture is 35%.

Example 44

When the composite is obtained by eutectic, the production method of Example 42 is repeated below the melting point of the composite itself (T < 45 ° C.) while maintaining the temperature of the system. The dispersion obtained is pre-uniformed with Silverson and then homogenized at constant temperature (45 ° C.) and high pressure (750 bar).

Example 45

Eutectic and cooling yields a complex mixture comprising 5% stearic acid and 95% Labrafil ^™ M2130CS polyhydroxylated triglycerides. To the molten composite mixture is added 40 ml of an aqueous solution of pH 5.5 containing 1 g of the composite mixture plus 1 g of ibuprofen and 240 mg of soy lecithin (Lipoid S75-35) and 1.2% Tween 20 to the molten composite mixture (75 ° C.). The emulsion thus formed is treated in a high pressure Rannie-MiniLab 8.30 homogenizer at a temperature of 70 ° C. and a pressure of 750 bar for 0-10 minutes. The dispersion was immediately cooled to 4 ° C. by constant stirring at 250 rpm in a thermostatically controlled bath to obtain solid nanoparticles. The average diameter is as follows:

Process time (minutes) Diameter (nm) Polydispersity Index 2 224 0.304 5 264 0.364 10 253 0.352

Example A (according to prior art European Patent No. 0526666A1)

4.2 g of stearic acid was melted and heated to about 72 ° C. After dissolving 2.6 g of sodium taurodioxycholate in 20 ml of water acidified to pH 3, the solution heated to 72 DEG C was added to the molten stearic acid, and then 2 ml of n-butanol was added thereto. The emulsion thus formed is added while 5 ml of Tween 20 is kept under stirring in a mixer to obtain a transparent, anisotropic microemulsion. The microemulsion heated to about 60 ° C. is dissolved in 5 volumes of water at pH 3 at about 3-5 ° C. with constant stirring (250 rpm) to form nanoparticles according to the technique used in EP0526666A1. The suspension is washed by ultrafiltration to remove excess surfactant. The average diameter of the nanoparticles is 209 nm and the polydispersity index is 0.155.

Example B

3.85 mg of Salmon calcitonin is added and the preparation of Example A is repeated. The calcitonin incorporation efficiency in the nanoparticles is 1.82%. The average diameter of the nanoparticles is 193 nm and the polydispersity index is 0.235.

Example C

The preparation method of Example B is repeated. The nanoparticles thus obtained are added to an amphiphilic substance (dimyristoyl phosphatidyl glycerol, DMPG) at a ratio of 1: 10 to the lipid mass of stearic acid while stirring in a mixer, and absorbed onto the surface of the nanoparticles themselves. The absorption efficiency of calcitonin on the nanoparticles is 1.75%. A direct comparison of such a composition according to the prior art with Example 5 of the present invention differs only in that it has an amphiphilic component that is absorbed on the surface and not as a component of the composite forming the nanoparticles. The suspension is washed by ultrafiltration to remove excess surfactant. The average diameter of the nanoparticles is 215 nm and the polydispersity index is 0.175.

Example D

1.7 g of stearic acid and 300 mg of ubidecarenone are mixed and melted by heating to about 70 ° C. 0.5 ml of n-butanol and 1.30 g of sodium taurodioxycholate were added to 10 ml of an aqueous solution of pH 3 heated to 70 ° C., followed by eutectic. While maintaining the emulsion thus formed under stirring, 3.25 g of Tween 20 is added to form a microemulsion. The microemulsion heated to about 70 ° C. is dispersed in 50 volumes of water at pH 3 at about 3-5 ° C. with constant stirring (250 rpm) to form lipidic nanoparticles according to European Patent No. 0526666A1 technology. The suspension is washed by ultrafiltration to remove excess surfactant. The percentage of incorporation of ubidecarenone in the nanoparticles is about 80%, the average diameter of the nanoparticles is 205 nm and the polydispersity index is 0.244.

Example E (according to Prior Art WO 93/05768)

7.5 g of stearic acid is melted at 75 ° C. 3 g of soy lecithin (Lipoid S75-35) are added and 300 ml of an aqueous solution of pH 5.5 containing 6 g of Tween 20 is added. The emulsion thus obtained is treated with a high pressure Rannie-MiniLab 8.30 homogenizer at a temperature of 70 ° C. and a pressure of 750 bar for 0-10 minutes. The dispersion is immediately cooled to 4 ° C. to obtain solid nanoparticles according to the technique disclosed in WO 93/05768.

The average diameter is as follows:

Process time (minutes) Diameter (nm) Polydispersity Index 5 190 0.254 10 178 0.308

Characteristic Experiment Example

-Incorporation of hydrophilic drugs (peptides)

One of the excellent features of the present invention is the possibility of improving the incorporation efficiency of the hydrophilic drug (peptide) and the distribution inside the composite nanoparticles. This point is indicated by fluorescence analysis.

According to a known technique (Biochem. J., 272, 713-719, [1990]), a calcitonin peptide is indicated by a fluorophore (7-nitrobenz-2-oxa-1,3-diazole, NBD), Subsequently, they were incorporated into nanoparticles as shown in Examples 1 to 8 and Comparative Examples B to C of the present invention. Samples were washed according to the ultrafiltration process shown in the examples.

The percentage of peptide incorporated into the nanoparticles

When fluorescence is measured before and after treatment with a protease trypsin, which can dissolve and decompose only the portion of the peptide adsorbed on its own surface, the percentage of peptide adsorbed on the surface to the total amount incorporated is standard. The curve and the fluorescence emission value for 100% fluorescence emitted prior to ultrafiltration were calculated.

The results disclosed in Example F, Table 1 show how the composite nanoparticles reduce the rate of their location on the surface (which can be adsorbed and attacked by proteolytic enzymes) and increase the incorporation effect of peptides while maintaining most of the interior of the composite matrix Show if you let it.

Example F

The efficiency of calcitonin incorporation in complex lipidic nanoparticles and the percentage of peptide adsorbed on the nanoparticles' own surface Example matter Furtherance Mixing efficiency Adsorbed Peptides (%) One Stearic Acid DMPG 99.50.5 10.5 n.d. 2 Stearic Acid DMPG 99.01.0 10.2 0.11 3 Stearic Acid DMPG 95.54.5 13.2 0.05 4 Stearic Acid DMPG 94.85.2 13.4 0.04 5 Stearic Acid DMPG 91.59.5 9.19 0.49 6 Stearic Acid DMPG 7525 9.51 3.98 7 Stearic acid DSPA 98.81.2 9.9 n.d. 8 Stearic acid DSPA 94.85.2 14.4 n.d. 43 Stearic acid PEG 8515 35 n.d. B Stearic acid 100 1.82 0.93 C Stearic Acid DMPG 90.010.0 1.75 0.95

-Modification of core characteristics

Another excellent feature of the products of the present invention is the alteration of the central properties of the composites forming the nanoparticles. The modification of the core properties is measured quantitatively by thermal analysis techniques using differential scanning calorimeter, DSC, Perkin Elmer mod. DSC7. The nanoparticles according to the invention can be formed by either a complex or a composite present in a separate phase having different properties. In the former case, it can be observed, for example, that the uniform composite exhibits modification of the thermal phenomenon (melting point) depending on the composition (Example G) (Table 2). In the latter case, in relation to the thermal transition or melting of the separated phase, the thermal development temperature and specific enthalpy change for each composition percentage appear distinct (Examples H and I) (Tables 3 and 4).

Modifications of the thermodynamic behavior of composite nanoparticles as a function of component percentage can also be observed for nanoparticles in suspension, for example by "laser light scattering". The phase transition of the composite nanoparticles can determine the scattering intensity change measured by the Brookhaven mod. Bl-90 particle size meter as a function of temperature. Using this technique, one can point out how composite nanoparticles exhibit phase transitions and melt at temperatures that vary depending on the composition of the material formed. See Example L, FIG. 1.

Example G

Melting Point (° C.) of Binary Complex Mixtures of Stearic Acid and Average Chain Fatty Acids (Amphiphiles) Partially Similar to Examples 26-28 Amphipathic% 0 10 20 30 50 65 70 75 85 90 100 Decanoic acid 71 60 56 50 43 35 28 26 32 Lauric acid 71 69 62 56 44 43 42 45 Myristic acid 71 68 64 56 50 51 53 54 Palmitic acid 71 68 66 60 56 60 61 63

Example H

Phase transition and enthalpy of complexes of stearic acid-dimyristoyl-phosphatidyl glycerol (DMPG) and some meaningful compositions, partially similar to Examples 1-8. DMPG% T ₁ (℃) ΔH (J / g) T _f (℃) ΔH (J / g) 0 - - 73.3 210.5 6 55.3 0.55 72.2 185.95 12 55.0 5.811 71.8 160.02 18 55.2 6.71 71.5 146.09 30 56.2 nd 70.2 nd 40 56.4 nd 68.8 nd 50 57.2 35.15 69.7 56.9 100 - - 125.21 nd

Example I

Phase transition and enthalpy of complexes of stearic acid-Labrafil 2130 CS polyhydroxylated triglycerides, similar to Examples 18-25. Labrador% T ₁ (℃) ΔH (J / g) T _f (℃) ΔH (J / g) 0 - - 73.9 210.5 2 24. 0.01 74.1 210.2 5 25.1 0.4 73.3 204.4 10 25.2 1.5 71.6 189.8 15 25.1 2.5 72.0 180.5 50 25.3 6.3 65.4 99.3 78 25.0 8.4 56.0 25.0 85 26.0 8.5 55.1 15.2 90 27.1 10.2 52.0 5.2 100 29.1 13.8 38.6 0.77

Example L

Figure 1. Phase transition of composite nanoparticles (stearic acid-DMPG), determined by scattering techniques, similar to the products described in Examples 3-5 and Comparative Example A. Phase transition (melting) is represented as a decrease in the intensity of "scattering" as a function of temperature. The onset of this change (reduction from curve platau) corresponds to the onset of metastasis. In the compositions according to the invention, it is clear how these transitions can be changed and controlled by changing the composition of the material forming the nanoparticles. The melting point of the particles (63 ° C. in Example A) actually decreases to 55 ° C., 53 ° C. and 49 ° C. in Examples 3, 4 and 5, respectively.

Modification of manufacturing process characteristics (technique 1): presence of microemulsions

The properties of the composites according to the invention provide compositions with low melting points, which are used as intermediates in the preparation of composite nanoparticles according to technique 1 (Example M) (FIG. 2) at temperatures much lower than the melting point of single component materials. Get and extend the thermal presence interval of the microemulsion. This spacing was determined by measuring the scattering frequency and is the minimum value (<10 KHz) in the presence of microemulsion. A clear and major advantage of the present invention is the ability to construct thermally labile active ingredients in composite nanoparticles; Another advantage is that some materials with temperatures below their melting point can be used in the manufacture of nanoparticles.

Example M

2. Thermal interval of the presence of a microemulsion consisting of stearic acid and decanoic acid in the oil phase (prepared in technique 1, reference examples 26 to 28 and example A).

-Modification of surface properties

One of the excellent properties of the products of the present invention is the possibility of controlling the surface properties of the composites forming nanoparticles and consequently the nanoparticles themselves. The surface properties of the nanoparticles obtained according to the techniques and materials of the present invention differ in practical ways compared to the surface adsorption of the amphiphilic component described in the prior art as they depend on the formation of the composite. The advantage of the present invention is therefore the possibility of obtaining composite nanoparticles with desirable properties of biocompatibility, hydrophobicity, hydrophilicity and polarity, depending on the therapeutic objective to be achieved.

The surface properties of the composites forming the nanoparticles can be measured by contact angle method using the Lorentzen & Wettre instrument. This method can calculate the surface energy of the material that can be associated with the wettability through the following equation (1).

γ _sv = γ _sl + γ _lv (cosθ) (1)

Where the indexes sv, sl, lv represent the surface energy γ of the solid-vapor, solid-liquid and liquid-vapor surfaces, respectively, and θ is the angle at which the liquid surface and liquid contact. The surface free energy of each material can be divided into polar components and dispersion components according to (2).

γ ^t = γ ^p + γ ^d (2)

In addition to the polarity of the surface expressed in the following percentages, it can be calculated from the experimental measurements obtained by a series of equations, the values of these components and the total free energy.

% P = (γ ^p / γ ^t ) * 100 (3)

From the surface energy measurements of the composite according to the invention, it was possible to determine that the amphipathic component is as follows:

1) preferably arranged on the surface of the nanoparticles formed by the composite ("separated on the surface")

2) preferably arranged inside the nanoparticles formed by the composite ("isolated inside")

3) The nanospheres are uniformly arranged on and within their surface.

Diagram of surface energy of the composite mixture: stearic acid-DMPG (Example N) to form nanoparticles as described in Examples 1-6, stearic acid-labrapak hydro to form nanoparticles as described in Examples 13 to 17 (Labrafac Hydro ^™ ) Polyhydroxylated Triglycerides (Example O), Stearic Acid-Labrapak Lipocaprylic-Capric Triglyceride (Example P) are shown for purposes of demonstration in FIGS. 3, 4 and 5 Indicates. Curves A, B and C in FIGS. 3, 4 and 5 represent the total surface free energy for the dispersion component and the polar component, respectively.

Example N

3. The trend of surface energy of stearic acid-DMPG compositions pertaining to Examples 1-6. It represents the "preferred surface separation" of the amphiphilic component DMPG in the composite mixture characterized by a decrease in total energy.

Example O

4. Surface energy trends of stearic acid-Labrapak hydro composition, belonging to Examples 13-17. The trend indicates the "preferential internal separation" of the amphipathic component Labrapak Hydro (and internal stearic acid component) in the composite mixture.

Example P

5. Surface energy trend of stearic acid-Labrapak lipo composition. The constant trend shows a uniform distribution of the amphipathic component Labrapak lipo in the composite mixture.

Changes in the surface properties of the composite nanoparticles as a function of the composite composition used can also be quantified by zeta potential measurements made with a laser light scattering electrophoretic analyzer on an aqueous suspension of the composite nanoparticles. Zeta potential (cross section potential) is a measure of the surface charge of nanoparticles in a suspension. It is evident in Examples Q-T that the zeta potential and the resulting surface properties of the nanoparticles appearing as a function of the percentage of amphipathic material in the composites tend to be appropriate for each composition.

The possibility of modifying the surface properties and zeta potential of the nanoparticles of the present invention also has the important advantage of increasing stability by reducing or eliminating the tendency of precipitation and aggregation of the suspension. This advantage can be obtained by modifying the composition of the material forming the nanoparticles themselves.

Table 5. (Examples Q to T)

Example Q

Zeta potential (mV) was measured on stearic acid-DMPG nanoparticles and prepared similarly to Examples 2-5 and A.

Example R

Zeta potential (mV) on stearic acid-DMPG nanoparticles was measured and prepared similarly to Examples 9-11 and A.

Example S

Zeta potential (mV) was measured on stearic acid-Labrapak hydropolyhydroxylated triglyceride nanoparticles and prepared similarly to Examples 13-16 and A.

Example T

Zeta potential (mV) was measured on stearic acid-Labrafil 2130CS polyhydroxylated triglyceride nanoparticles and prepared similarly to Examples 19-24 and A.

ingredient DMPG DMPC Labrapak Hydro Labrador 2130CS Example Q R S T % In composite Zeta potential (mV) Zeta potential (mV) Zeta potential (mV) Zeta potential (mV) 0 -30.3 (Example A) -30.3 (Example A) -30.3 (Example A) -30.3 (Example A) One -30.0 (Example 2) -30.1 -31.4 (Example 13) 1.5 -33.7 2 -34.1 2.5 -30.3 -34.0 (Example 14) 3 -31.7 -21.2 (Example 19) 4 -32.2 (Example 9) 4.5 -34 (Example 3) 5 -35.4 (Example 4) -34.2 (Example 15) -13.6 7 -38.2 -31.9 (Example 10) 10 -40.4 (Example 5) -33.7 (Example 11) -33.6 (Example 16) -11.3 (Example 21) 12 -34.3 15 -14.1 (Example 22) 50 -19.8 (Example 23) 75 -18.9 (Example 24) 100 -25.4

-Composite nanoparticle structure according to the present invention and the prior art

In order to identify the substantial differences between the nanoparticles according to the present invention and the prior art, the structure of some important samples was examined using electron transfer microscopy ("negative staining" (uracil citrate) processes for the preparation of nanoparticle samples. TEM) (corresponding to Example 6 and Example A). In FIG. 6, it is evident that the structure of the composite nanoparticles is different compared to those prepared according to the prior art (Example A, FIG. 6A): on the particles in 6a corresponding to the phase of the composite (“preferential separation”). In fact, structurally different, clear and dark areas are observed, while system 6b appears uniform, and the inclusion of the sample corresponding to Example 1 (FIGS. 7A and 7B) shows that the material present on the surface is not present on the surface of the particles. It suggests that it is different from the amphipathic material layer absorbed by, but this is part of the particle itself.

Improved oral absorption of peptides (drug bioreaction and treatment efficiency)

In order to demonstrate the important advantages of the present invention, complex lipidic nanoparticles containing Salmon calcitonin as active ingredients have been administered "in vivo" (Examples 2, 4, 5), and nanoparticles containing calcitonin are known from the prior art ( Prepared according to Examples A, B, and C).

Dose Protocols and Sample Analysis

A complex nanoparticle suspension (20 ml) according to the invention containing calcitonin (inactive: 4000 Ul / mg; typical dose: 600 Ul / kg, effective dose mixed with nanoparticles: 60-80 Ul / kg) Oral monkeys (Rhesus macaques) were administered orally. Blood samples (1.5 ml) were taken at defined times and analyzed immediately after collection.

Drug Bioresponse: Peptide concentration in plasma was determined by non-radioimmunoassay, expressed in milliunits / ml (mUl / ml).

Treatment Efficiency: Total calcium levels in the plasma were measured with a colorimetric kit (Cobas Mira, Roche, CH). Ionized calcium levels were measured by injection into a device equipped with an ion-selective membrane (IG Radiometer, Copenhagen, DK). Results are expressed as percent change in calcium level relative to baseline.

The results are set forth in FIGS. 8-11 relating to Examples U-Z1 described below.

Example U

8. The absorption kinetics of calcitonin formulated in a composition according to the invention (Examples 2, 4, 5) and a composition according to the prior art (Examples A, B, C) after oral administration.

Example V

9. Dynamics of total calcium change in blood after oral administration of calcitonin formulated in a composition according to the invention (Examples 2, 4, 5) and a composition according to the prior art (Examples A, B, C).

Example Z

10. Dynamics of ionized calcium changes in blood after oral administration of calcitonin formulated in a composition according to the invention (Examples 2, 4, 5) and a composition according to the prior art (Examples A, B).

Example Z1

11.AUC (0-8 hours) related to oral administration of calcitonin formulated in a composition according to the present invention (Examples 2, 4, 5, 7) and prior art (Examples A, B, C) (Plasma Mechanics) Bioavailability, expressed as area for the curve).

The bioavailability of calcitonin formulated and orally administered in the composite nanoparticles according to the present invention was markedly increased (13.5 fold) compared to the formulations of the prior art by the drug bioreaction test described. In addition, it is evident that the nanoparticles according to the present invention, in addition to increased absorption, allow for the modulation of peptide absorption present in the active form in the plasma for 8 hours after administration. Moreover, the therapeutic efficiency test shows that the effect of the calcium phase on the blood is greater for the present invention (Examples 2, 4, 5) for formulations according to the prior art (A, B, C), and a simple mixture of components Example C) rather than existing composites.

Claims (20)

A pharmaceutical composition in the form of a solid nanoparticle, comprising a composite consisting of at least one lipid material and at least one amphipathic material, and a water soluble, fat soluble or poorly soluble active ingredient. The method of claim 1, wherein the lipid material is present in an amount of 0.5 to 99.5% by weight, the amphiphilic material is present in an amount of 0.5 to 99.5% by weight, based on the sum of the lipid material and amphiphilic material Composition wherein the active ingredient is present in an amount of 0.001 to 99% by weight. The composition of claim 1, wherein the nanoparticles have a diameter of 1000 nm or less, preferably 50 to 500 nm. The method of claim 1, 1) a mixture having at least one lipidic material and at least one amphiphilic material forming a complex; 2) one or more surfactants and crude surfactants; 3) dispersed / suspended aqueous phase or non-aqueous phase; 4) one or more pharmaceutically active substances; Composition comprising a. The composition of claim 4 having a time interval of presence at temperatures below the melting point of the single component. The semi-synthetic and synthetic mono- of claim 1, wherein the lipidic material contains natural fats, partially or wholly hydrogenated vegetable oils, saturated and / or unsaturated fatty acids having aliphatic chains of C ₁₀ to C ₂₂ , Di- and tri-glycerides and their polyhydroxyethylated derivatives, isopropyl myristate, isopropyl caprinate, -caprylate, -laurate, -palmitate, -stearate selected from liquid wax, ethyl From solid esters selected from oleates, fatty acid esters selected from oleyl oleate, carnauba wax and beeswax, cetyl alcohol, stearyl alcohol, lauryl alcohol, cetylstearyl alcohol and polyhydroxyethylated derivatives thereof Selected aliphatic alcohols, decanoic acid, lauric acid, palmitic acid, stearic acid, docoic acid, oleic acid, linoleic acid and their polyhydroxyethylation induction Is selected from aliphatic carboxylic acids (C ₁₀ to C ₂₂₎ as a composition it characterized in that the member selected from the group consisting. The method of claim 1 wherein the amphiphilic material is phosphatidyl glycerol, phosphatidylcholine, phosphatidic acid, glyceryl monostearate, glyceryl monooleate, glyceryl palmitostearate, triglycerides and polyhydroxylated triglycerides, fatty acids And esters of medium chain fatty acids (C ₆ to C ₁₂ ), polyethylene glycol, poly- (propyleneoxide) poly- (ethyleneoxide) copolymer, poloxamer, polyvinyl alcohol, polyacrylate, poly- (methylvinyl ether)- Maleic anhydride copolymer, chitosan, hyaluronic acid, cellulose, starch, xanthan, scleroglucan, gellan, guar gum, locust bean gum, alginate, dextran, poly-ε-caprolactone, poly-hydroxy Butyrate, polylactic acid, polyglycolic acid and copolymer. 5. The copolymer of claim 4, wherein the dispersed phase is water itself, or buffered water at different pH or ionic strengths, polyethylene glycol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid, polyoxyethylene, polyoxy Aqueous solutions of hydrophilic, water soluble or water dispersible polymers selected from propylene, dextran, xanthan, scleroglucan, gum arabic, guar gum, chitosan, cellulose and starch derivatives, sorbitol, mannitol, xylitol, short-chain mono or polyhydride A composition comprising hydroxyl aliphatic alcohol, liquid polyethylene glycol, polyglycol glycerides, propylene glycol, tetraglycol, and ethoxydiglycol. The method of claim 4, wherein the surfactant is sorbitan-ester of fatty acid, polyoxyethylene sorbitan ester of fatty acid, copolymer of polypropylene oxide-polyethylene oxide, ester of polyethylene glycol-glycerol, polyethylene glycol and acid or long chain aliphatic Esters of alcohols, polyglyceride esters, esters of saccharides and fatty acids, sodium lauryl sulfate, sodium stearate, sodium oleate, sodium glycocholate, taurodioxycholate, taurocholate, ursodioxycholate, lecithin itself, A composition characterized in that it is selected from the group consisting of hydrogenated and partially hydrogenated lecithin, phospholipids and their semisynthetic or synthetic derivatives and sorbitan esters of fatty acids. The method according to claim 4, wherein the cosurfactant is ethanol, 2-propanol, n-butanol, isopropanol, butyric acid, valeric acid, capronic acid, benzyl alcohol, decanoic acid, lauric acid, caprylyl alcohol, lauryl alcohol, acid Or esters or ethers of medium-chain aliphatic alcohols with mono- or polyhydroxylated alcohols. The method of claim 1, wherein the active water-soluble component is calcitonin, somatostatin, somatotropin (GH), LH-RH analogue, desmopressin (DDAVP), interferon, molgramostin, epidermal growth factor (EGF), nerves A composition comprising growth factor (NGF), insulin, glucagon, toxin or toxoid, antigen factor of the protein or polysaccharide type, heparin, heparin and heparinoid with low molecular weight. The composition of claim 1, wherein said active fat soluble component comprises cyclosporin, leuprolide, taxol and derivatives and etoposide. The composition according to claim 1, wherein the poorly soluble active ingredients are acyclovir and grancyclovir. a) at least one lipid material together with at least one amphiphilic material is initially eutectic or covalently dissolved at its melting point or at the melting point of at least one of the two materials if the latter can be dissolved or dispersed in the former Preparing a composite; b) water, which is useful for forming a microemulsion or for forming the microemulsion itself, by adding an aqueous solution containing at least one surfactant and a cosurfactant to the molten composite by heating to the same temperature as the molten composite, Heating the lipid and amphiphilic component to a melting point in the presence of a surfactant and a cosurfactant to form a microemulsion; c) dissolving or dispersing the active ingredient in the initial eutectic material or adding the active ingredient to the microemulsion; d) dispersing the microemulsion in water or an aqueous medium in the temperature range of + 1 ° C to + 10 ° C or in a non-aqueous solvent that can be mixed with water in the temperature range of -15 to -30 ° C to obtain a nanoparticle suspension; e) optionally washing the nanoparticles with water or an aqueous solution using an ultrafiltration or dialysis system to remove surfactants, cosurfactants and glass chemicals present in the suspension, lyophilization, filtration, spray drying or evaporation of the solvent Method for producing a pharmaceutical composition according to claim 1 or 4, comprising the step of obtaining a dry powder form through. a) at least one lipid material with at least one amphiphilic material is eutectic or covalently dissolved at the melting point of at least one of the materials at the melting point of the material itself or if the latter can be dissolved or dispersed in the former Manufacturing; b) dispersing the composite in an aqueous solution containing a surfactant, stabilizer and / or viscous agent, using a high pressure homogenization technique at a temperature that maintains the composite's melting point or its "softening" temperature or at which the composite remains in the solid state. step; c) cooling the mixture in a cooling circulation process at a temperature of + 10 ° C. to −30 ° C. to produce nanoparticles; d) optionally, washing the nanoparticles with water or an aqueous solution using an ultrafiltration or dialysis system to remove the surfactants, cosurfactants and glass chemicals present in the suspension, and freeze drying techniques, filtration techniques, spray drying techniques or Recovering the composite nanoparticles as a solid using a solvent evaporation technique, wherein the active ingredient is mixed with itself or with a substance having surface activity which aids incorporation into or adsorbs onto these surfaces, A method for preparing a pharmaceutical composition according to claim 1 or 4, comprising the step of adding a covalent solution or in a subsequent step of the process. The composition of claim 1, wherein the nanoparticles are dispersed in an aqueous suspension at a concentration of 0.1-50% w / v. The composition of claim 1, wherein the nanoparticles are formulated as capsules or globules for pharmaceutical use comprising an aqueous suspension therein. The composition of claim 1, wherein the nanoparticles are formulated in syrup form. The composition of claim 1, wherein the nanoparticles are formulated as powders in capsules for pharmaceutical use. The composition of claim 1, wherein the nanoparticles are formulated in powders, tablets, pellets or granules for pharmaceutical use.