RU2362544C2 - Nano emulsion with biologically active substances - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: transparent or slightly opalescent nano emulsion of water in oil type for oral, transdermal application, for use in ophthalmologic practice, with biologically active bonds, characterised that it contains 35-80% of a hydrophobic phase, 17-43% of surface-active substance, 3-7% of a cosolvent and 1-15% of a water phase, admixtures of mono and di - triglycerides with mono- and di-ethers of saturated and non-saturated fatty acids are used as a hydrophobic phase, the surface-active component is chosen from the group of non-ionic surface-active substances - sorbitans in an admixture with auxiliary surface-active substance (from the group of polyhydroxyalkanes or monoatomic alcohols).

EFFECT: nano emulsion is a biologically compatible and well tolerated substance, provides uniform prolonged release of active substance.

8 cl, 3 tbl, 24 ex, 5 dwg

Description

The invention relates to the pharmaceutical, food industry and cosmetology, in particular to the field of creating nano-emulsion systems used as carriers of active substances in pharmaceutical compositions, as well as in the manufacture of food and cosmetic products.

Nanoemulsions are understood to mean systems that do not exhibit double refraction in the rays of polarized light, transparent or translucent, thermodynamically stable, consisting of extremely small droplets with a diameter in the range from 5 to 200 nm, for the formation of which oil, water, a surfactant or surfactant and, optionally, an auxiliary surfactant or co-surfactant with careful selection of the optimal ratio of surfactant to co-surfactant, as well as their total amount in the system IU, which is often difficult and time consuming.

Known nanoemulsion based on amphiphilic nonionic lipids and aminated silicones and its use (RF Patent No. 2142481, C08L 83/04, A61K 7/00, publ. 1999), oil globules of which have an average size below 150 nm, including an amphiphilic lipid phase containing at least one amphiphilic non-ionic lipid, liquid at room temperature below 45 ° C, at least one oil and at least one aminated silicone, also its use in cosmetics or in dermopharmacy.

It is an object of the present invention to provide readily obtainable storage stable, biocompatible compositions containing a relatively large amount of water, well-tolerated water-in-oil (W / O) nanoemulsions.

The problem is solved by creating a transparent or slightly opalescent water-in-oil nanoemulsions for oral, transdermal use for use in ophthalmic practice with biologically active compounds, characterized in that it contains 35-80% hydrophobic phase, 17-43% surfactant, 3 -7% cosolvent and 1-15% aqueous phase. As a hydrophobic phase, mixtures of mono-, di- and triglycerides with mono- and di-esters of saturated and unsaturated fatty acids are used, the surfactant is selected from the group of nonionic surfactants - sorbitans mixed with an auxiliary surfactant (from groups of polyhydroxyalkanes or monohydric alcohols).

In this case, the biologically active compounds of nanoemulsions are flavonoids, betulin, Boswellia extract, vitamins, trace elements, etc.

In addition, the nanoemulsion has a pH in the range between 5.0 and 7.5, and the ratio of surfactant to auxiliary surfactant is from 3: 1 to 9: 1.

Nanoemulsions make it possible to obtain formulations with prolonged release of hydrophilic active compounds. As a hydrophobic continuous phase, mixtures of mono-, di- and triglycerides with mono-and di-esters of saturated and unsaturated fatty acids (C ₁₆ -C ₂₀ ), for example labrafil, isopropyl myristate, etc., are used.

Suitable nanoemulsion surfactants are selected from the group of nonionic surfactants sorbitans (esters of anhydrosorbite and fatty acids), preferably sorbitan bis (polyoxyethylene) monooleate (n = 20).

The auxiliary surfactants present in the system are selected from the group of polyhydroxyalkanes, preferably propylene glycol, and monohydric alcohols, preferably ethanol.

The proposed nanoemulsions may also contain other biocompatible compounds that do not affect the stability of the nanoemulsion.

Various nanoemulsion compositions are prepared as follows.

Example 1. Obtaining a nano-emulsion of the type water in oil (w / o) containing rutin (flavonoid):

a) Preparation of the aqueous phase: 0.09% sodium chloride was dissolved in 9.91% water,

b) Preparation of the hydrophobic phase: 1.0% rutin was dissolved in 48.0% labrafil, mixed separately in a suitable vessel with a stirrer, thermostatted at 60-70 ° C until dissolved, 34.2% tween-80, 6.8% were added propylene glycol, again stirred, the result was a clear homogeneous solution.

c) Preparation of the nanoemulsion: the aqueous phase (solution a) was added with stirring to the hydrophobic phase (solution b) to obtain an optically transparent homogeneous nanoemulsion with an aqueous phase content of 10%. The pH value is about 6.

Example 2. Obtaining a nanoemulsion of the type water in oil (w / o) containing quercetin (flavonoid)

The procedure of Example 1 was followed, but instead of rutin, 1.0% quercetin was added to the composition of the nanoemulsion. The pH value is about 6.

Example 3. Obtaining a nanoemulsion of the type water in oil (w / o) containing taxifolin (flavonoid)

The procedure of Example 1 was followed, but instead of rutin, 1.0% taxifolin was introduced into the composition of the nanoemulsion. The pH value is about 7.

Example 4. Obtaining a nanoemulsion of the type water in oil (w / o) containing taxifolin

The procedure of Example 1 was followed, but 2.0% of taxifolin was dissolved in 47.0% of labrafil.

Example 5. Obtaining a nano-emulsion of the type water in oil (w / o) containing taxifolin

The procedure of Example 3 was followed, but sodium chloride was not added to the aqueous phase.

Example 6. Obtaining a nanoemulsion of the type water in oil (w / o) containing taxifolin

The procedure of Example 4 was followed, but the amounts of surfactant and auxiliary surfactant in the hydrophobic phase were changed by 25.0% Tween-80 and 5.0% propylene glycol, respectively. As a result, at a constant ratio between the two indicated components (5: 1), the total amount of the surfactant / auxiliary surfactant mixture decreased from 40.0% to 30.0% and the amount of labrafil increased by 10%.

Example 7. Obtaining a nano-emulsion of the type water in oil (w / o) containing taxifolin

The procedure of Example 4 was followed, but the amount of the aqueous phase was changed to 15% and the amount of labrafil was reduced by 5%.

Example 8. Obtaining a nano-emulsion of the type water in oil (w / o)

The procedure of Example 3 was followed, but taxifolin was not introduced into the hydrophobic phase.

Example 9. Obtaining a nano-emulsion of the type water in oil (w / o) containing betulin (triterpenoid)

a) Preparation of the aqueous phase: 0.09% sodium chloride was dissolved in 9.91% water,

b) Preparation of a hydrophobic phase: 0.1% of betulin substance was dissolved in 41.9% of labrafil, mixed separately in a suitable vessel with an agitator, thermostatted at 60-70 ° С, until dissolution, 41.7% of tween-80, 6 were added. 3% ethyl alcohol was mixed again, and a clear, homogeneous solution was obtained.

c) Preparation of the nanoemulsion: the aqueous phase (solution a) was added with stirring to the hydrophobic phase (solution b) to obtain an optically transparent, homogeneous nanoemulsion with an aqueous phase content of 10%. PH value is about 6

Example 10. Obtaining a nanoemulsion of the type water in oil (w / o) containing boswellic acids (triterpenoid)

a) Preparation of the aqueous phase: 0.09% sodium chloride was dissolved in 9.91% water,

b) Preparation of a hydrophobic phase: 10.0% of Boswellia extract was dissolved in 30.0% isopropyl myristate, stirred separately in a suitable vessel with a stirrer, thermostatted at 60-70 ° C, until dissolved, 41.7% Tween-80, 8 were added. 3% propylene glycol was mixed again, resulting in a clear homogeneous solution.

c) Preparation of the nanoemulsion: the aqueous phase (solution a) was added with stirring to the hydrophobic phase (solution b) to obtain an optically transparent homogeneous nanoemulsion with an aqueous phase content of 10%. The pH value is about 7.

Example 11. Obtaining a nanoemulsion of the type water in oil (w / o) containing boswellic acids

The procedure of Example 10 was followed, but 5.0% of Boswellia extract was dissolved in 37.0% of isopropyl myristate.

Example 12. Obtaining a nano-emulsion of the type water in oil (w / o)

The procedure of Example 7 was followed, but Boswellia extract was not introduced into the hydrophobic phase.

Example 13. Obtaining a nano-emulsion of the type water in oil (w / o) containing L-ascorbic acid

The procedure of Example 8 was followed, but 2% ascorbic acid was added to the aqueous phase.

Example 14. Obtaining a nano-emulsion of the type water in oil (w / o) containing selenium in organic or inorganic form (selenomethionine, sodium selenite, selexen)

The procedure of Example 12 was followed, but 0.1% of selexene was introduced into the hydrophobic phase.

Example 15. Obtaining a nanoemulsion of the type water in oil (w / o) containing chromium

The procedure of Example 12 was followed, but 0.1% chromium (III) sulfate was added to the aqueous phase.

Example 16. Evaluation in ovo of the irritating effect of nanoemulsions

Evaluated the irritating effect of nanoemulsions prepared according to the methods described in examples 4 and 8.

Irritation was evaluated using the test on the chorionallantoic membrane of the chicken embryo (HET-KAM test) in accordance with the Guidelines for assessing the irritating effect of pharmacological substances (Guidelines for the experimental (preclinical) study of new pharmacological substances. Moscow. 2000. - P.87-89 )

For the study, developing embryos of eggs of domestic chickens of the White Leghorn and Highsex Brown breeds aged 9-10 days were used. The eggs were kept in an incubator at a temperature of 37.8 ° C and a relative humidity of 62.5%. Before the experiment, the incubated eggs were kept in a stationary position for 24 hours with the blunt end up the day before the experiment; they were transferred from the incubator to the thermostat (37.8 ° C). 0.3 ml of the test substance was applied to the chorioallantoic membrane. Monitoring the state of the shell under the drop was carried out for 100 seconds through a binocular stereoscopic microscope (Labostemi-4 zoom). Registration was carried out every 10 seconds by photographing the shell under a drop using a specialized color television camera (Zenith TVK-MI-01C) connected to a binocular microscope.

An indicator of the effect on the chorioallantoic membrane for 100 seconds was used as an irritant criterion. The classification of substances according to the degree of irritation is shown in the table (table 1).

Table 1
Classification of substances by degree of irritation Class Observed picture 5th grade, very strong Coagulation and lysis of vessels of small and medium, multiple hemorrhages and thrombosis with stopping blood flow. 4th grade, strong Thrombosis in small vessels with a stoppage of blood flow and small point hemorrhages throughout the treated surface. Hyperemia. 3rd grade, moderate Redness of the membrane, slowing down of blood flow and thrombosis in individual capillaries. 2nd grade, weak The narrowing of blood vessels without stopping or with a temporary stop of blood circulation in separate capillaries. 1st grade, no exposure An undisturbed transparent thin shell with a normally functioning network of blood vessels and capillaries.

The experimental results are shown in the table (table 2)

table 2
The results of a study to determine the degree of irritation of the chorioallantoic membrane (M ± t) Sample The average degree of observed irritation at 10 time points M ± m, n = 5 Nanoemulsion according to example 4 1,545 ± 0,207 1,745 ± 0,093 2.091 ± 0.211 1,636 ± 0,152 1.727 ± 0.141 1.727 ± 0.141 Nanoemulsion according to example 8 1.455 ± 0.157 1,400 ± 0,167 2,000 ± 0.234 1.273 ± 0.141 1,000 ± 0,000 1.273 ± 0.141

Based on the data obtained during the experiment on the chorioallantoic membrane, it can be concluded that nanoemulsions can be classified as class 2 by irritation, which indicates the safety of their use.

Example 17. Evaluation of the vasodilating effect of nanoemulsions

The vasodilating effect of in ovo nanoemulsions containing taxifolin was evaluated, prepared according to the procedures described in examples 4 and 8.

The experimental conditions are described in example 16. Monitoring the state of the chorioallantoic membrane under a drop was carried out for 60 minutes through a binocular microscope. Registration was carried out every minute by photographing the shell under a drop.

The effect of the substance was evaluated by the presence or absence of an effect on the diameter of the vessels of the chorioallantoic membrane under the applied drop of a solution of the test substance. To assess the vasodilating effect, the diameter of the vessels was calculated. The “tape width” of the vessel of a certain order (2, 3, 4 order) was taken into account before and after (60 minutes) application of the test substance. The calculation of changes in the diameter of the vessels was calculated as a percentage. The initial diameter of the vessel was taken as 100%. The experimental results are shown in the table (table 3).

Table 3
Results of a study to determine vasodilating effects on blood vessels Try The diameter of the vessels of the second order,% The diameter of the vessels of the third order,% The diameter of the vessels of the IV order,% The initial diameter of the vessels 100 ± 0,0 100 ± 0,0 100 ± 0,0 Nanoemulsion according to example 4 109.2 ± 1.3 * 114.3 ± 3.5 * 118.6 ± 3.1 * Nanoemulsion according to example 8 103.0 ± 0.5 * 103.8 ± 0.9 * 110.6 ± 3.8 *

Note: * - the differences are statistically significant compared with the initial diameter, at p <0.05.

It was found that nanoemulsions have a vasodilating effect, which is manifested as much as possible on vessels of the fourth order.

Example 18. Evaluation of the rate of release of taxifolin from a nanoemulsion The release rate of taxifoline from a nanoemulsion prepared according to the procedure described in Example 4 was evaluated. The release from a nanoemulsion was determined on an Erweka DT 600 series solubility tester (Germany). For each experiment, the amount of the test sample equivalent to 10 mg of taxifolin (exact weight) was taken.

To study the release under local application modeling conditions, the Paddle over disk method was used. The experiment was carried out at a temperature of 32 ± 1 ° С, the blade rotation speed was 100 rpm, and the dissolution medium was water. The results are shown in FIG. 1, which shows the release of taxifolin from a nanoemulsion under conditions of local application modeling.

The release curve is linear in the first 2 hours, with about 95% of the substance passing into the dissolution medium. The dissolution rate constant calculated by the least squares method was 0.9306 h ^-1 .

The linearization of the curves of taxifolin release in coordinates: the amount of the drug that went into the solution — the square root of time — suggests that the release occurs through diffusion, and the release rate is described by the Higuchi theoretical model.

The study of the rate of release when simulating the conditions of the gastrointestinal tract (GIT) was carried out at a temperature of 37 ± 1 ° C, a nanoemulsion sample was placed in a hard gelatin capsule. The rotational speed of the blade is 100 rpm, the dissolution medium is a two-phase water-octanol system in a ratio of 300: 100 ml (Karlina M.V., Pozharitskaya O.N., Kosman V.M. Non-traditional dissolution media for studying drug release in vitro // Questions of biological, medical and pharmaceutical chemistry. - 2006. - No. 3. - P.42-46). For quantitative analysis, the direct spectrophotometry method in the UV region was used. The results are shown in figure 2, which shows the release of taxifolin in the simulation of the gastrointestinal tract.

When simulating the conditions of the gastrointestinal tract by the 3rd hour, about 90% of taxifolin passes into the lipophilic phase. The dissolution rate constant calculated by the least squares method was 0.8479 h ^-1 . A nanoemulsion containing 2% taxifolin provides uniform, sustained release of the active substance under simulation conditions for both local and oral use.

Example 19. Evaluation of the rate of release of boswellic acids from nanoemulsions

The release rate of boswellic acids from the nanoemulsion prepared according to the procedure described in Example 10 was evaluated. The release from the nanoemulsion was determined on an Erweka DT 600 series solubility tester (Germany). For each experiment, the amount of the test sample equivalent to 10 mg of Boswellia extract (exact weight) was taken. We studied the release of boswellic acids through a semipermeable membrane (local modeling conditions) using the “rotating basket” method. The experiment was carried out at a temperature of 32 ± 1 ° С, the basket rotation speed was 100 rpm, the dissolution medium was alcohol.

For the quantitative analysis of the released boswellic acids, the reverse phase HPLC method on a Waters high-pressure chromatograph (UV) with a UV detector and a Luna C ₁₈ column of 4.6 × 150 mm (particle size of the sorbent 5 μm) with a 20 mm long pre-column filled the same sorbent. Two chromatographic modes were used: isocratic elution with 100% acetonitrile and gradient elution from 75% acetonitrile in a 0.03% trifluoroacetic acid solution to 100% acetonitrile in 20 minutes. The flow rate of the eluent 1.0 ml / min, the volume of the injected sample 20 μl. Detection wavelengths of 254 and 210 nm. Identification and quantitative analysis were performed using the external standard method. The results are shown in figure 3, which shows the release of boswellic acids from the nanoemulsion in the conditions of modeling topical application.

The release curves of boswellic acids are linear, about 6% of 11-keto-β-boswellian (BCA), 50% of acetyl-11-keto-β-boswellian (ACBA) and acetyl-β-boswellic acid passes into the dissolution medium after 6 hours of the experiment β-ABA) acids and 60% β-boswellic R-BK, α-boswellic (α-BK) and acetyl-α-boswellic (α-ABA) acids.

The dissolution rate constants calculated by the least squares method were: KBK - 0.0892 h ^-1 ; AKBK - 0.1268 h ^-1 ; α-BK - 0.1874 h ^-1 ; α-ABA - 0.1624 h ^-1 ; β-BK - 0.1737 h ^-1 ; β-ABA - 0.1322 h ^-1 .

The linearization of the release curves in the coordinates: the amount of drug transferred to the solution is the square root of time, suggesting that the release occurs through diffusion, the release rate is described by the Higuchi theoretical model.

Example 20. Evaluation of the rate of release of rutin from a nanoemulsion

Estimated the rate of release of rutin from a nanoemulsion prepared according to the method described in example 1.

The release of rutin from a nanoemulsion was determined by modeling the conditions of the gastrointestinal tract, as described in example 18. Under the same conditions, the rate of release of rutin from a substance was evaluated.

The results are shown in figure 4, which shows the release of rutin in the simulation of the gastrointestinal tract.

By the 4th hour, about 35% of the rutin passes from the nanoemulsion to the lipophilic phase, while about 5% is released from the substance by the 4th hour. The dissolution rate constants calculated by the least squares method were 0.0133 and 0.1222 h ^-1 for the substance and nanoemulsion, respectively. Thus, the rate of release of rutin from a nanoemulsion is 9 times higher than that of pure substance.

The data obtained indicate that the introduction of rutin into a nanoemulsion can significantly increase its dissolution rate, increase bioavailability and ensure uniform release of the active substance under conditions of oral administration.

Example 21. Evaluation of the rate of release of quercetin from a nanoemulsion

The release rate of quercetin from a nanoemulsion prepared according to the procedure described in example 2 was evaluated.

The release of quercetin from a nanoemulsion was determined by modeling the conditions of the gastrointestinal tract, as described in Example 18. Under the same conditions, the rate of release of quercetin from a substance was evaluated.

The results are shown in figure 5, which shows the release of quercetin in the simulation of the gastrointestinal tract.

From the data shown in FIG. 5, it is seen that after 3 hours of the experiment, about 90% of quercetin passes into the dissolution medium. The first-order kinetics equation (r = 0.9805) is most suitable for describing the dissolution process, the dissolution rate constant calculated by the least squares method is 0.7266 h ^-1 . Release from the substance is much slower, by 5 hours about 40% of quercetin passes into the dissolution medium. The dissolution rate constant was 0.1253 h ^-1 .

The data obtained indicate that the introduction of quercetin into the nanoemulsion can significantly increase its dissolution rate and ensures uniform release of the active substance under the conditions of oral administration.

Example 22. Evaluation of the stability of the nanoemulsion

The stability of nanoemulsions prepared according to the procedures described in examples 1, 2, 4, 8, 9, and 10 was evaluated.

Stability was evaluated visually when stored under elevated (40 ° C) and low temperature (4 ° C) for 6 weeks, as well as when stored in natural conditions (20 ° C) for 12 months. All nanoemulsions during the experiment remained transparent, did not delaminate.

Example 23. Determination of particle size of nanoemulsions

The particle size of nanoemulsions prepared according to the methods described in examples 1, 2, 4, 8, 9, and 10 was determined by photon correlation spectroscopy on a Malvern Zetasizer 3000HSA instrument (Malvern instruments, Worcestershire, UK). The average particle size of the nanoemulsions was 10-170 nm.

Example 24 Sterilization of Nano-Emulsions

Nanoemulsions prepared according to methods 1, 2, 4, 9, 10 were sterilized by filtration through a membrane filter with a pore diameter of 0.22 μm.

The created compositions of nanoemulsions can be easily obtained, easily sterilized, do not have an irritating effect, are well suited for oral, transdermal use, as well as for use in ophthalmic practice, provide a prolonged effect of the active substances introduced into their composition.

The obtained nanoemulsions are especially suitable for use as carriers of polyphenolic, triterpene compounds, vitamins, microelements, etc., allow to obtain formulations with prolonged release of hydrophilic active ingredients.

Nanoemulsions, including polyphenolic compounds, can be used to obtain agents with antioxidant and capillaroprotective effects, and nanoemulsions containing triterpenoids can be used to obtain a medicament with anti-inflammatory and wound healing properties.

Claims (8)

1. A transparent or slightly opalescent water-in-oil nanoemulsion for oral, transdermal use, for use in ophthalmic practice with biologically active compounds, characterized in that it contains 35-80% hydrophobic phase, 17-43% surfactant, 3- 7% co-solvent and 1-15% aqueous phase, mixtures of mono-, di- and triglycerides with mono- and di-esters of saturated and unsaturated fatty acids are used as the hydrophobic phase, the surfactant is selected from the group of nonionic surfaces but-active substances - sorbitans in a mixture with an auxiliary surfactant (from the group of polyhydroxyalkanes or monohydric alcohols). 2. The nanoemulsion according to claim 1, in which the hydrophilic, biologically active compounds are flavonoids. 3. The nanoemulsion according to claim 1, in which the biologically active compound is betulin. 4. The nanoemulsion according to claim 1, in which the biologically active compound is a Boswellia extract. 5. The nanoemulsion according to claim 1, in which the biologically active compounds are vitamins. 6. Nanoemulsions according to claim 1, in which the biologically active compounds are trace elements. 7. The nanoemulsion according to claim 1 has a pH in the range between 5.0 and 7.5. 8. The nanoemulsion according to claim 1, wherein the ratio of surfactant to auxiliary surfactant is from 3: 1 to 9: 1.

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