Bilayer preparations


(57) Abstract:

The invention relates to pharmacology, specifically to dukaina lipid drug in the polar solvent, which comprises from 0.01 to 90 wt.%, preferably 0.1 to 50 wt.%, the material forming the bilayer, while the said material, forming a bilayer, is galactolipid material from cereals, consisting of at least 50% of digalactosyldiacylglycerols, and the remainder includes other polar lipids. The invention relates also to pharmaceutical compositions comprising a therapeutically active substance in a mixture with the abovementioned dvusloinye drug. Drugs have increased resistance and chemical stability. 3 S. and 9 C.p. f-crystals, 13 tab., 2 Il.

The invention relates to dvusloinye drugs of lipids in polar solvents. These drugs are used as carriers of active substances in pharmaceutical compositions, as well as in the production of food, cosmetics and agricultural products.

Natural amphiphilic lipid excipients are found only limited application in the production of pharmaceutical and cosmetic products. The reasons for this are the lack of="ptx2">

Among the natural polar lipids capable of forming a bilayer, i.e., amphiphilic lipids, often find application in pharmaceutical and cosmetic production of phospholipids. Due to its ability to form a double layer (bilayer) such polar lipids can be used to obtain various types of aggregates and particles such as vesicles and liquid crystals, which are used in various fields of technology.

However, the use of lipid phospholipid gels of nature is only limited pharmaceutical technology, mainly due to their lack of heliopause ability and poor chemical stability. Phosphatidylcholine from egg yolk or soy beans as the most frequently used natural lipid, forming a double layer (bilayer), has only a small lipophilic for optimum swell in water and education flexible double layers (bilayers), built from a liquid crystalline lamellar structures.

Because liposomes are dispersion bisloinoi or lamellar phases in excess of an aqueous solution, the method of use of lamellar phases of phospholipid nature through education lipoamide material for a suitable period of time requires a high expenditure of mechanical energy.

Natural phospholipids, such as phosphatidylcholine from egg yolk, are highly unsaturated substances. And unsaturation of acyl chains of the phospholipid is a condition for the formation of liquid crystalline lamellar phase at room temperature. In addition, this means that the double-layer membrane formed by natural phospholipids, have a high permeability for water-soluble drugs, because the acyl chains are disordered liquid state. Liposomes formed from natural phospholipids, are characterized by low kapsulirujushchej efficiency in connection with the course included medicines through the liposomal bilayers. Usually, if you spend the inclusion of medicines in natural phospholipid liposomes, they should be stabilized by the addition of a large amount of cholesterol in the amount of 30-50 mol.% of the total composition of the lipid composition.

The same is true also in respect of other active substances, not drugs, which are also different considerations may be incorporated into liposomes or other bilayer structure.

Vesicles and liposomes of phospholipid nature usually have a very short lifetime Polley system (RES) of the liver and spleen. To circumvent this difficulty, the liposomes should sterically stabilize the addition of gangliosides containing some carbohydrate units, or hydrophilic polymers such as polyethylene oxide or pullulan. The last agents usually associated covalently with phosphatidylethanolamine. In principle, this approach is very effective, as modified liposomes can avoid absorption of REFs. However, the practical implementation of this method is fraught with shortcomings as an economic nature, and from the point of view of security associated with the need to make liposomal formula additional components or natural and rare, which causes it to rise, or synthetic, which are not always biologically compatible.

The use of phospholipids and other polar lipids for the production of liquid crystals and liposomes is well known.

Patent EP-B1-0126751 reveals the composition of controlled release, which may include amphiphilic substance related, in particular, to galactolipids, phospholipids and monoglycerides. The above-mentioned patent relates to cubic and reversible hexagonal liquid crystalline phases, stabilnosti gel type. The alcohol represented by ethanol, 1-propanol or 2-propanol. The content of phospholipids varies from 15 to 30 wt.%. In addition, the patent discloses the use of phospholipid compositions for the preparation of liposomes upon dilution with an aqueous solution, and gelaterias drugs.

Patent WO 91/04013 discloses a hybrid nicolemarie lipid vesicles containing lipid bilayer phospho - or glycolipid and non-ionic, anionic or zwitter-ionic surfactant. For your preferred glycolipids include cerebrosides, gangliosides and sulfatide, each of which belongs to the family of glycosphingolipids.

Glycosylglycerols represent the views of glycolipids, which, as you know, are part of the cell membranes of plants. The most common two types, based on galactose: monogalactosyldiacylglycerol - MGDG (MGDG) and digalactosyldiacylglycerols - DGDG (DGDG), which make up 40% of the dry weight thylakoid membranes.

Plant glycolipids are carbohydrate units, mainly galactose associated with glycerin. In MGDG 1-position of galactose ring connects connection with glycerin and DGDG between the exact sulfoquinovosyldiacylglycerol - SQDG (SQDG), which contains rather sulphonate, and not the hydroxyl group, which is associated with the 6th carbon atom termination desoxyglucose balance. Most plant glycolipids can be described by the formula

< / BR>
where R1and R2independently of one another are saturated or unsaturated residues of fatty acids containing 2-24 carbon atoms and 0-6 double bonds, esterified with hydroxy acid that gives astride or hydrogen; carbohydrate is a monosaccharide unit; n = 1-5; and R3denotes hydroxyl or sulphonate group.

In interaction studies of glycosylglycerols with water and other polar solvents, the authors found that specific glycolipids material from cereal characterized by such features that simplify the use of the above material as a carrier, first and foremost in the case of pharmaceutical compositions, but also for the preparation of other drugs, in particular, cosmetic, agricultural, diet and food purposes.

It is well known that cereal lipids can interact with water due to the presence of some phase G. Jayasinghe et al. , J. Disp. Sci. Technol., 1991, vol. 12, 443-451).

In document SE 9400368-8 disclosed industrially applicable method of obtaining from plants, mainly of cereals, glycolipids material using solvent extraction and chromatographic separation. Thus prepared glycolipids material can then be used as the amphiphilic material in the production of pharmaceuticals, cosmetics and food products.

The invention relates to dukaina lipid drug in the polar lipid solvent consisting of 0.01 to 90 wt.%, preferably 0.1-50 weight. percent of forming a double layer (bilayer) of material in a polar solvent, which is characterized in that the said forming a double layer material is galactolipid material from cereals, consisting of at least 50% of digalactosyldiacylglycerols, and the remainder is filled with other polar lipids.

In the preferred drug galactolipid material consists of about 70-80% of digalactosyldiacylglycerols, and 20-30% from other polar lipids.

In another preferred drug galactolipid material is up to 100% of digalactosyl is B> and R2independently of one another are saturated or unsaturated residues of fatty acids containing 10 to 22 carbon atoms and 0-4 double bonds, or hydrogen; and R3denotes hydroxyl or sulphonate group.

As the preferred fatty acid residue, denoted by R1and R2you can mention natural acyl groups of fatty acids, such as, for example, residues of saturated palmitic (C15H31CO; 16:0) and stearic acids (C17H35CO; 18:0); monounsaturated oleic acid (C17H33CO; 18: 1); and polyunsaturated linoleic (C17H31CO; 18: 2) and linolenic acids (C17H29CO; 18: 3). Residues of fatty acids may also include oxyacids, United with glycerin in part through hydroxyl groups esterified with other fatty acids, the so-called astride.

Other polar lipids included in galactolipid material is a mixture of various Glyco - and phospholipids, such as MGDG and phosphatidylcholine. The composition depends on the source material and the production method of galactolipids.

The specific ratio of components in galactolipids the material when used in different purposes, the maximum effect is achieved with a high content of DGDG as the most important component, forming a bilayer.

Galactolipid material can be preextraction from almost any kind of plant material. Preferred plant material are seeds and grain from the grain and cereals, for example wheat, rye, oats, maize, rice, millet and sesame. Oat groats and wheat gluten are characterized by a high concentration of lipid and therefore they are very well suited to this method of obtaining. Digalactosyldiacylglycerols in galactolipid material can also, if applicable, be of synthetic origin.

Galactolipid material can be used as a polar lipid component in a variety of ordered solutions, where the lipid forms ordered particles dispersed in a diluted, mixed randomly solutions containing such polar solvents as water, ethanol, glycerin and other polar solvents and mixtures thereof. Molecular geometry DGDG resembles a truncated cone, which makes possible the creation of flexible bilayers, representing in aqueous solutions under physiological conditions lamellar liquid crystalline phase, and liposomes or vesicles.

The galactolipids is the addition of water to the galactolipids leads to the spontaneous formation of clear viscous gel. The gel consists of a lamellar liquid crystalline phase (Lain which of lipid bilayers alternating with water in the lamellar structure. Laphase and are easily detected in a polarizing light microscope, thermodynamically stable.

Swelling occurs relatively slowly due to the high viscosity of the lamellar liquid crystal structure of the gel; within 24 h of slow aggregation is possible to prepare a transparent homogeneous samples containing only 10 wt.% aqueous solution. Immediately after its formation, the gel has an extremely high stability to chemical and microbial degradation, which allows to maintain its physical integrity for a long period of time.

The alternating layers of galactosylceramide and polar solvent make the gel structure is suitable for inclusion as lipophilic and hydrophilic biologically active substances. Lamellar structure has a relatively low viscosity at high velocities, which allows to be injected gel with a syringe and fine needle. The drug can be used for the delivery of drugs in different parts of the body of the animal and man.

In its preferred embodiment the invention relates to a gel preparations, including 25-90 weight. % galactolipid material in a polar solvent.

The gels can be easily prepared by adding a polar solvent such as water or an aqueous solution, to the dry galactolipid material to achieve a final lipid concentration in the range of 25-90 weight. %. Mixture is left to swell for 1-24 h at room temperature with gentle stirring in an appropriate container, i.e., either in a glass flask, or in an open beaker. Gels can be prepared in a glass tube under stirring rod and further centrifugation at room temperature.

Gels are pseudoplasticity and are characterized by a remarkable stability in relation to its physical appearance and resistance to the action of microbes. The viscosity of these gels slightly exposed to moderate temperature changes, so they can be transferred directly from the refrigerator into the syringe or other device to call Eleusa the release of the active ingredient, and more effectively than gels derived from phospholipids.

It is shown, except that the gels of the present invention may include a wide range of therapeutically active components, including lipophilic components, hydrochloride, nitrates, amphiphile, proteins and peptides.

In accordance with another preferred variant of this invention relates to a liposomal preparation containing 0.01-25 wt.% galactolipid material in a polar solvent.

Best feature peculiar to galactosylceramide, is the presence of galactose units comprising a polar head group in each lipid molecule that is capable of sterically stabilized liposomes, thus ensuring a long lifetime in the blood stream.

Liposomes in the form of a multilayer vesicles receive by direct hydration. To dry galactolipid material type polar solvent, such as water or an aqueous solution, which leads to achievement of a final concentration of lipid in the range of 0.01-25 wt.%. The mixture is left for swelling and equilibration at room temperature with gentle stirring for 1-24 h, Polustrovo solvent to the gel, obtained in accordance with the foregoing, i.e., just when diluted gel.

Single-layer vesicles obtained by dispersion of multilayer vesicles, i.e., via extrusion through membranes or homogenization under high pressure.

The unusual ability galactolipid material to swell very easy to prepare on the basis of liposomal dispersions and water gels. For example, the possibility of spontaneous formation of liposomes in water suddenly turned out to be very useful for the preparation of liposomal dispersions even at large scales. This procedure is markedly different from those necessary for the formation of liposomes from phospholipids, which uses organic solvents, such as chloroform, ether, ethanol or a combination thereof. Well-known problems arising from the transfer of conventional treatments receiving liposomal drugs in large-scale production, which was developed many options. The present invention provides a simple and reproducible method of obtaining liposomal dispersions, the importance of which increases especially in the practical use of liposomes, for example as carriers persuade unexpectedly high encapsulation efficiency.

In addition, it was shown that liposomes of the present invention have an amazing pronounced ability to increase the life time of the active ingredient, even if the vesicles were formed in the solution of these components, and thus, only part of these components is encapsulated in vesicles.

It was shown further that the liposomes of the present invention reduce the toxicity powerful anti-cancer drug, without reducing its pharmacological effect.

Liposomes of the present invention have bioadhesive property and can therefore be useful to solve the adequate availability of the active component to certain biological surfaces, such as the cornea and mucous membrane.

Liposomes of the present invention can include active components in a very wide range, including lipophilic components, hydrochloride, nitrates, amphiphile, proteins, peptides, etc.

In accordance with the present invention can be used synthetic deglycosylation derived from galactose or any other monosaccharide units, such as glucose, as well as natural is genizah, other than galactose, such as glucose.

Galactolipid material

Galactolipid material obtained from different cereals on the following method and then used for preparation and pharmaceutical compositions of the present invention, as shown in the examples. In the description of the % denotes weight %, unless otherwise specified. The proportion of solvents in mixtures of solvents specified in volumetric units.

Galactolipid material from oats

200 kg of oat grain (Kungsornen AB, Sweden) milled and extracted with 1000 l of 95% ethanol at a temperature of 70oC for 3 h in the extractor with stirring. The slurry is centrifuged in a warm state and is separated from the solid particles. The liquid fraction is evaporated at a temperature of 60oC, receiving about 10 kg light brown oil.

The oil is applied to a stainless steel column with 6.25 kg of silica gel [Matrix of Silicone Si (Matrex Silica Si), particle size 20-45 mm, pore size 60 received from Amicon Bldg., USA]. The column temperature is 50oC. Then to remove all non-polar lipids column was washed with 30 l of a mixture of hexane: isopropanol, 90:10.

Galactolipid material elute with pariwana this fraction gives about 700 g DGDG the main class of lipids. Then galactolipid material is dispersed in water and subjected to freeze drying, receiving non-dusting powder.

Enrichment DGDG from galactolipids

50 g of galactolipids derived from oats, as described above, which have DGDG content of about 70%, is dissolved in 250 ml of a mixture of hexane:isopropanol, 70: 30, to a final volume of 300 ml. of the resulting solution contribute on a column of silica gel (110 g) and elute the less polar components of the mixture of 1 liter of hexane: isopropanol, 70: 30. Enriched DGDG fraction elute 2 l of acetone. Acetone fraction is evaporated and dried at a temperature below 0oC. Obtain 17 g of practically pure DGDG product.

Hydrogenation of galactolipids

200 g galactolipids mixture obtained from oats by the method described above, is dissolved in 2 liters of warm isopropanol. At the bottom under pressure reactor [model N M; Parr instruments Co., USA (Parr Instruments Co.)], equipped with two handles on the blades of the agitator, placed 15 g of the catalyst of palladium on coal [Pd 15%, humidity 53%, Engelhardt, Rome, Italy (Engelhardt Rome's.r.i.)]. After this, the solution is transferred into a sealed reactor, which created an atmosphere of nitrogen, to reduce the risk of fire. Resuh, and then three times under pressure is passed hydrogen [place 4.5, AGA Gas AB, Sweden (Plus 4.5, AGA Gas AB)]. The hydrogen pressure is maintained at the level of 6 bar, the stirrer is set at 600 rpm and heated the mixture up to a temperature of 70oC. it takes 14 minutes to the reaction mixture took the desired temperature. The process of hydrogenation is conducted for 6 h, after which the reaction product is filtered through a filter with pore size of 0.45 μm to remove particles of charcoal and palladium. The solvent is evaporated in a rotary evaporator, the residual material is dispersed in 1600 ml of deionized water and freeze-dried.

Output hydrogenated galactolipids after filtration and freeze drying is 155, the Process of hydrogenation is followed by gas chromatography; hydrogenated product are detected only saturated fatty acids.

The galactolipids from wheat gluten

1 kg of wheat gluten powder [AB Schonbrunner Sweden] is extracted with 4 l of 95% ethanol in a beaker at a temperature of 70oC for 3 hours and Then under a pressure of 400-500 kPa filtered slam, and the filtered cake was washed with 1 l of warm 95% ethanol. United atenolil put on a stainless steel column, containing 45 g of silica gel [Matrix of Silicone Si (Matrex Silica Si), particle size 20-45 μm, pore size 60 received from Amicon Bldg., USA]. Then the column was washed with 700 ml of a mixture of hexane:isopropanol, 90:10, to remove neutral lipids.

Removal of MGDG and other polar lipids column is sequentially washed with 1000 ml of a mixture of hexane:isopropanol, 70:30. The elution of DGDG carried out with 1000 ml of pure acetone. After evaporation receive approximately 4 g of practically pure product DGDG.

The galactolipids from rye

100 g rye flakes [Kungsleden AB (Kungsrnen AB), Sweden] stirred for 60 min in a mixture of industrial hexane and isopropanol, 90:10. Slam filtered and evaporated to obtain 0.5 g of the polar lipids. The residue is dissolved in 10 ml of a mixture of hexane and isopropanol, 70:30, bring on a series of three columns with silica gel [Sep-Pak of Silicone place (Silica Seppak plus) Millipor Bldg., USA], washed with 20 ml of the same solvent mixture and elute 15 ml of acetone. The eluate is evaporated and freeze-dried, receiving 47 mg of galactolipids.

Chemical and physical characteristics of different galactolipid materials

The analysis belonging to the lipid class

The analysis belonging to the lipid class opening, modified using diol (Lichrospher 100 DIOL, 5 µm, 250 x 4 mm - EXT. dia., E. Merck, Germany). Column placed in a water bath with a temperature of 75oC. Analytical system consists of a pump HPLC CM 4000 [LDC/Milton Roy LDC/Milton Roy, USA] and injector, model 7125, equipped with an injection loop of 20 μm [Readin Inc. (Rheodyne Inc.), USA] . Use of evaporative light-scattering detector sedex 45 (Sedex, S. E. D. E. R. E, France) equipped with a camera to spray sedex 55 installed on the temperature and pressure of the inlet air within respectively 97oC and 2.0 bar.

During the analysis the velocity of the moving phase is 1 ml/min Gradient binary solvent supported linear for 25 min, then it is replaced, starting with 100% A to 100% B, where A = hexane:isopropanol: n-butanol: tetrahydrofuran: isooctane: water, 64:20:6:4.5:4.5:1, and B = isopropanol: n-butanol: tetrahydrofuran: isooctane:water, 75:6:4.5:4.5:10. All solvents contain ammonium acetate, 180 mg/l

The data collection and processing were performed on the system Genkisoft date (GynkoSoft Data), version 4.22 (Softron GmbH, Germany). In a typical case, the injection quantity is 100 μg. Identification is based on comparing lie is), Sweden]. This system does not detect volatile components. Quantification is performed on the basis of the calculation of the peak area.

Zeta-potentials determined in dilute aqueous dispersions of galactolipid using the device Zetasizer 4, manufactured by Malvern instruments Ltd., UK (Zetasizer 4, Malvern Instruments Ltd.).

In table. 1, as in table. 2, uses the following abbreviations:

o-CH = galactolipids from oats;

o-d-CH = hydrogenated galactolipids from oats;

on-DGDG = enriched galactolipids from oats;

p-CH = galactolipids from wheat;

p-DGDG = enriched galactolipids from wheat;

R-CH = galactolipids from rye.

Analysis of fatty acids

The definition of the profile of fatty acids was carried out using a gas chromatograph after interesterification of lipids in methyl esters of fatty acids. These esters are separated and analyzed quantitatively by gas chromatograph with capillary column Varian 3500 (Varian 3500 Capillary Gas Chromatograph) using in this case capillary column 30 m x 0.25 mm EXT. dia. (DB-WAX, J&W Scientific, USA), column injector and a flame ionization detector. As the carrier gas used was helium. Vychisleny adding 1 mg of lipid sample to 2 ml of a mixture of dimethyl carbonate:isooctane, 1:1. Add 1 ml of a solution containing 2.3 g of sodium dissolved in 200 ml of ethanol and vigorously stirred under test tubes for 30 s, then leaving them at room temperature for 15 min to complete the reaction. Add 3 ml of water, mix tubes with the test compound, followed by centrifugation at 2 x g. Compliance with the following conditions required for separation, the chromatograph is injected with 0.5 μl of the organic layer. Thermostatic device column programmed at the following temperature: initial temperature 130oC (2 min), in the next 10 minutes the temperature is raised to 150oC (speed 30oC/min) and up to 220oC (speed 3.2oC/min). The injector temperature is 130oC and the detector temperature 250oC. the Source gas flow has a speed of 2.7 ml/min. and the Obtained results are expressed as the normalized weight percent using the method of external standard. Relatively minor components for which standards are either not available or not sufficiently clean, the correction factors are missing.

NMR spectroscopy of digalactosyldiacylglycerols

Spectrometer Bruker AM-4 is to determine the natural distribution13C, at a frequency of13C, equal 100.614 MHz. Angle pulse 36othe interval between repetitions of the pulse is equal to 1.0, and the resolution is 1.526 Hz at this point. The analysis used the extension line 3 Hz. Samples (10-40 mg) were diluted with a mixture of 730 ál DMSO-d6[Aldrich Chemical Corp. (Aldrich Chemical Corp., Inc.), USA] and 20 μl of D2O [Aldrich Chemical Corp. (Aldrich Chemical Corp., Inc.), USA] and transferred into a test tube (EXT. dia. 5 mm) for analysis by NMR (table. 3).


Example 1

Receiving water gel

Galactolipid material derived from oats, mixed with different amounts of water in order to determine the swelling properties in water. The water-lipid samples are prepared in parts by weight in a glass tube. Samples alternately stir stick and centrifuged at room temperature before the formation of a fully homogeneous systems. After keeping at room temperature for six months doing a physical examination of the samples. Lamellar liquid crystalline phase is determined using light polarization microscopy. The results are presented in table. 4.

The data show that when the content of galactolipids

According to the method of example 1 is prepared galactolipids gel containing 55% water. Viscosity measurements carried out using a rheometer Bohlin FAURE (Bohlin VOR Reologi AB, Sweden), equipped with a concentric cylinder (C14; torque element: 91 g/cm) at various shear rates and temperatures. The viscosity is measured after 24 h after manufacture. Viscosity values of the stationary flow (Pas) are given in table. 5.

It was concluded that the sample is soluble under the action of shear material (pseudoplastic), i.e., its viscosity depends on shear rate, which demonstrates the rheological behavior of lamellar liquid crystalline phases. Furthermore, increasing temperature leads to a slight decrease in viscosity, indicating the absence of phase transitions in the investigated temperature range. After keeping the gel at room temperature for 18 months spent his visual inspection. Was not observed microbial growth. Physical appearance when standing not changed, which is surprising, because it was not taken any precautions related to the storage temperature, the addition of antioxidants, preservatives, etc.

Privedennom call for example, the hydrolysis of acyl chains or microbial degradation.

Example 3

Testing release

In vitro galactolipids and phospholipid gels containing the initial state 60% lipid and 50 mm of water-soluble dye methylene blue, determine the release of the above-mentioned dye. Galactolipid material derived from oats. Phospholipid material is a chromatographically purified phosphatidylcholine from soybean (s-PC; Karlshamns Lipidemic AB, Sweden). Diffusion medium is filtered through the membrane water equilibrated at 37oC. the Diffusion cell consists of a membrane tubing made from regenerated cellulose (Spectrum/Long; segments with a molecular weight of 6000 - 8000; slice width 1.0 cm), which contains about 2.5 g of gel. The tube is immersed in a thermostatted glass (EXT. dia. 10 cm) containing 250 g environment when fixing its position at a distance of 3 cm from the bottom. The beaker placed on a magnetic stirrer with a speed of rotation 200 rpm at certain points In time are selected aliquots environment and examine them on the spectrophotometer at a wavelength of 665 nm. As standard proberaum the release of methylene blue which was notable release of the dye from galactolipids gel, while s-PC gel freed 1.3%. After 5 h galactolipids gel released only 0.13% of the contained dye that was approximately 12 times lower than the corresponding figure for the s-PC gel.

The results show that for galactolipids of the drug is characterized by slow release of biologically active material in the case of the introduction of its in vivo and in this regard it can be used as a drug, creating in the body of the depot.

Example 4

Test release

On galactolipids gel, initially containing 60% lipids, 15% remoxipride hydrochloride and 25% water in vitro test is done on the release of water soluble drugs - hydrochloride remoxipride monohydrate (Astra AB, Sweden).

The role of diffuse environment performs phosphate buffer (pH 7.4; ionic strength 0.05 M), balanced at a temperature of 37oC. the Diffusion cell consists of a membrane tubing made from regenerated cellulose (Spectrum/Then 3; molecular weight cut 3.500; width of cut of 1.0 cm, length 4 cm), which contains about 1 ml of gel. The tube is closed at both ends by clamps and placed on the bottom of thermostatted bath for dissolution (Sotax AT 6; EXT. dia. 10 cm), rpm At the specified time a sample is taken for analysis by volume of 1 ml. Selected sample replace 1 ml of buffer. The concentration remoxipride determined by liquid chromatography with treatment phases using column m-Bondapak C18 (m-Bondapak C18). Used eluent is a mixture of phosphate buffer, pH 1.8, and acetonitrile, 4: 1. Use spectrophotometric detector, and the measurement is carried out at a wavelength of 254 nm.

The gel was allowed to maintain a remarkably low level of release of incorporated drugs. After 2 h of galactolipids gel was released less than 2% of the incorporated drug. And about 3% is released after 4 hours

The data obtained suggest that galactolipids formula suitable for parenteral use, for the purpose of creating a body of depot biologically active material with subsequent long-term release, as it was shown in example remoxipride hydrochloride.

Example 5

The formation of liposomes

Liposomes in the form of a multilayer vesicles receive and investigate in the following way. The galactolipids of the oats, add water to obtain a final concentration of 1.0 ucheniem liposomal dispersions.

To obtain liposomes in the form of single-layer vesicles part of the dispersion is transferred into a glass test tube and destroy in nitrogen atmosphere at a temperature of 0oC using an ultrasonic disintegrator [XL-2020; Hit systems Inc. (XL-2020; Heat Systems Inc.), USA], provided with a device with a tip for the selection of microprobe. Use the following modes of operation: control output 3.5; processing time 3 x 2 min, pulse mode 2 x 4 minutes

The distribution of the received liposomal dispersions according to the size of the particles was determined by dynamic light scattering [Zetasizer 4, Malvern instruments (Zetasizer 4, Malvern Instruments, UK] at an angle of 90oand at room temperature, using a calibrated cell ZET5110 by multimodal analysis. The following results were obtained, which are presented as average values of Z in the table. 6.

Using an optical microscope [40-100x, Olympus CH-2 (40-100x, Olympus CH-2)] examine the obtained dispersion before and after sound. A small amount of the liposomal dispersion was placed on a glass slide. Then consider the sample between crossed polarizers. Found only neizvestnye liposomes, which have a characteristic shape of the Maltese to the Onna microscopy of frozen fractures.

These data demonstrate excellent ability of galactolipids to form a multi-layered vesicles using a simple method aimed hydration and without the addition of volatile organic solvents or co-surfactants. Small single-layer vesicles are easily formed and then when ultrasonic disintegration compliance with the above conditions or with the use of traditional methods, such as extrusion through a polycarbonate membrane.

Example 6

The efficiency of encapsulation

We study the efficiency of encapsulation of the dye included in the vesicles galactolipid material derived from oats. Galactolipid material is subjected to directed hydration in 20 mm aqueous solution of fluorescein at a concentration of 4.8%. The dispersion is left to swell at room temperature for 24 h

Loaded with dye vesicles are separated from those that are not dye by gel-filtration on a column of Sephadex G 50 (height 60 cm, EXT. dia. 1.5 cm) at room temperature. As eluent used EDTA buffer (1 mm EDTA, 5 mm Tris, 150 mm NaCl), brought to pH 7.4. Concentrated liposomal dispersion, segreteria on the absorption at the wavelength of 240 nm using UV-spectrophotometer, equipped with microcache and recorder, and then collect fractions on automatic collector. Two factions - loaded dye vesicles and the solution unabsorbed dye is collected separately and brought to a certain extent. Spectrophotometrically at a wavelength of 258.2 nm to determine the concentration of the dye and is calculated on the basis of these data the values of the absorbed amount and the efficiency of encapsulation. The following results were obtained: amount absorbed is 2.1 ál/mg lipid, and the efficiency of encapsulation is equal to 11%. The particle size is based on the definition of 509 nm (average Z-value).

Direct hydration of galactolipids leads to the formation of multilayer vesicles, as shown in example 5. The above data indicate that these particles are characterized by a much higher value of the absorbed amount and efficiency of encapsulation than conventional vesicles based on phospholipids, which, as follows from the messages that have the value of the absorbed volume of about 0.5 μl/mg lipid.

Method direct hydration used to produce multilayer vesicles, technically simple and fast and therefore suitable for priolsec values of the absorbed volume, which indicates a significantly higher efficiency of encapsulation, which previously using phospholipids to obtain a multilayer vesicles was low, which was considered as the main disadvantage of the method.

Example 7

The formation of aqueous dispersions

To test the swelling in water enriched galactolipid material DGDG of oats mixed with water. The water-lipid samples are prepared according to the method described in example 1. The results obtained are summarized in table. 7.

As in the case of raw material, the swelling properties in water enriched material was very good, it was a homogeneous dispersion.

Example 8

The formation of aqueous dispersions

Hydrogenated galactolipid material of oats mixed with water to determine the swelling properties in water. The water-lipid samples receive according to the method of example 1 (table. 8).

As in the case of dehydrogenation material, the swelling properties in water, hydrogenated material was extremely good. Specified material contains only the remains of saturated fatty acids that determines the ability to create highly ordered crystalline structure of the metry shows the melting point of the chain in the hydrogenated material, having a form of a dispersion in water is about 55oC. the Corresponding value for dehydrogenation material is sufficiently below the 0oC.

Example 9

Preparation of a viscous non-aqueous dispersion

Viscous dispersion receive in accordance with the following formulations

Ingredient - %

The galactolipids - 10.0

Glycerin, 99% - 90.0

Ingredient - %

The galactolipids - 20.0

Glycerin, 99% - 80.0

The galactolipids derived from oats, and glycerin alternately stir stick and centrifuged at room temperature until the formation of highly viscous, homogeneous and clearly isotropic liquids. Both fluids are composed of a dispersion of lamellar phases in glycerol, i.e., multilayer vesicles, as shown by the study of their structure in light polarizing microscope. After keeping the samples in a period of one year at room temperature, conducting a physical examination. Regardless of the concentration of galactolipid not found any precipitate that indicates the presence of a stabilizing effect of glycerol on the dispersion of vesicles.

Example 10

Preparation of non-aqueous prep the dust, such as DL-cysteine and N-acetyl-L-cysteine, can stimulate the treatment and to prevent the recurrence of duodenal ulcers in humans. Stomach ulcers can be caused by ischemia and harmful substances such as ethanol and acetylsalicylic acid, which impress and remove the mucosa of the duodenum.

Prepare the medication using the following ingredients:

Ingredient - %

The galactolipids of oats - 10.0

N-acetyl-L-cysteine - 10.0

Glycerin, 99% - 80.0

N-acetyl-L-cysteine dissolved in glycerol under mild stirring and heated to approximately 60oC in an open beaker. After that add galactolipid material, and the obtained clearly transparent liquid is transferred into a glass container with a plastic lid. The drug, which, as demonstrated by his research in light polarizing microscope, the dispersion was stored in a refrigerator for more than 6 months.

The solvent was chosen glycerin due to the fact that substances containing sulfhydryl groups, such as, for example, N-acetyl-L-cysteine, unstable in aqueous solution and can be converted into substances which are not pharmacologists who was shown in vivo studies on rats, he has a protective effect on gastric mucosa.

According to the methods described in examples 11-14 were prepared with various local drugs with anti-inflammatory agent is hydrocortisone. Galactolipid material derived from oats. All compositions are stable when stored for more than 2 months at room temperature.

Example 11

Ingredient - %

The galactolipids - 10.3

Hydrocortisone - 1.0

Water - 88.7

Hydrocortisone and galactolipids are thoroughly mixed using a vortex mixer. After adding water, the drug is subjected to vortex mixing, centrifuged and gently heated to obtain a fine dairy dispersion.

Example 12

Ingredient - %

The galactolipids - 22.1

Hydrocortisone - 1.2

1-propanol - 16.1

Water - 60.6

Hydrocortisone dissolved in 1-propanol at low heat and stirring. After adding water and galactolipids mixture is subjected to vortex mixing, centrifuged and gently heated to obtain an opaque very viscous gel.

Example 13

Ingredient - %

The galactolipids - 18.9

Hydrocortisone - 0.8

1,2-propandiol - 26.3

Water - 54.0
water and galactolipid material mixture is subjected to vortex mixing, centrifuged and gently heated to obtain a yellowish almost transparent gel.

Example 14

Ingredient - %

The galactolipids - 26.8

Hydrocortisone - 1.2

Ethanol - 20.7

Water - 51.3

Hydrocortisone dissolved in ethanol with gentle heating and stirring. After adding water and galactolipids mixture is subjected to vortex mixing, centrifuged and gently heat until a light brown transparent gel.

In examples 15-17 describes the various anhydrous local products. Here, just as before, as a model drug selected hydrocortisone. The galactolipids derived from oats. All preparations are stable if stored more than 2 months at room temperature.

Example 15

Ingredient - %

The galactolipids - 7.0

Hydrocortisone - 0.8

Glycerin, 99% vol. - 92.2

The galactolipids is dispersed in the glycerin. After obtaining a homogeneous gel phase add hydrocortisone. The mixture is slightly heated and then mixed using a vortex mixer. The resulting preparation-suspension is a yellowish viscous gel containing melkodispersionnyy solids hydrocortisone.

Example 16

Example 17

Ingredient - %

The galactolipids - 21.8

Hydrocortisone - 1.1

Propanol - 14.5

Glycerin, 99% vol. - 62.6

Hydrocortisone partially dissolved in propanol with gentle heating and stirring. After adding galactolipids, followed by vigorous stirring contribute glycerol. Then the composition is subjected to vortex mixing, centrifuged and heated to obtain a yellowish gel phase. The gel contains fine particles of hydrocortisone.

Example 18

An antifungal drug for vaginal introduction

Ingredient - %

The galactolipids - 45.6

Miconazole nitrate - 1.8

Water - 52.6

Miconazole nitrate and galactolipids derived from oats, mix well in a vortex mixer. After adding water the composition is subjected to vortex mixing and shaken to obtain a brownish homogeneous very viscous gel.

Example 19

Antibacterial drug in the form of a bandage on the wound

Ingredient - %

The galactolipids - 42.8

Doxiciclina hydrochloride - 1.7

Water - 55.5

Doxiciclina guy who sa the mixture is stirred in a vortex mixer and shaken until a light brown very viscous gel.

Example 20

Antibacterial drug for insertion into the ear canal

Ingredient - %

The galactolipids - 2.0

Doxiciclina hydrochloride - 2.0

Water - 96.0

Doxiciclina hydrochloride is dissolved in water to obtain a yellow solution. After adding galactolipids derived from oats, the mixture is stirred in a vortex mixer to obtain a yellow breast dispersion of vesicles loaded with doxiciclina.

Example 21

Anti-diabetic composition for nasal injection

Ingredient - %

The galactolipids - 3.5

A solution of insulin, 100 IU/ml [Actrapid human, Novo Nordisk AS (Human Actrapid, Novo Nordisk AS), Sweden] - 96.5

The galactolipids derived from oats, hydratious in the solution of commercial insulin with gentle stirring for 24 h the resulting dispersion is transferred into a normal bottle with a pump for the intranasal route, which can be obtained thin aerosol jet.

Example 22

Spermatocidal track

Ingredient - %

The galactolipids - 22.5

Nonoxynol - 5.0

Water - 72.5

The mixture of the three components is placed brownish transparent gel.

Example 23

Analgesic composition for rectal injection

Ingredient - %

The galactolipids - 43.4

Paracetamol - 2.9

Water - 53.7

Derived from oats galactolipids and paracetamol mix well. After adding water, the drug is alternately stir stick, slightly heated and then centrifuged at room temperature until a yellow-brown very viscous gel.

The viscosity galactolipids compositions have only a minor impact moderate changes in temperature. The composition stored in the refrigerator, you can easily instantly transferred into the syringe or similar device and then enter rectal, when this composition is heated to body temperature, does not lose its characteristic viscosity and consistency.

Example 24

Antiglaucoma composition for ocular injection

Ingredient - %

The galactolipids - 1.00

Thymol maleate - 0.34

Water - 98.66

Derived from oats galactolipids is dispersed in one part of water and leave overnight to swell with moderate stirring at room temperature. Then add dissolved in the remaining amount of water thymol maleate, transfer received liposome 0oC using an ultrasonic disintegrator [XL-2020, Hit systems Inc., USA (XL-2020; Heat Systems Inc.), control output 4], provided with a device with a tip for the selection of microprobe.

Get in the transparent dispersion containing a small single-layer vesicles and drug pharmacologically effective amount, which can be used as eye drops. While the galactolipids increase the viscosity of the obtained composition and improve its bioadhesives that can lead to improved bioavailability of drugs due to increased detention time in the cornea.

Example 25

The inclusion of the lithium salt of gamma-linolenic acid in galactolipids liposomes

Liposomal galactolipid composition comprising a lithium salt of gamma-linolenic acid, which is used as an anticancer agent, is prepared as follows:

Ingredient - %

Li-GLA - 1.5

Enriched galactolipid - 10.0

Glycerin in water of 2.3% To 100.0

Li-GLA content of gamma-linolenic acid in 75% receive from Callanish Ltd., Scotland (Callanish Ltd.). Enriched galactolipid material derived from oats, and Li-GLA mixed Diya). After a very strong stirring at 12000 rpm for 30 with liposomal dispersion is homogenized under a pressure of 86 MPa for 3 min [Emulsiflex-C30, Avestin Inc. Canada (EmulsiFlex-C30, Avestin Inc.)]; 1.5% concentration of Li-GLA corresponds to 53 mm.

Hemolytic effect of liposomal dispersions determined in vitro according to the method described in test 6.

Example 26

Obtaining a dispersion containing 10% L-tyrosine

The dispersion is prepared as follows:

Ingredient - %

The galactolipids of oats - 6.4

L-tyrosine - 10.0

Water - 83.6

All the ingredients are mixed and subjected to intense stirring at 15,000 rpm for 4 min before the formation of a homogeneous dispersion. The resulting dispersion is stable for several weeks after preparation.

In Fig. 1 shows a photomicrograph under polarized light of liposomes obtained in example 9 out of 10 vol.% of galactolipids in glycerol at magnification X100. For liposomes characteristic spherical shape with Maltese crosses.

In Fig. 2 shows the neuromuscular-blocking effect of local anesthetic on the model rats with long vnutriobolochechnoe implant in accordance with the data of the test 3.

Biologicalsystems invention conducted the following tests.

The galactolipids of oats mixed with water for injection to obtain a 10% gel and put it in the form of a dose corresponding to 0.5 ml per animal, on the intact skin of male 6 new Zealand white rabbits (New Zealand White rabbits) and keep it under semi-hermetic bandage for 4 hours Examination of the skin for the presence of erythema and edema carried out after 1, 24, 48 and 72 h after removal of the dressing. Then calculate the average values based on the evaluation of skin lesions after 24, 48 and 72 hours the Results are shown in table. 9.

Based on the above data we can conclude that the application of galactolipids gel does not cause significant irritation to the skin.

Test 2

Assessment clearance galactolipids liposomes in vivo

Getting DGDG labeled with3H-fatty acid

500 mg of galactolipids from oats have been labelled with tritium in the catalytic reduction of double bonds in fatty acids with gaseous tritium [Amersham Tritium Labeling Service (Amersham Tritium Labelling Service), UK] . Specific activity is 30-60 Ci/mmol per restored double bond.3H-DGDG cleaned using two-dimensional thin-layer chromatography on plates with silica gel 60. Moving phase in the first direction of the pre is the first acid: water, 85: 15:10:3.5 (volume/volume). Spot DGDG elute successively with mixtures of chloroform:methanol:water, 65:25:4 (volume/volume) and 50:50:10 (volume/volume), pure methanol, and finally with a mixture of methanol:water, 1:1 (volume/volume). Ratio3H in the lipophilic part galactolipid material was determined as follows: 18 nCi3H-labeled DGDG and 2 mg of unlabeled material is subjected to alkaline hydrolysis in 1 ml of 1 M KOH at a temperature of 60oC for 4 h After neutralization with 0.2 ml of 5 M HCl add 5 ml of chloroform, 1.5 ml of ethanol and 2.5 ml of water. In the specified two-phase distribution system 97% of radioactivity is recovered in the lower (chloroform) phase.

The unlabeled galactolipid3H-DGDG at a total concentration of 2.0 vol.% was dispersed in 2.5% vol. glycerin in water. Liposomal dispersion balance within 36 hours at a temperature of 4oC. After that, her voice at a temperature of 0oC in nitrogen atmosphere with the use of the device with a tip for the selection of microprobe for 3 x 2 min, 4 min pulse.

Tests on rats

Starving male rats sprag Share (Sprague Dawley) weighing about 250 g undergo anesthesia with diethyl ether. 0.5 ml voiced dispersion level radioactively the Horta. Lipids from samples of liver and blood plasma extracted with a mixture of chloroform:methanol, 1: 1 (volume/volume). The extracts are dried in nitrogen atmosphere, pererastayut in chloroform and subjected to chromatographicaliy in a thin layer on the plates with silica gel 60, implemented in the system chloroform:methanol:water: acetic acid, 65:25:4:4 (volume/volume). Spot DGDG clean off in ampoules for the account; then add 1 ml of a mixture methanol:water, 1:1 (volume/volume), then add 10 ml of a mixture of toluene: Install [Packard instruments C. V. (Instagel, Packard Instruments C. V.), the Netherlands], 1:1 (volume/volume), the resulting mixture is subjected to vortex mixing. The radioactivity determined in a liquid scintillation counter Packard Tricarb (Packard TriCarb). Data expressed as % of injected dose3H-DGDG in 10 ml of plasma (4% of total body weight) and the whole liver at different times, which are summarized in table. 10.

The results suggest that vesicles comprising DGDG, by intravenous injection to rats have a half-life of about 30 minutes in Addition, it is obvious that galactolipids vesicles derived from the blood stream and are effective degradation, mainly in the liver.

Test 3

Compositions with lokalnie song of bupivacaine hydrochloride is prepared as follows (table. 11).

The ability of the compositions to increase the duration of local anesthetic for spinal cord and nerve roots was investigated in a controlled experiment on rats with long-term implanted vnutriobolochechnoe catheters.

Four groups of male rats sprag Share (Sprague Dawley) (weight 235-300 g) were examined one week after implantation vnutriobolochechnoe catheter according to the method of yaksh and Rudy (T. L. Yaksh and T. A. Rudy, Physiol. Behav., 1976, vol. 17, 1031-1036). Two groups of rats were injected bupivacaine in galactolipids formula (composition A), the third group was given bupivacaine in aqueous solution (composition B), and the fourth group was given galactolipids formula without any local anesthetic (composition), in accordance with the regime, are presented in table. 12.

Rats were placed randomly into one of four groups. The test substance is introduced into the lower lumbar area in the dural SAC through the implanted catheter. The effect of the analyte on the motor function of the track at regular intervals of time and distribute on a scale of 0, 1, 2, 3 and 4, where 0 is the absence of motor disorders, and 4 - paralysis of both rear and both front legs. Rats were observed in mechanistic, show severely impaired motor function quickly after a dose. However, the duration of this action is markedly different for the three different groups, with the largest duration observed in the group receiving the high dose and the shortest duration of paralysis in the control group. To the 90th min, all animals recovered fully. At 20 min of differences relative to the control were statistically significant for the groups treated respectively high dose and low dose [ranking on Wilcoxon (Wilcoxon signed rank test)]. The group who took a high dose differed from the control group at the 30th min of the placebo-treated Group, did not show any effect at any time. It was not marked or unexpected toxic effects, and all observed effects were reversible.

Conclusion. Galactolipid composition with bupivacaine can significantly increase the duration of action of bupivacaine by blocking the nerves in rat models with vnutriobolochechnoe chronic implant. In addition, these data confirm the usefulness of galactolipid composition for longer interaction of biologically military following procedure investigated the hemolytic action of liposomal Li-GLA composition, obtained by the method of example 25 by comparing it with three other Li-GLA using whole blood.

Blood from healthy volunteers was collected in 10-ml tubes of Vacutainer [Becton Dickinson, Canada (Vakutainer, Becton Dickinson, Canada)], containing 143 units of sodium heparin (U.S. Pharmacopeia, USP).

Blood samples of 2 ml is transferred into a 25-ml Erlenmeyer flask, each of them mixed with 1.0 ml of the investigated compositions diluted in 0.9% saline to the desired concentration. Then the samples are incubated at a temperature of 37oC in enriched oxygen atmosphere:carbon dioxide, 95:5, 3 l/min for 1 h with constant gentle stirring. By the end of the incubation, 10 μl of a mixture of blood transferred to 500 ál of salt solution in a 1.5-ml appendectomy vitro. Demonstrating 100% hemolysis standards are prepared by adding 10 ál of blood to 500 ml of pure water in a 1.5-ml appendectomy vitro. Then all the samples out for 2 min in microcentrifuge Eppendorf [Brinkmann instruments Ltd. (Brinkman Instruments Ltd.), Canada] and finally explore the centrifugal analyzer [Kobal Bioanalyzer; Hoffman-La Roche Ltd. (Cobal Bioanalyzer; Hoffman-La Roche Ltd.), Canada] in the ultraviolet region at a wavelength of 540 nm. In table. 13 presents obtained the AI is about 4-5 mm. Thus, the hemolytic activity of the Li-GLA in liposomal form is significantly reduced. Reduced hemolytic activity of liposomal Li-GLA was confirmed by preliminary in vivo studies on rats: hemolysis is reduced by 50-80%, if instead of free Li-GLA uses a liposomal composition. Liposomal composition in contrast to the free-GLA does not lead to the formation in rats "red urine". It was moreover shown that the anticancer activity in vitro in a free Li-GLA and liposomal Li-GLA almost identical. Described tests suggest that liposomal galactolipid composition reduces hemolysis, severe adverse reactions of the medicinal product without affecting its pharmacological efficacy.

In conclusion, it should be noted that the present invention provides evidence that lipid preparations in polar solvent, based on galactolipid material, has improved in comparison with phospholipid preparations properties on all aspects such as physical stability of drugs, the effectiveness of the inclusion of medicines, as well as the ability to slow the release cluck and 0.01 - 90 wt.%, preferably 0.1 to 50 wt.% galactolipids from a cereal that contains at least 50% of digalactosyldiacylglycerols and the rest from other polar lipids, as a carrier of active substances in the pharmaceutical, cosmetic or food products.

2. Application under item 1, where the galactolipids of cereal contain 70 - 80% of digalactosyldiacylglycerols and 20 - 30% of other polar lipids.

3. Application under item 1, where the galactolipids of cereals consist of up to 100% of digalactosyldiacylglycerols.

4. Application under item 1, where the drug is in the form of a gel containing 25 to 90 wt.% galactolipids from cereals and the rest - polar solvent.

5. The use according to any one of paragraphs.1 to 3, where the drug is in the form of liposomes, containing from 0.01 to 25 wt.% galactolipids from cereals and the rest - polar solvent.

6. Pharmaceutical composition comprising a therapeutically active substance and DL lipid preparation containing a polar solvent and 0.01 to 90 wt.%, preferably 0.1 to 50 wt.% material, forming a double layer, characterized in that the material forming the double layer, represents the galactolipids of cereal that contains, at m the position on p. 6, characterized in that the galactolipids of cereal contain 70 - 80% of digalactosyldiacylglycerols and 20 - 30% of other polar lipids.

8. The pharmaceutical composition according to p. 6, characterized in that the galactolipids of cereals consist of up to 100% of digalactosyldiacylglycerols.

9. The pharmaceutical composition according to any one of paragraphs.6 to 8, comprising a therapeutically effective amount of a therapeutically active substance, 1 to 50 wt.% from the whole composition of the galactolipids of cereal, a polar solvent, and as a result of isotopically effective amount of an isotonic agent.

10. The pharmaceutical composition according to any one of paragraphs.6 - 9, designed for oral, enteral, parenteral, rectal, vaginal, local, ocular, nasal or ear of the administration.

11. The pharmaceutical composition according to any one of paragraphs.6 to 9, characterized in that therapeutically active substance is a salt or ester of linoleic acid and the number of galactolipids from cereals is less than 25 wt.% of the total composition.

12. A method of obtaining a pharmaceutical composition according to any one of paragraphs.6 to 8, characterized in that 0.01 - 25 wt.% from the whole composition of galactolipids from cereal, soda is an active substance in a polar solvent in a quantity reaching up to 100 wt.%.


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