Liposome suspension stabiliser and method for production thereof
SUBSTANCE: stabiliser includes modified chitosan which is obtained by modifying chitosan particles located in an emulsion of an organic solvent - water, with pH 6.0-6.5, by first reacting a mixture consisting of a carboxylic acid in an organic solvent and a condensing agent, and then with an organic base, wherein the carboxylic acid used is either palmitic acid or stearic acid or dodecanoic acid, the condensing agent used is a mixture of hydroxysuccinimide and an aliphatic carbodiimide or formaldehyde and an aliphatic isocyanide, and the organic base used is triethylamine.
EFFECT: effective liposome composition stabiliser which can be obtained using a simple method.
8 cl, 3 tbl, 5 ex, 7 dwg
The claimed group of inventions relates to the biotechnology and pharmaceutical industry and can be used to create systems, in particular, liposomal compositions with enhanced stability and intended for implementation of targeted transport of physiologically active substances with the aim of improving therapeutic activity of drugs.
Liposomes are widely used as carriers of various drugs, including antiviral drugs. Since that time, have been revealed both advantages and disadvantages liposomal dosage forms. Liposomes can carry any compound structure (polar, non-polar, amphiphilic, with big and small molecular weight). In addition, they have high biocompatibility and Biodegradability.
In addition, in many cases using liposomes can provide targeted delivery of drug substances through passive targeting or conjugation of liposomes with biomarkers.
At the same time the disadvantages of liposomes as drug form is the difficulty associated not only with their production, but also occur during long-term storage of liposomal suspensions.
Liposomal suspensions are thermodynamically unstable colloidal system is neither, they quite quickly, sometimes within a few hours, stratified with the formation of two phases. Another disadvantage of liposomal compositions is the high rate of uptake of liposomes by macrophages, which does not allow to achieve a specific effect of liposomal dosage forms.
Summarizing data on the use of liposomal forms, we can conclude that many of these shortcomings are fundamental and cannot be solved without a fundamental change in the technology of production of liposomes.
One of the solutions to address some of the shortcomings of liposomal forms without losing all of the benefits available, is to obtain liposomes with composite shells. An example liposomes, membrane which is implemented, for example, polysaccharide, polypeptide, or cross-linked synthetic polymer. When this is achieved a significant increase in the strength of the membrane of the liposomes, as in this case, the polymeric compound performs the function of a kind of framework.
In addition, the resulting additional hydrophilic shell can reduce the interaction with proteins in the blood plasma of the patient and, accordingly, to increase their stability.
Thus, the composite liposomes are similar to organisms that have cell wall, the more the positive shell can be fixed biomarkers and other functional groups with the aim of further targeted drug delivery to the lesion.
An extremely important feature of liposomes with composite shell is their increased stability for long periods of storage under normal conditions, and the possibility of implementation in the form of various dosage forms, such as mortars, plasters, transdermal therapeutic systems or capsules.
The increased stability of the composite liposomes can significantly extend the list of drugs that can be applied in the form of liposomal forms.
Thus, the development of effective ways to obtain a stable liposomal compositions is an important task.
The prior art method of obtaining a stable sterile liposomal compositions for delivery of pharmaceutical agents, comprising the following stages:
a) providing a suitable water environment;
b) providing a suitable phospholipid;
C) providing at least one pharmaceutical agents capable of at least partially encapsulated in liposomes and selected from the
i) lipophilic amine and pharmaceutically acceptable acid, where the pharmaceutically acceptable acid selected from organic or inorganic acids, and
ii) pharmaceutically acceptable salts are organic acid lipophilic amine, it is not necessary, the pharmacist is Cesky acceptable acid includes pharmaceutically acceptable organic acid;
where the amount of pharmaceutically acceptable acid present in the composition is such that the pH of the liposomal composition is less than or approximately equal to the PKandamino group pharmaceutically active lipophilic amine;
d) the Association of the water environment, phospholipid and pharmaceuticals with the formation of liposomal composition; and
e) autoclaving the specified composition (see RF patent №2369384 to the invention Stable liposomal compositions", filing date 22.11.2004, published 10.10.2009,).
Liposomal compositions obtained in accordance with the known method, autoclaved to increase retention. In addition, stable liposomal suspension according to this invention do not settle, undergoing a phase change, they are resistant to oxidation and hydrolysis.
A method of obtaining membranes based on chitosan and salts of alginic acid for microcapsules containing phospholipid micelles characterized by sequential exposure of the cores of the microcapsules in 0,5+1,0% (weight/volume) solution of chitosan of medium or low viscosity in 1.0%acetic acid, 2.5-+3,0%solution of a chloride of the alkali earth metal, 0.5 to+1.0 percent (weight/volume) solution of sodium alginate and, again, 2.5-+3,0%solution of a chloride of the alkali earth metal (see RF patent №2411077 invention is a Method obtain membranes based on chitosan and salts of alginic acid for microcapsules, containing phospholipid micelles", filing date 09.06.2009, published 27.09.2011,).
Chitosan is a (1→4)-linked copolymer of glucosamine and acetylglucosamine obtained N-dezazetilirovanie of chitin in the primary environment (see figure 1).
Chitosan causes permanent interest thanks to its outstanding biological properties such as Biodegradability, bioactivity and biocompatibility.
Known for the invention is directed to creating technological process, which allows you to get the shell of microcapsules on the basis of alginate calcium or barium alginate and chitosan with different viscosity, affecting the relative percentage yield of phospholipid micelles from the core of the capsules in media simulating conditions in the gastrointestinal tract of man.
This invention allows you to create the shell of microcapsules based on chitosan and salts of alginic acid, through which the phospholipid micelles penetrate a certain speed due to the use of chitosan with different viscosity and different nature of the cation that forms a salt with alginic acid.
In addition, there is a method of stabilization of liposomal compositions by the addition of stabilizing substances, which ecovalence associated with lipid and is present in a quantity sufficient to cover whether the IDA, but insufficient to increase the viscosity of the composition, at the same time as stabilizing substances used polymers in which the repeating units contain one or more hydroxyl groups (polyhydroxyalkane); polymers in which the repeating units contain one or more amino groups (polyamine); polymers in which the repeating units contain one or more carboxyl groups (polycarboxylates); and polymers in which the repeating units contain one or more sharidny residues (polysaccharides). The molecular weight of the polymers can vary, and typically ranges from about 50 to about 5000000, preferably from about 100 to about 50,000. Preferred polymers with molecular weights from about 150 to about 10,000 and most preferably from about 800 to about 8000. Among the proposed polymers include natural and modified polysaccharides resin, such as xanthan gum, resin from the beans Robinia, guar or carageenan (from red algae Chondrus crispus); methoxycarbonyl pectin; starch; agar; cellulose and semisynthetic cellulose, such as methylcellulose, hydroxyethylcellulose, methoxycinnamate and hydroxypropylcellulose; agar; chitosan; carboxymethyl cellulose; alginic acid salts, such as sodium alginates and calcium; salts of glycosaminoglycans, including salts of hyaluronic key is lots; phosphorylated and sulfonated derivatives of carbohydrates (see RF patent №2181998 for the invention of "New compositions of lipids and stabilizing materials", filing date 27.03.1996, published 10.05.2002 year).
Such compositions have a high stability. In their application reduces the blockage of small vessels.
The closest technical solution to the claimed invention is a method of stabilization of liposomal compositions, which use a modified biopolymer chitosan.
Known stable liposomal composition refers to the target chitosan-derivatives containing octreotide, and more specifically N-succinyl-M-alkylate chitosan derivative and to N-alkylate carboxymethyl chitosan. In addition, a method of obtaining the composition (see patent CN 101396563 "Chitose derivates using octreotide as terget ligand and use thereof in medicalament", publication date 01.04.2009 year).
The disadvantages of this invention due to the use of complex multi-stage modification of chitosan, as well as applying carboxymethyl chitosan, impairing the load liposomes anionic drugs.
The technical result, which is aimed by the invention is the simplification of the mode of production (synthesis) of the modified chitosan, as well as improving the efficiency of the camera is the implementation of the liposomal compositions due to the introduction of the stabilizer based on modified chitosan.
This technical result is achieved by the fact that the stabilizer for liposomal suspensions, including the modified chitosan, according to the invention as a modified chitosan using chitosan, obtained by modification of chitosan particles, mixed with an emulsion of organic solvent - water with a pH of 6.0 to 6.5, by exposing the first mixture consisting of a carboxylic acid in an organic solvent and condensing agent, and then the organic base, the carboxylic acids used or palmitic, or stearic or dodecanoyl acid as the condensing agent is a mixture of hydroxysuccinimide and aliphatic carbodiimide or of formaldehyde and aliphatic isocyanate, as the organic base is triethylamine.
As the organic solvent used or dichloromethane, or chloroform, or toluene, or benzene.
In the invention using chitosan with molecular weight of 20-150 thousand daltons and a degree of deacetylation 85-98%.
As fatty acids are used carboxylic acids with a chain length of C10-C18preferably stearic and palmitic.
The degree of substitution of residues of fatty acids is 0.5 to 4.0%.
The optimum concentration of the stabilizing agent is 0.5-2.0 g/L.This technical result is achieved in that a method of obtaining a stabilizer for liposomal suspensions, including modification of chitosan according to the invention the modification of chitosan is carried out by alkalizing solution of chitosan hydrochloride prior to the formation of a colloidal solution with a pH of 6.0 to 6.5, which is affected by a mixture consisting of carboxylic acid in an organic solvent and condensing agent, after which the composition is intensely stirred and incubated, and then injected into the emulsion of the organic base to achieve a pH of 8.5, and then removing the organic solvent, the carboxylic acids used or palmitic, or stearic or dodecanoyl acid as condensing agent is a mixture of hydroxysuccinimide and aliphatic carbodiimide or of formaldehyde and aliphatic isocyanate, and as the organic base is triethylamine.
The organic solvent is removed by distillation or separation of the phases.
Thus, the essence of the proposed method consists in the synthesis of hydrophobically-modified chitosan on the phase boundary of the organic solvent is water.
The invention is illustrated by drawings, where
Figure 1 - structure of unmodified chitosan.
Figure 2 - structure of the modified chitosan, recip is spent by the reaction of acylation.
Figure 3 - structure of the modified chitosan, obtained by reaction IGO.
4 is a graph showing the distribution of particle size in the sample treated with a solution of unmodified chitosan.
5 is a graph showing the distribution in size of the particles in the sample liposomes sustained in solution modified with palmitic acid chitosan with a viscosity of 45 cps.
6 is a graph showing the distribution in particle size of the source (Record 3) and coated (Record 4) samples.
Fig.7 is a graph showing the change of medium size and polydispersity liposomes with a stabilizer (1) and without it (2).
In the process of synthesis of the chitosan molecules are oriented interphase and therefore the modification reaction proceeds more selectively obtaining derivatives with pronounced surface-active properties. Such molecules are actively interact with liposomes having a higher specific surface. So, hydrophobic fragments of modified chitosan dissolved in the lipid surface layer, while the glucosamine residues are oriented towards the aqueous phase. Hydrated chains of chitosan provide high steric stabilization of liposomes.
In addition, the polymer carries a charge, leading to electrostatic repulsion between them. Introduction of modified chitosan allows the ussite mechanical properties of liposomal membrane, including to increase the elasticity and viscosity of the lipid layer. In General, all these effects lead to a substantial increase in the stability of the liposomal dosage forms. For example, liposomes with a stabilizer in aqueous solutions is not affected maculele and the formation of conglomerates in the weeks and even months. Furthermore, the reduced rate of uptake of liposomes by macrophages, which leads to a more pronounced medicinal effect of liposomal drug. In particular, stabilized liposomes are able to pass through the mucous membranes without destroying the lipid layer, which opens up great possibilities for practical use in medical practice.
In the invention, a mixture of carboxylic acids in the presence of condensing agents - phenols and aliphatic carbodiimides or formaldehyde and aliphatic isocyanides. In both cases, the output of the modified product reaches 75-80%. The interaction is performed as follows. A solution of chitosan hydrochloride alkalinized to the formation of a colloidal solution, and then adds it to the solution of the initial reagents in organic solvent, which is used or dichloromethane, or chloroform, or toluene, or benzene. Both phases are mixed to obtain a stable emulsion with an effective IU Alki or homogenizer. Upon completion of the reaction, the emulsion breaks down spontaneously, otherwise the separation of the phases is achieved by adding diluted hydrochloric acid or solvent removal.
The end products in all cases are hydrophobic modified polymers based on chitosan (see figure 2), the structure of which is confirmed by PMR spectra. The reaction mechanism involves the formation of an activated ester and subsequent nucleophilic substitution with the formation of amide linkages. Thus obtained derivative showed a pronounced ability to stabilize emulsions of organic solvents in water at low (less than 2 g/l) concentrations. The result is biocompatible and biodegradable stabilizers of emulsions sensitive to changes in pH and ionic strength of the solutions, which are used to stabilize the liposomal suspensions and as components of liposomal compositions in the food and medical industries.
When using the initial reagents - palmitinic acid, hydroxysuccinimide and dicyclohexylcarbodiimide (<0.5 mmol/l) was selected product, soluble in water and hydrochloric acid solutions. In the IR spectrum was observed intense absorption bands in the region 1613 and 1550 cm-1. In NMR1N observed peaks of 1.55 was 2.76 and to 2.85 ppm, These peaks corresponds to the signals palmitic sour is s, that speaks of the passage of the acylation reaction.
Similar data were obtained with the use of stearic and dodecanol acids. At low concentration of initial reagents in organic solvent particles of chitosan easy desorbers with the interface and the reaction takes place in aqueous phase. The high concentration of initial reagents particles cannot easily decorrelates from the surface, so they accumulate therein, and the reaction occurs at the boundary of water and the organic phase.
If you use a different combination of the condensing agent is formaldehyde and aliphatic isocyanides, formed reaction products IGO. The advantage of this variant consists in the absence of side reaction products. At the same time, the main product of the reaction, the modified chitosan (see figure 3) has a pronounced surface-active properties.
Modification of chitosan aliphatic acids can improve its ability to stabilize the liposomal suspension.
For example, unmodified chitosan (figure 1) has weak adhesion to liposomal membranes (membranes) due to which the main content of the polymer is in solution and only a small fraction is deposited on the membranes.
Figure 4 shows the graph of the distribution of particle size in the sample processed what astora unmodified chitosan.
The sample is polydisperse, the index of polydispersity averaged 0,46 that is significantly higher than 0.2, which is accepted as the upper value for monodisperse samples. The distribution clearly shows two major peaks: one around 320 nm and the second about 37 nm. The peak in the region of 30-40 nm is characteristic for the samples of the original polymer, which allows to conclude that a significant portion of chitosan remains in solution and does not participate in the formation of the shell. In addition, the particle size increased significantly (from 243 to 320 nm), which indicates the formation of loose shell on the surface of liposomes.
Figure 5 shows a graph of the distribution of particle size in the sample of liposomes sustained in solution modified with palmitic acid chitosan with a viscosity of 45 cps. The sample is monodisperse, and the index of polydispersity on average amounted to 0.17, which is slightly different from the sample in figure 4.
In the distribution there is one peak at 302 nm, which indicates the increasing size of liposomes by forming a shell (see Fig.6). At the same time, the increase is not so significant as in the case of unmodified chitosan, which confirms the formation of a thicker shell.
Crucial for this method are two factors: the rate of deposition of chitosan on membrane l is bosomy and stability of the resulting suspensions. Both values can be determined by the rate of disappearance of small particles in the range of 30-80 nm in the spectra of light scattering samples.
The results of the deposition rate samples of chitosan with different viscosity (10, 20, 45 and 120 cps), are shown in Table 1.
|The deposition rate of the chitosan modified with 2%palmitic acid, depending on the viscosity of chitosan.|
|The deposition rate, h||0,5||1,5||5,0||14,0|
As can be seen from Table 1, the viscosity increase rate of deposition of chitosan on the surface of liposomes decreases rapidly. At the same time chitosan with a higher viscosity provides better stabilization of liposomes. Thus, the inventive stabilizer provides reliable stabilization of liposomal membrane.
Technical solutions, which coincides with the set of essential features of the claimed invention, is not revealed, which allows to make a conclusion about compliance and the claimed invention to such a condition of patentability as "novelty".
Declare the essential features that determine the receipt of the indicated technical result that is explicitly not follow from the prior art, which allows to make a conclusion on the compliance of the claimed invention to such a condition of patentability as "inventive step".
The condition of patentability "industrial applicability" is confirmed by examples of specific applications.
The inventive stabilizer for liposomal suspensions received and used as follows.
Carry out the synthesis of the modified chitosan, wherein the chitosan in the amount of 250 mg dissolved in 100 ml of 0.2%solution of hydrochloric acid in water. The resulting solution was titrated with 0.2%solution of sodium hydroxide in water until the turbidity of the solution (pH 6.0 to 6.5). To the resulting solution was added the calculated amount of fatty acid or palmitic, or stearic or dodecanol) and a condensing agent (mixture of hydroxysuccinimide and dicyclohexylcarbodiimide or aliphatic isocyanate and formaldehyde) in 20 ml of organic solvent (or dichloromethane, or chloroform, or benzene, or toluene).
The mixture is intensively stirred until a homogeneous emulsion. Then the pH of the mixture was adjusted to 8.5 by the addition of triethylamine and maintain the emulsion for 1 hour. After that, the organic solvent from onaut by heating and vigorous stirring. The aqueous phase is filtered and concentrated to 10-15 g/l by evaporation on a rotary evaporator. The resulting solution can be directly used for deposition on the surface of liposomes.
Surface modification of liposomes. To the liposomal suspension is added an aqueous solution of hydrophobically-modified chitosan with a concentration of 10-15 g/l to establish its concentration in suspension up to 1 g/L. the resulting solution was incubated for 3 hours at room temperature.
The parameters obtained liposomes with stabilizer was evaluated using dynamic light scattering spectrophotometer Malvem Zetasizer Nano SL. For the experiment liposomal composition was diluted with distilled water to a concentration of the hydrophobic-modified chitosan 1 g/l and was placed in the cuvette of the spectrophotometer. The results of the evaluation confirmed that the inventive liposomal suspension, which entered the stabilizer obtained by the claimed method, has the necessary medical use characteristics, in particular, up to 95% of the liposomes have a diameter in the range of inlet 150 up to 450 nm.
The main parameters of the inventive liposomal compositions are shown in Table 2.
|C is Uchenie|
|The total lipid concentration in solution, g/l||10-50|
|The content of the antioxidant, g/l||0-4,0|
|The stabilizer content, g/l||0,5-2,0|
|Shelf life at t=20°C||up to 6 months|
|The average particle size, nm||Inlet 150 up to 450|
In addition, comparative studies on the evolution of the average and polydispersity liposomes, which was introduced by the inventive stabilizer, and liposomes without stabilizer.
The data obtained are given in Table 3.
|Modify the sample stabilized liposomes depending on storage time|
|Time, months||The average size of liposomes, mi||Index polydispersity|
Analysis of the data given in Table 3, allows to conclude that the change of size and polydispersity liposomes does not exceed 2% per month.
Comparative analysis of changes of parameters of samples of liposomes with the stabilizer (1) and without (2) is shown in Fig.7. The analysis showed that the average size of liposomes without stabilizer increased within 20 days from 264 to 336 nm, which corresponds to the aging rate of about 35% per month. At the same time, the index polydispersity has increased by half, allowing you to use the data suspension only for 3-4 days after receipt. At the same time the parameters of liposomes with stabilizer did not significantly change during storage at room temperature for 5 months.
All of the above confirms the possibility of practical use of the proposed stabilizer for liposomal suspensions.
The implementation of the claimed invention is confirmed by examples of specific performance.
Example No. 1.
Getting stabilizer liposomal suspensions using the acylation reaction.
Synthesis of modified chitosan carry out follow the way. Chitosan in the amount of 250 mg dissolved in 100 ml of 0.2%aqueous solution of hydrochloric acid in water. The resulting solution was titrated with 0.2%solution of sodium hydroxide in water until the turbidity of the solution (pH 6.0 to 6.5). To the resulting solution was added the calculated amount of the mixture from hydroxysuccinimide, dicyclohexylcarbodiimide and palmitic acid in 20 ml of dichloromethane. The mixture is intensively stirred until a homogeneous emulsion. Then the pH of the mixture by adding the organic base is triethylamine adjusted to 8.5 and incubated for 1 hour. Then dichloromethane is distilled off by heating and vigorous stirring. The precipitate is filtered off, dicyclohexylamine and the aqueous phase is concentrated to 10-15 g/L. the resulting solution can be used for deposition on the surface of liposomes.
Example No. 2.
Getting stabilizer liposomal suspensions using the acylation reaction.
Synthesis of modified chitosan as follows. Chitosan in the amount of 250 mg dissolved in 100 ml of 0.2%aqueous solution of hydrochloric acid in water. The resulting solution was titrated with 0.2%solution of sodium hydroxide in water until the turbidity of the solution (pH 6.0 to 6.5). To the resulting solution was added the calculated amount of the mixture of hydroxysuccinimide, dicyclohexylcarbodiimide and dodecanol acid in 20 ml of benzene. The mixture is intensive stirred until a homogeneous emulsion. Then the pH of the mixture by adding triethylamine adjusted to 8.5 and incubated for 1 hour. Thereafter, the benzene solution is separated in a separating funnel. The aqueous phase is filtered from the resulting precipitate of dicyclohexylamine and concentrated to 10-15 g/L. the resulting solution can be used for deposition on the surface of liposomes.
Example No. 3.
Getting stabilizer liposomal suspensions using reaction IGO.
Synthesis of modified chitosan as follows. Chitosan in the amount of 250 mg dissolved in 100 ml of 0.2%aqueous solution of hydrochloric acid in water. The resulting solution was titrated with 0.2%solution of sodium hydroxide in water until the turbidity of the solution (pH 6.0 to 6.5). To the resulting solution was added the calculated amount of the mixture of methoxyacridine, formaldehyde and stearic acid in 20 ml of toluene. The mixture is intensively stirred until a homogeneous emulsion. Then the pH of the mixture by adding triethylamine adjusted to a value of 8.5, and then the resulting emulsion was incubated for 1 hour. During this time there is a spontaneous separation of the emulsion. After the reaction, the toluene is separated in a separating funnel, and the aqueous phase is concentrated to 10-15 g/L. the resulting solution can be used for deposition on the surface of liposomes.
Example No. 4.
Receiving the stabilizator liposomal suspensions using reaction IGO.
Synthesis of modified chitosan as follows. Chitosan in the amount of 250 mg dissolved in 100 ml of 0.2%aqueous solution of hydrochloric acid in water. The resulting solution was titrated with 0.2%solution of sodium hydroxide in water until the turbidity of the solution (pH 6.0 to 6.5). To the resulting solution was added the calculated amount of the mixture of cyclohexyldiamine, formaldehyde and palmitic acid in 20 ml of chloroform. The mixture is intensively stirred until a homogeneous emulsion. Then the pH of the mixture by adding triethylamine adjusted to a value of 8.5, and then the resulting emulsion was incubated for 1 hour. After chloroform is distilled off by heating and vigorous stirring. The aqueous phase is filtered and concentrated to 10-15 g/L. the resulting solution can be used for deposition on the surface of liposomes.
Example No. 5.
The stabilizer of liposomal suspensions.
Carry out the surface modification of liposomes to the liposomal suspension is added an aqueous solution of hydrophobically-modified chitosan with a concentration of 10-15 g/l to establish its concentration in suspension up to 1 g/L. the resulting solution was incubated for 3 hours at room temperature.
The claimed group of inventions allows you to get a stabilizer for liposomal suspensions having high stabiles is updated and intended for implementation of targeted transport of physiologically active substances.
1. Stabilizer for liposomal suspensions, including the modified chitosan, characterized in that the modified chitosan using chitosan, obtained by modification of chitosan particles, mixed with an emulsion of organic solvent - water with a pH of 6.0 to 6.5, by exposing the first mixture consisting of a carboxylic acid in an organic solvent and condensing agent, and then the organic base, the carboxylic acids used or palmitic, or stearic or dodecanoyl acid as the condensing agent is a mixture of hydroxysuccinimide and aliphatic carbodiimide or of formaldehyde and aliphatic isocyanate, as well as organic bases - the triethylamine.
2. Stabilizer for liposomal suspension according to claim 1, characterized in that the organic solvent used or dichloromethane, or chloroform, or toluene, or benzene.
3. Stabilizer for liposomal suspension according to claim 1, characterized in that the use of chitosan with molecular weight of 20-150 thousand daltons and a degree of deacetylation 85-98%.
4. Stabilizer for liposomal suspension according to claim 1, characterized in that the degree of substitution of residues of carboxylic acids is 0.5 to 4.0%.
5. A method of producing a stabilizer for liposomal suspension according to claim 1, on the expectation modified chitosan, characterized in that the modification of chitosan is carried out by alkalizing solution of chitosan hydrochloride prior to the formation of a colloidal solution with a pH of 6.0 to 6.5, which is affected by a mixture consisting of carboxylic acid in an organic solvent and condensing agent, after which the composition is intensely stirred and incubated, and then it is injected organic base to achieve a pH of 8.5, and then removing the organic solvent, the carboxylic acids used or palmitic, or stearic or dodecanoyl acid as the condensing agent is a mixture of hydroxysuccinimide and aliphatic carbodiimide or of formaldehyde and aliphatic isocyanate and as the organic base is triethylamine.
6. A method of producing a stabilizer for liposomal suspension according to claim 5, characterized in that the use of chitosan with molecular weight of 20-150 thousand daltons and a degree of deacetylation 85-98%.
7. A method of producing a stabilizer for liposomal suspension according to claim 5, characterized in that the degree of substitution of residues of carboxylic acids is 0.5 to 4.0%.
8. A method of producing a stabilizer for liposomal suspension according to claim 5, characterized in that the organic solvent is removed by distillation or separation of the phases.
SUBSTANCE: invention relates to the field of organic synthesis. A method of obtaining a water-insoluble sulphur-containing chitosan-based biopolymer includes interaction of chitosan with a thiomethylating agent, preliminarily obtained by saturation of a formaldehyde solution with gaseous H2S, with molar ratio chitosan: formaldehyde: hydrogen sulphide 1:6:4, at a temperature of 60°C for 20-25 hours.
EFFECT: invention ensures obtaining the water-insoluble sulphur-containing chitosan-based biopolymer, which possesses a complexing activity to ions of noble metals (Pd, Pt).
1 ex, 1 tbl
SUBSTANCE: skins of pond fish are flushed with cold flushing water during 10-15 minutes. They are crushed to the size of 2-3 mm. Water extraction is performed at the temperature of 40-45°C during 40-50 minutes at the ratio of crushed skins to water, which is equal to 1:1 at periodic mixing. Then, they are filtered; liquid fraction is dried in a spraying drier at the drier outlet product temperature of 60-65°C during 15-25 minutes so that hyaluronic acid is obtained. Solid fraction is subject to bleaching during 12 hours with hydrogen peroxide-salt solution that is prepared by mixing of 1 l of 3% hydrogen peroxide and 20 g of sodium chloride. Treatment of bleached solid fraction is performed with 1.0-1.2% solution of sodium hydroxide during 24 hours at the temperature of 20-25°C with further neutralisation of the obtained mixture with 3% boric acid solution. Treatment of swollen solid fraction is performed with Pancreatin ferment preparation solution taken in the quantity of 0.5-0.6% to the weight of solid fraction during 1.5-2.0 hours at the temperature of 37-40°C. Flushing of solid fraction is performed with cold flushing water for removal of Pancreatin residues so that collagen is obtained. The obtained collagen, depending on the purpose, is supplied for drying in drying chambers with forced air circulation at the temperature of 18-20°C during 12 hours and storage in dry ventilated rooms at the temperature of not higher than 20°C during 24 months or frozen to the temperature of minus 18 - minus 20°C and stored at the temperature of minus 18 - minus 20°C during 24 months. The liquid fraction dried in the spraying chamber is stored at the temperature of 0-4°C during 12 months or dissolved in physiological buffer solution.
EFFECT: improvement of the method.
2 dwg, 1 tbl, 1 ex
SUBSTANCE: production method of glucan-chitosan complex from yeast biomass of brewing waste involves mechanical and ultrasonic treatment of yeast biomass, destruction of proteins by treatment of the obtained suspension using alkali reagents with further extraction of a target product. As biomass, Saccharomyces living yeast is used. First, yeast is frozen to -15°C during 24 hours. After mechanical destruction, biomass is treated for 15 minutes at 20°C in an ultrasonic bath with frequency of an emitter of 35 kHz and voltage of 285 W. Biomass is acidified with chlorhydric acid till pH=5.5 and treated with ferment preparation in the amount of one pellet containing lipase - 3500 units of Ph.Eur., amylase - 4200 units of Ph.Eur. and protease - 250 units of Ph.Eur. per kilogramme of biomass in terms of dry substance; then, lipid components of yeast are removed. Fermentation is performed at t=20-29°C during 30-60 minutes. Destruction of proteins is performed at 55°C by means of a water bath during 60 minutes by treatment using 4% water solution of caustic soda at the ratio of yeast biomass and alkali, which is equal to 1:4. The medium is neutralised and hydrosol of glucan-chitosan complex is deposited by centrifugation during 10 minutes. The deposit is dried at t=55°C during 48 hours.
EFFECT: invention allows improving the quality of the obtained complex and its biological activity.
SUBSTANCE: invention relates to production of hydroxyalkyl derivatives of polysaccharides. The method of producing 2,3-dihydroxypropyl chitosan involves reacting chitosan with glycidol in the presence of hydrochloric acid with ratio glycidol:chitosan:hydrochloric acid = (2-6):1:1, at room temperature until a gel forms. The mixture is then heated at 55-65°C for 12-14 hours and the reaction mass is treated with water. The mixture is then deposited, subjected to hot extraction with water-soluble alcohols or ketones and dried.
EFFECT: invention simplifies the method of production and output of the end product and improves sorption properties of the compound.
1 tbl, 3 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: there are presented: using benzophenanthridine alkaloid salts for preparing therapeutic agents for treating tumours, wherein the alkaloid salt is found in the form luteic, phosphatidic or hyaluronic acid, the benzophenanthridine alkaloid salt with phosphatidic acid or hyaluronic acid, and a based pharmaceutical composition for treating tumours.
EFFECT: what is shown is cytotoxic activity of the sanguinarine salts according to the invention at least twice increased in all studied tumour cell lines in relation to the chloride salt; it is suggested to be caused by higher absorption by the tumour cells.
12 cl, 8 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to a recovered imidised biologically compatible polymer functionalised by an imide group. The above polymer is selected from the group consisting of polyethylene oxide, partially or completely hydrolysed by polyvinyl alcohol, polyvinylpyrrolidone, polyethyloxazoline, polyoxypropylene oxide block copolymers (poloxamers and meroxapol), polyethylene oxide and poloxamine copolymer, carboxymethyl cellulose and hydroxyalkylated cellulose, polypeptides, polysaccharides, carbohydrates, polysaccharose, hyaluronic acid, dextran, heparin sulphate, keratan sulphate, chondroitin sulphate, heparin, alginate, gelatin, collagen, albumin, ovalbumin, complex polyphosphoesters, polylactides, polyglycolides, polycaprolactones, polyamides, polyurethanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, polymaleic acids, polyamino acids, polyvinyl alcohol, polyvinylpyrrolidone, polyhydroxy cellulose, chitin, chitosan, and copolymers, ternary copolymers, or combinations or mixtures of the aforementioned materials. Also, the invention refers to a composition for a tissue adhesive, a medical device and a pharmaceutical composition.
EFFECT: invention represents additionally modified or functionalised imidised polymers.
9 cl, 2 ex, 20 dwg
SUBSTANCE: fish roe is homogenised. Fish roe hydrolysis is carried out with a ferment preparation "Collagenase" in presence of an inhibitor for 10-12 hours. Chitosan is added to the produced hydrolysate at the ratio of 0.5-1.0:1.0. Components are mixed.
EFFECT: invention makes it possible to accelerate process of chitosan-nucleic complex production.
1 dwg, 1 tbl, 3 ex
SUBSTANCE: method includes depolymerisation of a high-molecular chitosan with hydrogen peroxide. The process of chitosan depolymerisation is carried out in a double-phase system. The solid phase is activated chitosan with Mav = 450-650 kDa and the average particle size of 0.05-0.20 mm. The liquid phase is a water solution of H2O2 with concentration of H2O2 in a reaction system equal to 1-7%. The reaction is carried out for 120-180 minutes at 70°C. Then phase separation of produced chitosan homologs is carried out by means of filtration via paper or textile surface of the produced reaction mixture. The produced filtrate contains water-soluble chitosan oligomers.
EFFECT: invention makes it possible to quantitatively control extent of conversion of an initial high-molecular chitosam into oligomer and low-molecular structures of its homologs.
1 tbl, 1 ex
SUBSTANCE: invention relates to a method of purifying chondroitin sulphate and can be used in food and cosmetic industry and in medicine. The method involves electrochemical deposition to obtain a hydrogel of chondroitin sulphate, stabilisation, removal from the electrode, washing and drying. The chondroitin sulphate is dissolved in a 0.01-0.1 n alkali solution in ratio of 1:50-1:200 and deposited in an alkaline medium with constant cooling and stirring. The solution is stirred at a rate of 10-20 rpm. Current density is equal to 1-10 A/m2. Voltage is preferably not lower than 2.7 V. The hydrogel of chondroitin sulphate is stabilised in a 0.05-0.5 n HCl solution.
EFFECT: invention enables to obtain chondroitin sulphate with high weight ratio of the basic substance and increases output of the end product.
5 cl, 1 ex
SUBSTANCE: present invention relates to a method of producing a N,S-cyclo-containing chitosan derivative. Described is a method of producing a chitosan-based N,S-cyclo-containing polymer (I) which contains in the macrochain 1-oxa-6-thia-4,8-diazocycloundecane fragments: I, by reacting chitosan with formaldehyde and a S-containing compound, characterised by that the S-containing compound used is hydrogen sulphide, the formaldehyde solution is pre-saturated with H2S and the reaction is carried out with molar ratio chitosan: formaldehyde: hydrogen sulphide of 1:2:1, at temperature of 0-60°C in a chloride medium for 24 hours.
EFFECT: obtaining modified chitosan which exhibits properties of a highly efficient heavy metal sorbent for waste water treatment, an extractant for separating rare, noble and precious metals and a complexing agent for biological molecules.
1 tbl, 3 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to a carrier applicable for the local drug delivery. A drug is enclosed in a carrier, and the carrier comprises a coating able to release an enclosed drug as a result of a local stimulus. The coating additionally surrounds the contrast agent MR 19F which changes its detectability after being released from the carrier. The invention refers to a method for the drug delivery to an MRT-controlled individual, wherein the method involves administering the above carrier into the individual enabling the carrier releasing the drug, and forming MR 19F images with the use of a contrast produced by the contrast agent MR 19F.
EFFECT: invention enables monitoring the beginning of the drug release from the carrier.
18 cl, 11 dwg, 2 tbl, 2 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to pharmaceutics and concerns irinotecan liposomes or its hydrochloride containing irinotecan or its hydrochloride, neutral phospholipid and cholesterol, wherein the weight ratio of cholesterol to neutral phospholipid makes 1:3-5, and a method for preparing them.
EFFECT: liposomes have higher stability.
15 cl, 3 dwg, 10 tbl, 10 ex
SUBSTANCE: disclosed is a vesicle-containing composition which is characterised by that it contains: (A) a silicone-based surfactant which is silicon modified with polyoxyalkylene, (B) one or more anionic surfactants selected from polyoxyethylenealkyl(12-15)ether-phosphate, acylmethyltaurate and acylglutamate in amount of 0.001-0.2 wt %, (C) polar oil having IOB of 0.05-0.80 and/or silicone oil, and (D) water, which contains a water-soluble medicinal agent, in amount of 0.5-5 wt % of the weight of the composition, where the silicone-based surfactant (A) forms vesicles; the anionic surfactant(s) (B) is attached to the surface of the vesicles; and the polar oil and/or silicone oil (C) is present inside the bilayer membrane of the vesicles.
EFFECT: disclosed composition has excellent stability even in the presence of high concentrations of a water-soluble medicinal agent.
6 cl, 8 tbl, 10 ex
SUBSTANCE: liposomal pharmaceutical composition includes a medicinal substance, lipids in form of phosphatidylcholine and cholesterol, and additionally contains minor positively charged component in form of cetylpyridinium chloride or stearyl ethanolamine.
EFFECT: high bioavailability of the medicinal substance through active transport to body organs and tissue.
10 cl, 3 dwg, 5 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to medicine and deals with pharmaceutical composition for local application in form of gel, which contains enzyme deoxyribonuclease (DNA-ase) and/or ribonuclease (RNA-ase) and liposomes, which can be applied in medicine for treatment and prevention of viral infection.
EFFECT: invention ensures obtaining stable pharmaceutical compositions.
1 cl, 7 ex, 1 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to a method for preparing a magnet-sensitive lipid composition. The declared method involves preparing a therapeutic agent containing cisplatin, a source of phospholipids and a magnetism carrier representing pirocarbon-coated ferric nanoparticles to be exposed to UV radiation. The above phospholipid source is the pharmacopeial drug 'Phosphogliv' with the composite prepared in the micellar form.
EFFECT: providing the uniformity and thermodynamic stability of the composite prepared over a short optimum period of time with using no organic solvents.
1 dwg, 2 ex
SUBSTANCE: liposomes are used as a matrix for activated ferment - horseradish peroxidase. To 5 mg of horseradish peroxidase oxygenated with a periodate method there added is 1 ml of liposome suspension in 0.01 M solution of carbonate-bicarbonate buffer at pH 9.5, and subjected to ultrasonic treatment during 1 min. Then it is incubated for 1 hour; immobilised with immunoglobulins in concentration of 5 mg during 2 hours at the temperature of 22±4°C; stabilised with 5 mg of sodium borane with further gel-chromatographic cleaning.
EFFECT: invention allows obtaining liposomal-immunoperoxidase conjugate for indication of infectious agents in an immunoenzymometric analysis and increasing service life of a preparation up to 6 years.
1 tbl, 3 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to pharmaceutical industry and represents a thermally-sensitive liposome for treating cancer in a subject, containing at least one phosphatidylcholine, at least one phosphatidylglycerol, monostearoyl phosphatidylcholine, pegylated phospholipid and an active substance specified in a group consisting of taxotere, docetaxel and carboplatin wherein the liposome has a gel to liquid transition point from approximately 39°C to approximately 45°C.
EFFECT: invention provides storage-stability for a long period of time of one month or more at low temperatures.
9 cl, 8 ex, 8 dwg
FIELD: medicine, pharmaceutics.
SUBSTANCE: group of inventions refers to medicine, and concerns a liposomal drug preparation containing a polyvalent ionic preparation as an active substance with one or more dissociating groups at dissociation constant 4.5-9.5, wherein a liposome has a size of about 30-80 mm and a phospholipid bilayer has a phospholipid with a phase transition temperature above the body temperature so that the phase transition temperature of the liposome is above the body; a method of treating a tumour in a patient involving administering the above liposomal drug preparation.
EFFECT: group of inventions provides the higher therapeutic efficacy index of the liposomal preparation, especially more effective control of liposomal drug preparation recovery after targeted on the involved area, reduced loss of the drug preparation during the blood circulation of the liposomal drug preparation and more effective drug release into the target tissues.
17 cl, 21 ex, 3 dwg, 9 tbl
SUBSTANCE: there are presented: a liposomal preparation for the pulmonary delivery, wherein the liposomes a surface of which is modified by at least one polymer specified in a group consisting of terminally hydrophobisated polyvinyl alcohols and chitosan, an encapsulated gene, a method for preparing and a method of treating a pulmonary tissue disease involving the stage of administering the above liposomal preparation into the patient's lung. It has been shown that the liposome modified by terminally hydrophobisated polyvinyl alcohol may be kept on the pulmonary tissue surface for a long time.
EFFECT: high effectiveness of the pulmonary delivery of the claimed liposomal preparation for a relatively short time.
10 cl, 1 dwg
SUBSTANCE: invention relates to a haemostatic anti-burn and wound-healing composition in the form of a hydrogel, which contains an active gel-forming component, a plasticiser, active and auxiliary components, namely a water-soluble heteropolymer of chitosonium salt in an amount from 1.0 to 10.0 wt %, a dexpanthenol substance and/or a 2-allyloxyethanol substance in an amount from 1.0 to 10.0 wt %, immobilised medicinal substances of aminocaproic or tranexamic acid in an amount from 0.1 to 5.0 wt % and calcium chloride in an amount from 0.05 to 2.0 wt %, immobilised medicinal substances of lidocaine or anylocaine in an amount from 0.1 to 5.0 wt % and chlorhexidine in an amount from 0.005 to 0.1 wt % and water.
EFFECT: extension of exploitation possibilities in stopping external capillary bleedings, local treatment of injured skin, non-healing wounds and local burns.