RU2669374C2 - Bis-naphthazarin based agent and method for production thereof - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: invention relates to pharmaceutical industry, in particular to a method for producing a bis-naphthazarin based agent for ophthalmic use in the form of liposomes, possessing anti-inflammatory and antiallergic activities. Method for producing a bis-naphthazarin based agent for ophthalmic use in the form of liposomes, possessing anti-inflammatory and anti-allergic activities, in which the aqueous and lipid phases are separately prepared, aqueous phase is prepared by mixing 0.01 M of phosphate buffer with a pH of 7.4 and dissolving a trehalose therein, lipid phase is prepared by melting a mixture of phosphatidylcholine and cholesterol with Tween-80, then ethylidene-6,6'bis(2,3,7-trihydroxynaphthazarin), dissolved in an alcoholic solution of hydrochloric acid is added to the resultant melt and stirred until a clear solution is obtained, followed by the addition of an aqueous phase, resultant mixture is stirred, the ethyl alcohol is removed in vacuo, the volume is adjusted with water, the resultant suspension is sonicated, are purified from a non-encapsulated substance and filtered through a membrane filter under certain conditions (options). Bis-naphthazarin based agent for ophthalmic use in the form of liposomes, possessing anti-inflammatory and antiallergic activities.

EFFECT: said solution allows producing a liposomal structure ensuring the solubility of bis-naphthazarins, their stability and ophthalmic availability in installations in the form of eye drops.

4 cl, 2 dwg, 9 ex, 2 tbl

Description

The invention relates to medicine, the pharmaceutical industry, and in particular to a method for producing an ophthalmic agent based on bisnaphthazarin in the form of liposomes that has anti-inflammatory and anti-allergic effects. The inventive liposome composition as an active component contains pigment bisnaphthazarin-ethylidene-6,6'-bis (2,3,7-tri-hydroxy-naphthazarin), lipids in the form of phospholipids and sterols, co-solvent, isolated from the needles and shell of the green sea urchin Strongylocentrotus droebachiensis aqueous phase and cryoprotectants. The liposomal structure ensures the solubility of bisnaphthazarines, their stability and ophthalmic bioavailability during installations in the form of eye drops.

Ophthalmic dosage forms for topical application are quickly removed from the conjunctival sac, therefore, the absorption time of the drug is only a few minutes, determining the low bioavailability of the drug, usually less than 5%. The cornea is the next significant barrier to drugs. The cornea is not only a barrier to mechanical damage, but also provides selective permeability to chemical compounds in order to maintain homeostasis in the tissues of the anterior chamber of the eye. Ensuring the delivery of substances necessary for life determines the presence in the corneal epithelium of developed systems of facilitated and active transport. In order to increase efficiency and increase local bioavailability, numerous controlled release systems of ophthalmic preparations based on soluble polymers have been proposed [Breton P., Larras V., Roy D. et al. Biocompatible poly (methylidene malonate) made materials for pharmaceutical and biomedical applications. Eur. J. Pharm. Biopharm. 2008; 68: 479-495]. For a number of reasons (complexity of use, annoying effect), these systems could not displace the local use of drugs. In this regard, the optimization of existing ophthalmic medicines for local use remains an urgent task. The use of substances that increase the permeability of the cornea is most effective against hydrophilic and large molecules. In order to increase the transport of drugs through the cornea, specific delivery systems are used. The dual nature of the lacrimal barrier from the physicochemical point of view, as well as the heterogeneous structure of the cornea, cause considerable interest in liposomes as a drug delivery system. The unique structure of the phospholipid bilayer of liposomes makes them equally tolerant of hydrophilic and lipophilic media. Along with this, liposomes can transport lipophilic substances by dissolving in the lipid membrane [Alyautdin R.K., Iezhitsa I.N., Agarval R. Transport of drugs through the cornea of the eye: prospects for the use of liposomal dosage forms. Bulletin of Ophthalmology. 2014; 4: 117-122].

An important advantage of the use of liposomal forms is that pharmacologically active compounds contained in liposomes retain their active form and remain stable without being exposed to various body systems and do not have toxic effects on healthy body cells.

One of the promising directions for the creation of liposomal forms is the creation of water-soluble dosage forms based on hydrophobic substances, which helps to increase their bioavailability and effectiveness (Pharmaceutical development: concept and practical recommendations. Scientific and practical guide for the pharmaceutical industry. Edited by S. N. Bykovsky and et al., 2015.472 s.).

Encapsulation of substances in liposomes can be carried out in a number of ways, the main of which are the following:

- the film method involves the formation of a thin film of phospholipid upon evaporation of the organic solvent, followed by its dispersion in a suitable aqueous medium;

- the ultrasonic method allows liposomes to be obtained by suspending a suitable lipid in an aqueous medium, followed by sonication of the mixture;

- solvent-based methods: quick mixing of a lipid in ethanol and water by injecting the lipid into an aqueous solution.

In addition to the rather laborious technology for the production of liposomes, some difficulties may arise in their use due to phase instability over time, which is expressed in sedimentation and phase separation, merging and agglomeration of liposomes during storage.

Often, liposomes include cholesterol, because this substance is capable of simulating the physicochemical properties of a lipid bilayer. Its addition in the ratio to phospholipid from 1: 1 to 2: 1 increases the stability of the bilayer and reduces its permeability. The action of cholesterol is based on the fact that the oxygen of its hydroxyl group binds the methyl groups of phosphotidylcholine, and the hydroxyl group of cholesterol interacts with the C = O / P = O groups of phosphatidylcholine (Arshinova O. Yu. Et al. Features of lyophilization of liposomal drugs (review). Chem .-Pharmac. J. 2011; 46 (4): 29-34).

Bisnaphthazarines are dimeric representatives of polyhydroxynaphthoquinone pigments and are capable of exhibiting significant antioxidant activity, for example, they are able to neutralize the action of the main initiators of the non-enzymatic process of oxidation of membrane lipids - iron cations that accumulate in the area of ischemic tissue damage (Kuwahara R. et al. Antioxidant shell n of purple sea urchin Anthocidaris crassispina. LWT-Food Sci. Technol. 2009, 42 (7): 1296-1300). It is known that binaphthoquinone pigments from the shells of green sea urchins due to the presence of a large number of hydroxyl groups have anti-radical activity against the DPPH radical, which exceeds that of the monomeric echinochrome A (Pozharitskaya O. N. et al. Evaluation of free radical-scavenging activity of sea urchin pigments using HPTLC with post-chromatographic derivatization. Chromatographia. 2013; 76 (19-20): 1353-1358).

The isolated pigment complex of bisnaphthazarines from the needles and carapace of the green sea urchin Strongylocentrotus droebachiensis enriched with ethylidene-6,6'-bis (2,3,7-tri-hydroxy-naphthazarin) showed significant anti-inflammatory activity in the rat carrageenin air sac model and models of allergic conjunctivitis in rabbits (RF Patent 2545692, publication date 04/10/2015).

Despite the high pharmacological activity, the applicability of naphthoquinone-type pigments in the form of solutions in medical practice is complicated due to their low solubility and instability in aqueous solutions and, as a consequence, low bioavailability (He J. Complex of shikonin and β-cyclodextrins by using supercritical carbon dioxide. J. Inclusion Phenomena Macrocyclic Chem. 2009; 63 (3-4): 249-255; Li DM et al. Extraction, structural characterization and antioxidant activity of polyhydroxylated 1,4-naphthoquinone pigments from spines of sea urchin Glyptocidaris crenularis and Strongylocentrotus intermedius. Eur. Food Research Technol. 2013; 237 (3): 331-339).

Increasing the solubility of poorly soluble substances can be achieved by creating salts based on them. From the existing level of technology there is known a tool - the closest analogue - the drug "Histochrome", exhibiting antioxidant and cardioprotective effects, used to treat myocardial infarction, coronary heart disease, as well as injuries and burns of the eyes, and which is an aqueous solution of di- and tri- sodium salts of echinochrome (2,3,5,6,8-pentahydroxy-7-ethyl-1,4-naphthoquinone) isolated from flat sea urchins (RF Patent No. 2137472, publication date 09/20/1999).

Another way to obtain aqueous solutions of hydrophobic substances is the creation of liposomal forms. A known method for the preparation and use of liposomes based on the plant pigment shikonin (5,8-dihydroxy-3 (1'-hydroxy-4-methylpent-3-enyl) -1,4-naphthoquinone), which has wound healing, antimicrobial, anti-inflammatory and antioxidant effects (CN patent No. 102091037 (A), publication date 06/15/2011).

Closest to the claimed invention is a tool that is a molecularly encapsulated form of echinochrome from flat sea urchins, obtained by creating conditions during molecular encapsulation, under which the binding of echinochrome with the polar part of the surfactant molecules, which allows stabilizing echinochrome in an aqueous medium at a concentration of up to 0 5% or more. Structurally diphilic surfactants are chosen as surfactants, namely Cremophor RH-40, Cremophor EL, Cremophor A 25, Cremophor A 6, Soluplus® (grafted copolymer of polyvinylcaprolactam, polyvinyl acetate and polyethylene glycol), tween-80, or a mixture of monomeric and polymeric surfactants . Surfactants from the above list differ in the structure of molecules, but have a common feature: the value of hydrophilic-lipophilic balance (HLB) for these surfactants is in the range of 12-18. As additional stabilization, in order to prevent premature oxidation of the active component, the presence of a structurally diphilic additive, an antioxidant (for example, ascorbic acid, ascorbyl palmitate), is allowed (RF Patent 2500396, publication date 12/10/2013).

This approach allows you to stabilize echinochrome. However, the disadvantage of this method is that the possibility of using the composition to create drugs is not described. Moreover, the effect of stabilization of an aqueous solution of echinochrome A due to the introduction of polymers may be temporary: data on the stability time of the obtained compositions are absent in the patent.

The available information on liposomal forms relates to a wide range of substances of various nature: from inorganic ions to proteins and nucleic acids (RF Patent 2514000, publication date 04/27/2014; RF Patent 2516893, publication date 05/05/2014; RF Patent 2494729, publication date 10.10. 2013; RF Patent 2492863, publication date 09/20/2013; RF Patent 2477632, publication date 03/20/2013).

However, despite the numerous developments of liposomal preparations, to date, there is no technological solution for creating an acceptable and stable liposome form of the dimeric pigments of the polyhydroxynaphthoquinone series. And on the modern market, drugs based on the liposomal form of pigments of the polyhydroxinaphthoquinone series of plant and animal origin are not represented.

Patent information on the liposomal form of eye drops based on pigments of bisnaphthazarines with anti-inflammatory, anti-allergic effects, as well as on methods for producing such liposomes and preparations based on it.

Thus, it is an object of the present invention to provide a liposome composition based on bisnaphthazarin pigment isolated from the needles and carapace of the green sea urchin Strongylocentrotus droebachiensis, ethylidene-6,6'-bis (2,3,7-tri-hydroxynaphthazarin), with a high capture coefficient and the stability of the retention of the active compound, in order to obtain an ophthalmic agent with increased bioavailability and anti-inflammatory activity.

The task of obtaining a liposomal form based on bisnaphthazarin is achieved in the following way: using an organic solvent, a solution of phospholipids with a pigment complex and a co-solvent is prepared, followed by dispersion of the lipid fraction in an aqueous medium to obtain a suspension of lipid vesicles with a biologically active substance included in them, then a suspension of lipid vesicles is homogenized liposomes with a size of not more than 250 nm are separated with the biologically active substance included in them. Eye drops based on the liposome composition may further comprise cryoprotectants.

The pigment bisnaphthazarin-ethylidene-6,6'-bis (2,3,7-tri-hydroxy-naphthazarin) used as a biologically active component is obtained according to the previously developed technology (RF Patent 2545692, publication date 04/10/2015). The content of the active component in the composition of liposomes to create an ophthalmic agent in the form of eye drops varies in the range of 0.01-0.50 mass%.

The phospholipid component of liposomes can be represented either individually by phospholipids of animal or plant origin, and / or their mixture, taken in any combination and ratio. The most preferred lipid components of the membranes of the present invention are phospholipids and / or derivatives of phospholipids. It has been experimentally shown that from 10 to 80% of a mixture of phosphotidylcholine and cholesterol is preferably used as the lipid phase.

Ethanol is used as an organic solvent. It is preferable to use acidified ethanol, namely a 0.16% solution of hydrochloric acid in 95% ethanol. The organic solvent is removed by distillation under vacuum and a temperature of 30-40 ° C using a rotary evaporator.

According to the claimed invention, as the aqueous (internal) phase of the liposomes, it is preferable to use buffer solutions, such as phosphate buffer solution, nitrate buffer solution and phosphate-saline buffered saline solution, physiological saline solution and others. Most preferred is the creation of an aqueous phase using a buffer solution, with concentrations from 5 to 200 mm, and more preferred is a concentration of 10-100 mm. There are no particular restrictions on the pH of the internal phase of liposomes, however, values from 3 to 11 are preferable, and from 6 to 8 are more preferable.

Most preferably, 0.01 M phosphate buffer with a pH of 7.4 is used as the aqueous medium for dispersing the lipid fraction.

A nonionic surfactant is used as a co-solvent of the pigment complex. According to experimental data, it is preferable to use tween-80.

According to the claimed invention, the grinding of liposomes is carried out by sonication. Most preferably by sonication at a frequency of 15-30 kHz for 1-60 minutes in an ice bath, most preferably within 10-40 minutes.

Purification of liposomes is carried out using sequential filtration of the resulting solution through membrane filters with pore sizes of 0.45 μm and 0.22 μm.

Removal of the active component not included in the composition of the visicles is carried out by dialysis with stirring on a magnetic stirrer and at 4 ° C for 2 hours using dialysis bags (OrDial D-Clean, 10 kDa, 25 kDa, manufactured by Orange Scientific). As the receiving solution, the most acceptable is the use of a 5% glucose solution. For the greatest effect, after 2 hours a fresh portion of the receiving solution is poured, and dialysis is repeated under the same conditions. As a result of the cleaning procedures, the liposome preparation thus obtained is characterized by an average particle diameter of 101 ± 5 nm.

The stability of the liposome composition is enhanced by the addition of cryoprotectants. Most preferably, trehalose is used as a cryoprotectant.

Despite the absence of restrictions on the content, it is preferable that the concentration of sugar is from 2 to 20% (weight / volume), and more preferably from 5 to 10% (weight / volume).

The invention is illustrated by the following examples.

Example 1

a) preparation of the aqueous phase: 500 ml of 0.01 M phosphate buffer (pH 7.4) is prepared and 12.5 g of trehalose are dissolved in it.

b) preparation of the lipophilic phase: pre-dissolved in 50 ml of a 0.16% hydrochloric acid solution in 95% ethyl alcohol 50 mg of the pigment complex bisnaphthazarin. A phospholipid mixture of phosphatidylcholine and cholesterol in an amount of 5.25 g is prepared in a 5: 1 ratio (mass ratio), then 3.75 g of Tween-20 co-solvent are added. The mixture is melted at a temperature of 55 ° C.

The alcohol solution of the pigment complex is quantitatively transferred to the obtained melt and mixed until a clear solution is obtained. After that, the resulting solution is quickly transferred to 500 ml of an aqueous medium (0.01 M phosphate buffer, pH 7.4 with trehalose) at a temperature of 55 ° C and alter for 30 minutes. Ethanol is distilled off under vacuum at a temperature of 35-40 ° C on an IKA rotary evaporator (Germany), and the resulting solution is brought up to 500 ml with water. The prepared suspension is treated with ultrasound at a frequency of 15 kHz for 20 minutes, continuously cooled with ice water (4710 Cole-Parmer Instruments, USA). Liposomes with a size of not more than 220 nm are separated, successively filtered through sterile membrane filters with pore sizes of 0.45 and 0.22 μm.

The resulting liposomes are Packed in bottles with a capacity of 5 ml.

Example 2

a) preparation of the aqueous phase: 250 ml of 0.01 M phosphate buffer (pH 7.4) is prepared and 12.5 g of trehalose are dissolved in it.

b) preparation of the lipophilic phase: pre-dissolved in 250 ml of a 0.16% hydrochloric acid solution in 95% ethyl alcohol 500 mg of the pigment complex bisnaphthazarin. A phospholipid mixture of phosphatidylcholine and cholesterol in an amount of 3 g is prepared in a ratio of 4: 1 (mass ratio), then 2 g of tween-80 co-solvent are added. The mixture is melted at a temperature of 55 ° C.

The alcohol solution of the pigment complex is quantitatively transferred to the obtained melt and mixed until a clear solution is obtained. After that, the resulting solution is quickly transferred to 250 ml of an aqueous medium (0.01 M phosphate buffer, pH 7.4 with trehalose) at a temperature of 55 ° C and alter for 30 minutes. Ethanol is distilled off under vacuum at a temperature of 30-35 ° C on an IKA rotary evaporator (Germany), and the resulting solution is adjusted with water to 500 ml. The prepared suspension is treated with ultrasound at a frequency of 30 kHz for 30 minutes, continuously cooled with ice water (ultrasonic disperser 4710 Cole-Parmer Instruments, USA). Liposomes with a size of not more than 220 nm are separated, successively filtered through sterile membrane filters with pore sizes of 0.45 and 0.22 μm.

The resulting liposomes are Packed in bottles with a capacity of 5 ml.

Example 3

a) preparation of the aqueous phase: 100 ml of 0.01 M phosphate buffer (pH 7.4) is prepared and 5 g of trehalose are dissolved in it.

b) preparation of the lipophilic phase: previously dissolved in 100 ml of a 0.16% solution of hydrochloric acid in 95% ethyl alcohol 100 mg of the pigment complex bisnaphthazarin. A phospholipid mixture of phosphatidylcholine brand lipoid C100 (LIPOID GmbH (Germany)) and cholesterol in the amount of 1.05 g is prepared in the ratio of 6.22: 1 (mass ratio), then 0.75 g of tween-80 cosolvent is added. The mixture is melted at a temperature of 55 ° C.

The alcohol solution of the pigment complex is quantitatively transferred to the obtained melt and mixed until a clear solution is obtained. After that, using a syringe, the resulting solution is quickly transferred; the resulting solution is quickly transferred to 100 ml of an aqueous medium (0.01 M phosphate buffer, pH 7.4 with trehalose) at a temperature of 55 ° C and alter for 30 minutes. Ethanol is distilled off under vacuum at a temperature of 30-35 ° C on an IKA rotary evaporator (Germany), and the resulting solution is adjusted with water to 100 ml. The prepared suspension is treated with ultrasound at a frequency of 30 kHz for 40 minutes, continuously cooled with ice water (ultrasonic disperser 4710 Cole-Parmer Instruments, USA). To remove a substance that is not encapsulated in a liposome, dialysis is performed at 4 ° C for 2 hours using 25 kDa dialysis bags (OrDial D-Clean, manufactured by Orange Scientific), and a 5% glucose solution as an external solution, s stirring on a magnetic stirrer. After replacing the external solution, another dialysis is carried out for two hours with simultaneous stirring. Liposomes with a size of not more than 220 nm are separated, successively filtered through sterile membrane filters with pore sizes of 0.45 and 0.22 μm.

The weighted average particle size of liposomes is determined by dynamic light scattering using a dynamic light scattering device (model Zetasizer Nano ZS manufactured by Malvern Instruments Ltd.). Liposomes prepared in this way have an average particle diameter of 101.0 ± 5.0 nm.

The resulting liposome composition is packaged in 5 ml vials.

Example 4

a) deionized water is used as the aqueous phase

b) preparation of the lipophilic phase: pre-dissolved in 50 ml of a 0.16% hydrochloric acid solution in 95% ethyl alcohol 50 mg of the pigment complex bisnaphthazarin. A phospholipid mixture of phosphatidylcholine brand lipoid C100 (LIPOID GmbH (Germany)) and cholesterol in the amount of 1.05 g is prepared in the ratio of 6.22: 1 (mass ratio), then 0.75 g of tween-80 cosolvent is added. The mixture is melted at a temperature of 55 ° C.

After that, the alcohol solution is distilled off under vacuum on an IKA rotary evaporator (Germany) at a temperature of 30-3 5 ° C until a thin film is obtained. The resulting film is dispersed in 100 ml of deionized water for 1 hour until the complete transition of the dried substances into the aqueous phase. The resulting suspension is treated with ultrasound at a frequency of 30 kHz for 30 minutes, continuously cooled with ice water (ultrasonic disperser 4710 Cole-Parmer Instruments, USA). Liposomes with a size of not more than 220 nm are separated using filters with pore sizes of 0.45 and 0.22 microns.

The resulting liposome composition is packaged in 5 ml vials.

Example 5

a) preparation of the aqueous phase: 250 ml of 0.01 M phosphate buffer (pH 7.4) is prepared and 6.25 g of trehalose are dissolved in it.

b) preparation of the lipophilic phase: previously dissolved in 50 ml of 0.16% hydrochloric acid solution in 95% ethyl alcohol 100 mg of the pigment complex bisnaphthazarines. A phospholipid mixture of phosphotidylcholine and cholesterol in an amount of 2.5 g is prepared in a ratio of 4: 1 (mass ratio), then 1 g of Tween-80 co-solvent is added. The mixture is melted at a temperature of 55 ° C.

The alcohol solution of the pigment complex is quantitatively transferred to the obtained melt and mixed until a clear solution is obtained. After that, using a syringe, the resulting solution is quickly transferred; the resulting solution is quickly transferred to 100 ml of an aqueous medium (0.01 M phosphate buffer, pH 7.4 with trehalose) at a temperature of 55 ° C and alter for 30 minutes.

Then ethanol is distilled off on a rotary evaporator IKA (Germany) under vacuum at a temperature of 30-35 ° C, and the resulting solution is brought with water to 250 ml. The resulting suspension was treated with ultrasound at a frequency of 15 kHz for 15 minutes, continuously cooled with ice water (4710 Cole-Parmer Instruments, USA). To remove a substance that is not encapsulated in a liposome, dialysis is performed at 4 ° C for 2 hours using 25 kDa dialysis bags (OrDial D-Clean, manufactured by Orange Scientific), and a 5% glucose solution as an external solution, s stirring on a magnetic stirrer. After replacing the external solution, another dialysis is carried out for two hours with simultaneous stirring. Liposomes with a size of not more than 220 nm are separated, successively filtered through sterile membrane filters with pore sizes of 0.45 and 0.22 μm.

The resulting liposome composition is packaged in 5 ml vials.

Example 6. To assess the coefficient of capture of the active substance proposed in the present invention, the liposome composition was dialyzed. The concentration of the active substance in the receiving solution was measured spectrophotometrically at a wavelength of 340 nm, determining the amount of active compound not encapsulated in liposomes. The capture coefficient of the pigment complex of bisnaphthazarines was at least 85%.

Using the method of dynamic light scattering by measuring the average particle size in nm, the stability of the obtained liposome samples during long-term storage was confirmed: the average size is maintained for a long time, which indicates the absence of liposome destruction, adhesion or aggregation. It was found that after 12 months of storage at a temperature of + 2-8 ° C, the sizes of the obtained liposomes containing the pigment complex of bisnaphthazarines did not change. The data obtained indicate that the inventive liposome form is stable during storage for at least 1 year.

Example 7. The antihistamine effect was evaluated on a model of allergic conjunctivitis (AK) induced by topical application of the Compound 48/80 solution to the rabbit eye conjunctiva.

In the control group, pathology was induced on the right eye, the left eye remained intact. In the experimental groups, an inductor was applied to both eyes. On the day of induction, drugs were administered 60 minutes before the induction of pathology and after 15, 30, 45 and 60 minutes, 1 drop in each eye. A clinical examination of the rabbit's eye was performed daily. 2 hours after the induction of pathology, swabs were taken from each eye of the rabbit on a glass slide to assess the cellular composition. On the 5th day of the experiment, the animals were euthanized. A right eyeball with conjunctiva and lacrimal gland was isolated for histological examination and a left eyeball with conjunctiva and lacrimal gland for histamine analysis by HPLC.

According to the determination of histamine by HPLC-UV (Fig. 1), a statistically significant increase in histamine level was detected in the lacrimal gland during pathology induction compared to the histamine content in the lacrimal gland of the intact eye. In all groups receiving the studied drugs, a statistically significant decrease in histamine in the lacrimal gland was observed in relation to the histamine level of the lacrimal gland of the right eye (AK) of the control group receiving placebo.

Example 8. Anti-inflammatory effect on a model of rat carrageenin edema.

Acute inflammation was induced on day 14 of the study by administering 50 μl of a 0.5% λ-carrageenin solution subcutaneously into the plantar surface of the rat's right hind (“experimental) paw (subplantar) [Current Protocols in Pharmacology. Models of inflammation. Carrageenan induced paw edema in the rat // John Wiley & Sons, Inc. Contributed by Phyllis E. Whiteley and Stacie A. Dalrymple. 1998. Unit 5.4.1-5.4.3.]. Saline was injected into the left hind (control) paw in a volume of 50 μl.

3, 6 and 24 hours after the induction of inflammation with carrageenan, the degree of severity of tactile allodynia was assessed using calibrated microfilaments of Fry. The average tactile reactivity threshold was determined by the Chaplan method ("up-down method"; [Chaplan SR, Bach FW, Pogrel JW et al. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods. 1994; 53: 55-63 ]). For the test, animals were placed in individual plastic boxes on a grating floor. After a twenty-minute period of adaptation to the plantar surface of the hind legs of animals, calibrated microfilaments were applied in order to increase the cross-sectional area of the filament. 3 hours after the induction of inflammation with carrageenan, the degree of severity of tactile allodynia was assessed using calibrated microfilaments of Fry. 24 hours after the induction of inflammation with carrageenan, the temperature of the hind legs of animals was measured. The degree of development of the inflammatory process was judged by the temperature difference between the experimental and control paws. The data are presented in table 1.

In experimental groups, after 3 hours, the development of tactile allodynia was recorded, which manifested itself in the presence of statistically significant differences in its indicators (50% sensitivity threshold) in these groups from the control group. 24 hours after the induction of inflammation, statistically significant differences between all groups in this parameter were not observed. The temperature of the paws in the control and experimental placebo group was statistically significantly increased compared with the intact group. In the experimental groups receiving the drug based on bisnaphthazarin, a dose-dependent decrease in the temperature difference between the experimental and control limbs was observed. Moreover, the effect observed when using diclofenac at a dose of 7 mg / kg was comparable to the effect when using an agent based on bisnaphthazarin at a dose of 1.5 mg / kg. The administration of a bisnaphthazarin-based agent reduced nitric oxide and normalized catalase activity after 24 hours, comparable to the effect of diclofenac upon reaching the level of intact animals. The change in all the studied parameters under the influence of the agent based on bisnaphthazarin occurred dose-dependently.

Example 9. The study of the retinoprotective effect of funds based on bisnaphthazarin in a model of diabetic retinopathy in rats

Modeling of diabetic retinopathy was carried out using two subcutaneous injections of an aqueous solution of alloxan at a dose of 75 mg / kg to starving animals during the day. The duration of the development of pathology is 90 days. Pathology was monitored by determining the level of glucose and glycosylated hemoglobin in the blood of experimental animals.

The method of administration is local (in the conjunctival sac), the duration of administration was 90 days (from the first day of the induction of pathology).

At the end of the study, the animals were euthanized, the eyeball was enucleated. An eyeball with conjunctiva and lacrimal gland was placed in a 10% solution of neutral formalin for 24 hours, after which the material underwent standard processing in increasing alcohols (70-95%), xylene and paraffin for the manufacture of histological preparations with a thickness of serial paraffin sections 3- 7 microns. For microscopic examination, sections were stained with hematoxylin and eosin. The expression of endothelial nitric oxide synthase, which catalyzes the synthesis of NO, was determined by immunohistochemical analysis using monoclonal antibodies to eNOS manufactured by Novocastra at a 1:80 dilution according to the standard protocol. An immunohistochemical study of CD31 (PECAM-1) was performed using monoclonal antibodies (clone 1A10) manufactured by Novocastra in a 1: 100 dilution according to the standard protocol. Ki67 proliferation marker was determined using monoclonal antibodies (clone RTU-Ki67-MM1) manufactured by Novocastra, a proapoptotic marker p53 was also determined using monoclonal antibodies (clone PAB) manufactured by DakoCytomation at a 1:80 dilution to evaluate the expression of anti-apoptotic protein Mcl-1 monoclonal antibodies (clone 38G3, RTU form) manufactured by Novocastra were used.

Morphological examination and microphotography of histological preparations was carried out using a Leica DM LS light-optical microscope. Micromorphometric study was performed using a Reichert eyepiece micrometer.

When using the studied drug in a therapeutic dose (TD), as well as in doses 3 times lower, and 3 times more, a decrease in the diameter of the capillaries of the retina was observed (Table 2). In an immunohistochemical study of 3 doses of the test drug, eNOS expression increased sharply and was observed in the vascular endothelium and axon layers of photoreceptors, bipolar neurons, and ganglion cells. The expression of CD31 in vascular endothelium was markedly increased with the use of the drug in 3 doses. The change in all the studied parameters under the influence of the agent based on bisnaphthazarin occurred dose-dependently.

Example 10. Pharmacokinetics of a bisnaphthazarin-based agent when instilled into a conjunctival sac in rabbits.

Using the developed methods, the analysis of blood plasma and eye tissues (cornea, lens, vitreous, retina) obtained from laboratory animals after administration of a drug based on bisnaphthazarin was performed.

It has been established that a bisnaphthazarin-based agent, when administered once and administered at a higher daily dose, does not have systemic availability. The data obtained indicate that upon instillation of the studied drug, the maximum concentration of bisnaphthazarin in the cornea of the eye is reached in half an hour and amounts to 200.8 ± 13.3 μg / g. The average retention time of bisnaphthazarin in the cornea and the elimination period are low (less than 2 hours), which indicates its rapid excretion (Fig. 2).

Thus, the tool in the form of liposomes based on bisnaphthazarin is effective for use in ophthalmology.

Claims (5)

1. The method of obtaining funds for ophthalmic use based on bisnaphthazarin in the form of liposomes with anti-inflammatory and anti-allergic effects, in which the aqueous and lipid phases are separately prepared, the aqueous phase is prepared by mixing 500 ml of 0.01 M phosphate buffer with a pH of 7.4 and 12.5 g of trehalose are dissolved in it, and the lipid phase is prepared by melting 5.25 g of a mixture of phosphotidylcholine and cholesterol in a mass ratio of 5: 1 with 3.75 g of Tween-80 at 55 ° C for 30 minutes, then the resulting melt is transferred to 50 mg of ethylidene-6,6'- bis (2,3,7-trihydroxinaphthazarin) dissolved in 50 ml of a 0.16% alcohol solution of hydrochloric acid and stirred until a clear solution was obtained, followed by the addition of an aqueous phase, the resulting mixture was stirred for 30 minutes at 55 ° C, removed ethanol under vacuum, bring the volume of water to 500 ml, the resulting suspension is sonicated at a frequency of 15-30 kHz for 15-40 minutes in an ice bath, cleaned of unencapsulated substance by dialysis with 5% glucose solution using dialysis bags with stirring and at 4 ° C through membranes with a pore size of 10 kDa, 25 kDa, it is filtered through a membrane filter with a pore size of 0.45 and 0.22 μm. 2. A method of obtaining an ophthalmic agent based on bisnaphthazarin in the form of liposomes with anti-inflammatory and anti-allergic effects, in which the aqueous and lipid phases are separately prepared, the aqueous phase is prepared by mixing 250 ml of 0.01 M phosphate buffer with a pH of 7.4 and 12.5 g of trehalose are dissolved in it, and the lipid phase is prepared by melting 3 g of a mixture of phosphotidylcholine and cholesterol in a 4: 1 mass ratio with 2 g of Tween-80 at 55 ° C for 30 minutes, then 500 are transferred to the obtained melt mg ethylidene-6,6'-bi (2,3,7-trihydroxinaphthazarin) dissolved in 250 ml of a 0.16% alcohol solution of hydrochloric acid and stirred until a clear solution is obtained, followed by the addition of an aqueous phase, the resulting mixture is stirred for 30 minutes at 55 ° C, ethyl is removed alcohol under vacuum, bring the volume of water to 500 ml, the resulting suspension is sonicated at a frequency of 15-30 kHz for 15-40 minutes in an ice bath, cleaned from unencapsulated substance by dialysis with 5% glucose solution using dialysis bags with stirring When 4 ° C through a membrane with a pore size of 10 kDa, 25kDa, filtered through a membrane filter with a pore size of 0.45 and 0.22 microns. 3. A method of obtaining an ophthalmic agent based on bisnaphthazarin in the form of liposomes with anti-inflammatory and anti-allergic effects, in which the aqueous and lipid phases are separately prepared, the aqueous phase is prepared by mixing 100 ml of 0.01 M phosphate buffer with a pH of 7.4 and 5 g of trehalose are dissolved in it, and the lipid phase is prepared by melting 1.05 g of a mixture of phosphotidylcholine and cholesterol in a mass ratio of 6.22: 1 with 0.75 g of Tween-80 at 55 ° C for 30 minutes, then into the melt is transferred 100 mg of ethylidene-6,6'- bis (2,3,7-trihydroxinaphthazarin) dissolved in 100 ml of a 0.16% alcohol solution of hydrochloric acid, and stirred until a clear solution was obtained, followed by the addition of an aqueous phase, the resulting mixture was stirred for 30 minutes at 55 ° C, removed ethanol under vacuum, bring the volume of water to 100 ml, the resulting suspension is sonicated with a frequency of 15-30 kHz for 15-40 minutes in an ice bath, cleaned of unencapsulated substance by dialysis with 5% glucose solution using dialysis bags with stirring at 4 ° C through a membrane with a pore size of 10 kDa, 25kDa, filtered through a membrane filter with a pore size of 0.45 and 0.22 microns. 4. The tool obtained by the method according to any one of paragraphs. 1-3 for ophthalmic use based on bisnaphthazarin in the form of liposomes, which has anti-inflammatory and anti-allergic effects. 9. The method of claim 6, wherein the ophthalmic agent is obtained by sequential filtration through membrane filters with pore sizes of 0.45 μm and 0.22 μm.

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