RU2463056C1 - Composition for drug substance integration into lipid matrix, drug composition with phospholipid fatty acid system and method for preparing them - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and pharmaceutics, and concerns a storage-stable composition in the form of nanoparticles of size 10-20 nm containing a fatty acid salt, phosphatidyl choline and maltose for the integration of biologically active substances, particularly, drugs, as well as drug compositions containing the fatty acid salt, phosphatidyl choline, maltose and a drug substance, and a method for preparing them.

EFFECT: development of the storage-stable composition.

4 cl, 5 tbl, 5 ex

Description

The invention relates to medicine and pharmacology, and relates to a storage-stable composition in the form of nanoparticles with a size of 20-80 nm based on salts of fatty acids stabilized by phosphatidylcholine and intended for inclusion in the nanoparticle of biologically active compounds, namely medicinal substances, proteins, peptides, etc. and increase bioavailability and therapeutic efficacy of insoluble drugs.

Currently, one of the areas of application of nanotechnology in medicine is associated with the development of the pharmaceutical industry, which can be explained by the unique properties of new nanomaterials and nanoparticles that have appeared recently. This application of nanotechnology has contributed in recent years to the development of new pharmaceutical strategies, aimed primarily at creating systems that enhance the therapeutic efficacy of drugs, their bioavailability, and reduce side effects. Among these strategies, an important place is occupied by the systems of drug transport to organs, tissues, and target cells.

The supply of drug compounds with transport systems eliminates many of the shortcomings of developed and existing drugs - low solubility in water, rapid sorption or metabolism in the body, difficulty in crossing natural barriers (cell membranes, blood-brain barrier, etc.), side effects. The intensive development of delivery systems based on nanotechnology leads not only to an extension of the “life cycle” of known drugs in the pharmaceutical market, but also to the appearance of drugs with improved pharmacological and pharmacokinetic properties. The development of drugs equipped with transport systems does not require large investments, and the effects achieved are very significant for health and the economy. In the Russian Federation, there is still no production of drugs equipped with nanosystems of transport, which determines the relevance of conducting our own, domestic developments. This is especially important for drugs, the use of which in free forms is limited by their pronounced toxicity.

The most common nanosystems in the modern pharmaceutical market are currently liposomes. Of greatest applied interest are liposomes as in vivo drug delivery systems, since it has long been known that they have relative selectivity, which contributes to a fairly high accumulation of the drug at the delivery site.

The most effective approach to the construction of liposomal drugs is the development of technologies to obtain liposomal drugs in the form of nanoparticles with a diameter of less than 50 nm, because it is known that reducing the size of liposomes by 5 times increases its circulation time in the blood by 40 times.

An original technology is known that makes it possible to obtain a phospholipid transport nanosystem with a particle size of less than 30 nm, and it is shown that it is possible in principle to obtain medicinal compositions in which the active substance is supplied with a transport nanosystem based on plant phospholipids (RF patent No. 2373924). It was shown that as a result of this incorporation, the pharmacokinetics of drug substances changed when introduced into the body, their bioavailability and therapeutic effectiveness increased.

The presence of a hydrocarbon region in phospholipid structures, including micelles and liposomes, allows hydrophobic compounds to be included in it. The inclusion of a number of medicinal substances with amphipathic, lipophilic and hydrophobic properties in phospholipid nanoparticles can be based on this principle. An example of such substances is chlorin E6, diclofenac, lipoic acid, budesonide, etc.

Known storage stable nanosystem with a particle size of up to 10-30 nm, including phosphatidylcholine and maltose, intended for inclusion in the phospholipid nanoparticle of drugs (RF patent 2391966).

Due to its chemical structure, the above phospholipid transport nanosystem can serve as a carrier of both soluble and insoluble (hydrophobic) bioactive drugs in biological fluids, but, unfortunately, far from all medicinal substances can be integrated due to their physicochemical properties and chemical structure to the transport system based on phosphatidylcholine alone.

The aim of the invention is to create a composition based on salts of fatty acids and plant phospholipids, for the incorporation of biologically active compounds and the possibility of their subsequent transport in the body.

In accordance with the invention, a composition is described for incorporating drug substances into a lipid matrix in the form of lyophilized dried nanoparticles 10-20 nm in size, including plant-derived phosphatidylcholine (73-94%), a fatty acid salt and maltose in the following ratio, wt.%:

Phosphatidylcholine 20-25 Fatty acid salt 1,5-10 Maltose 70-78

Also described is a composition of a drug with a phospholipid fatty acid system in the form of nanoparticles of 20-80 nm in size, including a fatty acid salt, plant-derived phosphatidylcholine (73-94%), maltose and a drug substance in the following ratio, wt.%:

Phosphatidylcholine 19-25 Fatty acid salt 1,5-10 Maltose 67-78 Drug substance 1-10

In accordance with the invention, a method is described for preparing a composition for incorporating drug substances into a lipid matrix in the form of lyophilized dried nanoparticles 10-20 nm in size, including plant phosphatidylcholine (73-94%), a fatty acid salt and maltose in the following ratio, wt.% :

Phosphatidylcholine 20-25 Fatty acid salt 1,5-10 Maltose 70-78

consisting in the fact that the fatty acid salt, plant phosphatidylcholine and maltose are emulsified in water and the resulting emulsion is subjected to several homogenization cycles under high pressure of 800-1500 bar at a temperature of 40-45 ° C followed by lyophilization.

Also described is a method of obtaining a composition of a drug with a phospholipid-fatty acid system in the form of nanoparticles of 20-80 nm in size, including a fatty acid salt, plant-derived phosphatidylcholine (73-94%), maltose and a drug substance in the following ratio, wt.%:

Phosphatidylcholine 19-25 Fatty acid salt 1,5-10 Maltose 67-78 Drug substance 1-10

consisting in the fact that in the case of a drug substance of protein nature, the composition for incorporating drug substances described above is dissolved in deionized water, a drug of protein nature is added, stirred at room temperature, filtered and dried by lyophilization, and in the case of a drug substance of non-protein nature, a fatty acid salt , maltose and drug substance are emulsified in water, phosphatidylcholine is added and the resulting suspension is subjected to several cycles of (1-10) homogen izations under high pressure of 800-1500 bar at a temperature of 40-45 ° C with the subsequent lyophilization.

A distinctive feature of the developed composition from the known phospholipid system (RF patent No. 2391966) is the nature of the interaction with the drug substance. In contrast to the hydrophobic interaction in the phospholipid system, in the composition based on fatty acids stabilized by plant phospholipids, the connection between the fatty acid and the biologically active substance occurs due to electrostatic interaction, and the hydrophobic interaction of the phospholipid part of the composition can also make a significant contribution.

A significant advantage of nanoparticles formed by salts of fatty acids stabilized by phosphatidylcholine is that in addition to the ability to incorporate hydrophobic drug substances into hydrophobic regions of phospholipids, it is possible to bind due to the electrostatic interaction of charged groups / regions of the drug with the COO group of the fatty acid. It is important to note that this interaction is pH-dependent and in this regard, with a change in pH, a controlled release of the drug substance is possible. The resulting negatively charged nanophospholipid particles with fatty acid can be used to plant positively charged drug substances or positively charged peptides and proteins on them at pH 4.5-5.0.

It is known that water-soluble compounds are limitedly integrated into the phospholipid transport system (not more than 10%), due to the limited internal volume of liposomes. A fatty acid-based system binds to an active substance through electrostatic interaction and can thus transfer water-soluble compounds. In the free state, many drugs based on water-soluble substances can be quickly washed out of the body. By electrostatic interaction, they are retained; while, importantly, a smaller dose of the drug is needed. In addition, water-soluble compounds included in the inner space of liposomes, quickly "flow out" of this system, not having time to reach the target tissues.

The forces of electrostatic interaction make the composition with the included biologically active compound stable enough and do not give it the ability to quickly collapse in the body.

In the proposed composition, when using fatty acids, compounds with an excess positive charge are transported.

The resulting micelles based on FA in water aggregate and, when the pH of the solution decreases, spontaneously form an oil. When stabilized with phosphatidylcholine, particles with a size of 10-20 nm are formed.

Many surface-active substances (SAS) can be used as stabilizers: twins, poloxamers, cyclodextrin, and other compounds. Since the use of many of them is limited for toxicity reasons, plant phospholipids, in particular phosphatidylcholine, have proven to be the most suitable stabilizer from a large set of surfactants.

When obtaining a composition of proteins with a transport system based on FA stabilized by phosphatidylcholine, it is necessary to take into account that many technological operations, in particular sounding or homogenization under high pressure, are unsuitable for maintaining the protein in its native state. Therefore, particles of a transport system or composition must first be obtained, including a salt of a fatty acid in a solution of sugar stabilized by phosphatidylcholine, to which a protein can be attached later, subject to special precautions (see example 3).

Sugars are traditionally used as a cryoprotectant during lyophilization of the final product: maltose, lactose, trehalose, etc.

The main stages of the technology for producing nanocompositions based on fatty acids stabilized by plant phospholipids can be illustrated by the following example: weighed portions of plant phospholipid and fatty acid salt in a 10: 1 ratio in a 10% aqueous solution of maltose (phosphatidylcholine concentration 25 mg / ml), the mixture is homogenized using sonication or homogenization under high pressure. The pH of the resulting solutions was adjusted with a solution of 1M HCI to a pH of 7.0-7.5.

This nanosystem is almost universal for the incorporation of substances of various nature. In addition, it is possible to obtain a transport system in a dry, compact, convenient form for storage and further use.

An important characteristic of the developed composition is the percentage of incorporation of a biologically active substance (drug substance, etc.) directly into the nanoparticles of the system. For this purpose, the ultrafiltration method was used. The main stages of the quantitative determination of a drug substance in a preparation are as follows:

1. Place 200-250 μl of the test sample in a special cartridge, the bottom of which is a calibrated membrane.

2. Centrifuge the cartridge for 20-40 minutes at 10,000 rpm until the complete passage of the solvent and low molecular weight drugs into the lower part of the cartridge.

4. Determine the availability of medication. The difference in the obtained values determines the part of the active component that did not pass into the filtrate, because it is bound to lipid particles.

It should be noted that this procedure makes sense for medicinal substances or their salts / hydrochloride derivatives, which are soluble in water. For substances whose solubility in water is extremely small, this step can be skipped because the whole medicine is in a thermodynamically favorable state for itself, namely, in one way or another, it turns out to be embedded in the nanoparticles of the transport system.

Table 1 shows examples of the incorporation of drug substances into a transport system based on oleic acid and stabilized by plant phospholipids.

Table 1 Substance Percentage of inclusion Particle size Ambroxol 96 fifteen Doxorubicin HCI 99 fifteen Amlodipine 96 twenty Remantadine 99 twenty Dexamethasone 28.5 eleven Prednisone 73 19 Protionamide 37 12 Indomethacin 76 eighteen Lovastatin 48 42 Resveratrol one hundred twenty Insulin 99 73

The presented results confirm that the described laboratory technology allows to obtain a composition of drugs in the form of nanoparticles based on salts of fatty acids stabilized by phosphatidylcholine.

Upon receipt of a transport system based on only plant phospholipids, nanoparticles with a size of less than 30 nm are formed. The transport system based on salts of fatty acids stabilized by phosphatidylcholine has a particle size of 10-20 nm. When embedding drug substances in a phospholipid-fatty acid composition, the particle size increases. For example, for the drug lovastatin up to 41-44 nm, for insulin up to 70-75 nm.

The present invention is characterized by the following examples:

Example 1. Obtaining a composition in the form of nanoparticles based on salts of fatty acids stabilized by plant phospholipids for the incorporation of drug substances

To a solution of 20 g (78.7%) of maltose monohydrate in 150 ml of deionized water, with intensive stirring, 0.4 g (1.6%) of sodium salt of oleic acid, 5 g (19.7%) of plant phospholipid (phosphatidylcholine content) are successively added. 73-94%), bring the volume of the solution with deionized water to 200 ml and mix to the state of a homogeneous coarse emulsion.

The resulting emulsion is homogenized at a pressure of 800 bar, maintaining the product temperature at the outlet of not more than 45 ° C. With a 1 M HCI solution, the pH of the solution is adjusted to 7.3. The solution is filtered through a 0.22 μm filter, poured into 10 ml vials and dried by lyophilization.

Get 20 vials containing 1.26 g of dry white crystalline powder with a residual moisture content of 2.41%, after dissolving which in 8.5 ml of water, a solution is formed with a light transmission at 660 nm of 87.1%, particle size 17 nm, pH 7 , 3.

Similarly, a dry composition in the form of nanoparticles based on plant phospholipids and salts of fatty acids is obtained: linoleic, caprylic, capric, lauric, myristic, palmitic (table 2).

Table 2. lcd The ratio of phospholipid / LCD / Maltose (f / LCD / M) PARAMETER Particle size, nm T ₆₆₀ % Note Oleic 19.7: 1.6: 78.7 17 87.1 Linoleic 21.3: 8.5: 70.2 fourteen 83,2 Caprylic 24.0: 3.1: 72.9 twenty 79.6 Capric 20.7: 5.0: 74.3 16 84,4 Lauric 22.0: 2.0: 76.0 eighteen 80.3 Myristine 20.1: 1.9: 78.0 fifteen 82,4 Palmitic 23.2: 4.3: 72.5 19 78.9

Example 2. Obtaining a composition based on indomethacin, sodium salt of linoleic acid and plant phospholipid

To a solution of 20 g (77.5% by weight of dry weight) of maltose monohydrate in 150 ml of deionized water, with intensive stirring, 0.4 g (1.5% by weight) of linoleic acid sodium salt, 0.4 g (1 , 5% by weight) indomethacin and 5 g (19.5% by weight) of plant phospholipid (phosphatidylcholine content 73-94%), the solution volume is adjusted with deionized water to 200 ml and mixed to a state of a homogeneous coarse emulsion.

The resulting coarse emulsion is homogenized at a pressure of 1000 bar, maintaining the product temperature at the outlet of not more than 45 ° C. In the resulting nanoemulsion, a pH of 7.4 was adjusted with 1M HCI.

The solution is filtered through a 0.22 μm filter, poured into 10 ml vials and dried by lyophilization.

The resulting preparation was characterized by the following parameters: average particle size in the multimodal distribution by volume, light transmission at a wavelength of 660 nm (T ₆₆₀ ), the amount of built-in substance in% of the initial amount, residual moisture, pH of the solution. For this, the contents of the vial are dissolved in 10 ml of deionized water and the solution is analyzed.

Get 20 vials containing 1.29 g of dry slightly yellow crystalline powder with a residual moisture content of 1.63%, after dissolving in 8.5 ml of water gives a solution with a transmittance at 660 nm 86.5%, particle size 24 nm, pH 7.4.

Compositions in the form of nanoparticles based on indomethacin and salts of fatty acids: oleic, caprylic and capric, stabilized by plant phospholipids, and indomethacin are similarly prepared. The measurement results are presented in table 3.

Table 3 Characterization of compositions based on indomethacin, salts of fatty acids and plant phospholipids LCD The ratio of the components of the composition f / LCD / Indus / M PARAMETER Particle size, nm T ₆₆₀ % % inclusion of drug substance from ref. Oleic 19.5: 1.5: 1.5: 77.5 eighteen 88.4 76.0 Linoleic 24.2: 4.4: 2.7: 68.2 24 86.5 68.0 Caprylic 21.0: 3.2: 2.1: 73.7 fifteen 87.2 90,4 Capric 23.3: 4.8: 3.0: 68.9 19 84.6 71.6 Lauric 20.0: 2.5: 7.2: 70.3 23 81.3 74.5 Myristine 22.1: 2.1: 6.4: 69.4 17 78.9 82.1 Palmitic 20.9: 3.0: 5.8: 70.3 27 79.7 73.0

Example 3. Obtaining a composition in the form of nanoparticles based on lovastatin, sodium salt of caprylic acid and plant phospholipids

To a solution of 25.4 g (98.5% by weight) of the composition obtained in Example 1, where the sodium salt of caprylic acid is used as the fatty acid salt, 0.20 g (1.5%) are added to 100.0 ml of deionized water ) lovastatin, mix to a homogeneous coarse emulsion.

The resulting coarse emulsion is homogenized at a pressure of 1200 bar, maintaining the product temperature at the outlet of not more than 45 ° C. In the obtained nanoemulsion, the pH was adjusted to 7.4 with a 1M HCI solution, filtered through a 0.22 μm filter, poured into 10 ml vials and dried by lyophilization.

Get 10 bottles containing 2.58 g of dry white crystalline powder with a residual moisture content of 2.41%, after dissolving in 10 ml of water a solution is formed with a light transmission at 660 nm of 79.6%, particle size 27 nm, pH 7.3 .

Compositions in the form of nanoparticles based on plant phospholipids (phosphatidylcholine content 73-94%) and fatty acid salts: oleic, linoleic, capric, lauric, myristic, palmitic and indomethacin are similarly prepared.

The measurement results are presented in table 4.

Table 4 Characterization of compositions based on lovastatin in the transport system LCD The ratio of the composition of lovastatin F / LCD / L / M PARAMETER Particle size, nm T ₆₆₀ % Embedding,% of ref. Oleic 19.4: 1.6: 1.5: 77.5 42 79.0 87.0 Linoleic 20.4: 8.1: 4.1: 67.4 44 85.1 91.9 Caprylic 23.6: 3.1: 1.5: 71.8 27 79.6 82.5 Capric 20.0: 4.8: 3.5: 71.7 thirty 84.3 94.1 Lauric 20.6: 1.8: 6.5: 71.1 25 87.0 88.3 Myristine 19.1: 1.7: 75.1: 4.1 29th 81.1 69.9 Palmitic 21.3: 3.9: 8.4: 66.4 31 83.3 73,2

Example 4. Obtaining a composition in the form of nanoparticles based on insulin and phospholipid-fatty acid system

To a solution of 20 g (77.5% by weight of dry weight) of maltose monohydrate in 150 ml of deionized water, 5 g (19.5%) of plant phospholipid (phosphatidylcholine content 73-94%) and 0.4 g are successively added with vigorous stirring. (1.5%) of the sodium salt of oleic acid, stirred until a homogeneous coarse emulsion.

The resulting coarse emulsion is heated to 42-45 ° C and homogenized at a pressure of 1500 bar, maintaining the product temperature at the outlet of not more than 45 ° C. The resulting alkaline nanoemulsion (pH 9-10) is neutralized to pH 5.0 with solutions of KH ₂ PO ₄ or 1M HCI.

The solution was successively filtered through a 0.22 μm filter, poured into 10 ml vials and dried by lyophilization.

Get 20 vials containing 2.5 g of dry white crystalline powder, light transmission at 660 nm 88.4%, particle size 28 nm, residual moisture 1.40%, pH 5.0.

The resulting negatively charged fatty acid particles stabilized by plant phosphatidylcholine can be used to plant positively charged drug substances or positively charged peptides and proteins on them at pH 5.0.

25.0 g of the dry residue is dissolved in 80 ml of deionized water, 100 mg of NOI-5500 insulin are added, the volume of the solution is adjusted to 100 ml, and gently stirred at room temperature.

The solution is filtered through a 0.22 μm filter, poured into 10 ml vials and dried by lyophilization.

The percentage of insulin incorporation is determined by ultrafiltration. For ultrafiltration use a special cartridge (cartridge) with an integrated calibrated membrane (Ultrafree-MC Filters NMWL per mol. Wt. 10000). During ultrafiltration, phospholipid nanoparticles are retained on the membrane. A substance bound to phospholipid particles also lingers on the membrane. A substance not bound to particles is filtered and detected in the filtrate. The determination of insulin is carried out by HPLC.

Get 10 bottles containing 2.51 g of a dry white crystalline powder with a residual moisture content of 1.37%, after dissolving which in 8.5 ml of water, a solution is formed with a transmittance at 660 nm of 86.5%, particle size 74 nm, pH 6 , 5 and the embedding percentage of 98%.

Animal experiments were performed to determine the bioavailability of the preparations obtained.

Example 5. A comparative study in animals of the bioavailability of free lovastatin and in the form of a composition based on plant phospholipids and FAs with different chain lengths

The number of rats is 5 males weighing 320 ± 10 g.

The lyophilisates of the finished preparations (from Example 2) are dissolved in 1 ml of distilled water, then the volume of the solution is adjusted to:

3.3 ml - for the drug NFOle-Love;

3.2 ml - for the drug NFLin-Love;

4.6 ml - for the drug NFKil-Love;

2.0 ml - for the drug NFKin-Lov.

A suspension of 8.1 mg of lovastatin in 3 ml of water is also prepared (control).

1 ml of suspension is administered orally to a rat (dose 2.7 mg / rat) and ~ 200 μl of blood is taken from the tail after 30, 60, 120, 180, 240, 300 min. The selection is carried out in test tubes with 1.5 ml of eppendorf, pre-moistened with a 15% solution of EDTA.

Whole blood is centrifuged for 20 min at 8000 rpm and a temperature of 4 ° C. 100 μl of plasma are collected. Dilute the plasma 10 times with methanol. Mixed. Centrifuge for 10 minutes under the same conditions. 400-500 μl of sample is taken into vials for analysis by LC / MS.

Similarly, a comparative study is carried out on animals of free indomethacin and in the form of a composition based on plant phospholipids and FAs with different chain lengths.

Table 5 presents the obtained values of the concentrations of lovastatin and indomethacin in blood plasma, taking into account the dilution of the samples.

Table 5 Comparative bioavailability in vivo of a drug substance administered in a free form and in the form of compositions based on plant phospholipids and FAs with different chain lengths Name of FA in the system Phospholipid / FA Blood indomethacin 30 minutes after per os administration, mcg / ml plasma The content of Lovastatin in the blood 30 minutes after the administration of per os, ng / ml plasma Sodium oleate 6.6 13.7 Sodium linoleate 11,2 38.7 Sodium caprylate 14.6 19.9 Sodium caprine 16,2 13,2 Free drug 0.6 7.5

Thus, in a comparative study of the in vivo bioavailability of indomethacin and lovastatin, introduced in equivalent quantities in the free state and in the form of a composition based on fatty acids stabilized by phosphatidylcholine, a significant increase in the content of drugs in the blood of animals was shown (from 2 to 27 times).

Claims (4)

1. Composition for embedding medicinal substances in the lipid matrix in the form of lyophilized nanoparticles 10-20 nm in size, including plant-derived phosphatidylcholine (73-94%), fatty acid salt and maltose in the following ratio, wt.%:
Phosphatidylcholine 0-25 Fatty acid salt 1,5-10 Maltose 70-78 2. The composition of the drug with a phospholipid fatty acid system in the form of nanoparticles with a size of 20-80 nm, including a salt of a fatty acid, phosphatidylcholine of plant origin (73-94%), maltose and a drug substance in the following ratio of components, wt.%:
Phosphatidylcholine 19-25 Fatty acid salt 1,5-10 Maltose 67-78 Drug substance 1-10 3. The method of obtaining the composition according to claim 1, which consists in the fact that the salt of a fatty acid, phosphatidylcholine of plant origin and maltose are emulsified in water and the resulting emulsion is subjected to several homogenization cycles under high pressure of 800-1500 bar at a temperature of 40-45 ° C followed by lyophilization. 4. The method of obtaining the drug composition according to claim 2, which consists in the fact that in the case of a drug substance of protein nature, the composition according to claim 1 is dissolved in deirmed water, a drug substance of protein nature is added, stirred at room temperature, filtered and dried by lyophilization, and in the case of a non-protein drug substance, the fatty acid salt, maltose and drug substance are emulsified in water, phosphatidylcholine is added and the resulting suspension is subjected to several cycles (1-10) homogenization under high pressure of 800-1500 bar at a temperature of 40-45 ° C followed by lyophilization.