RU2599505C1 - Agent with liposome containing albumin and propolis extract, exhibiting reparative activity in anaemias haemorrhagic - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to agent exhibiting reparative activity in haemorrhagic anaemia. Agent exhibiting reparative activity in hemorrhagic anaemia, containing 10% aqueous solution of albumin and 10% alcohol extract of propolis, included into liposomes, obtained by dissolving egg lecithin in a mixture of diethyl ether and chloroform in ratio 2:1, at temperature 37°C, with further distillation of organic layer to a dry residue, adding 10% aqueous solution of albumin and double processing mixture with ultrasound frequency 22 kHz and power of 500 W to formed emulsion is added 10% alcohol extract of propolis, wherein liposomes are enclosed in capsules for oral administration, in certain proportions.

EFFECT: described above agent exhibits reparative, antioxidant, antispasmodic, anti-inflammatory, regenerative and antibacterial actions, as well as uniform release of active substances and manifested prolonged action, convenient for use by patients independently.

3 cl, 1 dwg, 3 tbl, 3 ex

Description

The present invention relates to medicine, namely to pharmacy, and for the development of medical capsules containing albumin and propolis extract, and can be used in the complex treatment of diseases associated with hemorrhagic anemia.

The well-known drug albumin, produced under various trade names - the drug "Plazbumin 20" - 20% solution for infusion in bottles of 20 g in 100 ml, manufactured by Talekris Bioteralutiks Inc. USA.

Albumin preparations known are Albumin Dry Postab Albumin, Dessau Albumin, Donor Albumin, Placental Albumin, Human Albumin, Zelnab, Postab, Uman albumin in the form of infusion solutions - 5%, 20%, 25%. Albumin is obtained by fractionating blood, plasma, placenta, blood serum from healthy donors. The therapeutic effect of albumin is due to its physiological functions - high oncotic activity [htxp: //www.piluli.com.ua/notes.php? Lek = 21028]. The main active ingredient is albumin. It is a plasma substitute, a natural protein, a blood product. It is part of the human protein fraction. In human plasma in a healthy state, albumin constitutes 60% of all protein proteins. Albumin contains 20 amino acids. It is synthesized in the liver. The main task of albumin is to maintain oncotic colloidal blood pressure. Solutions of the drug are prepared from the placenta, blood plasma by fractionation. Albumin is a clear liquid, the color of which varies from light brown to yellow [http://medside.ru/albumin-drug].

Albumin is effectively used to correct hypoalbuminemia of various etiologies, restore oncotic, colloidal blood pressure, water-electrolyte balance, with impaired hemodynamics (central, peripheral), improves the absorption of drugs, has a detoxifying effect. Albumin transports and binds fatty acids, pigments, drugs, metal ions, and inactivates toxins.

It is known a medicine with albumin and propolis extract for the treatment of burn diseases in the form of an ointment containing dry albumin and alcohol propolis extract, and as an ointment base, petroleum jelly, pentol and purified water in the following ratio, wt.%: Dry albumin - 10; alcohol propolis extract - 5; medical vaseline - 38; pentol - 2; purified water to 100, additionally containing a preservative (mixture of nipagin and nipazole 7: 3) - 0.1 [patent RU 2381804, 2010].

Known means Ferroplex, containing as active substance iron sulfate and ascorbic acid in the amount of 50 mg and 30 mg in the form of dragees. Ferroplex is used in the treatment of anemia of various origins (hypochromic anemia, chronic gastritis, the condition after resection of the stomach, acute and chronic bleeding, peptic ulcer of the stomach and duodenum) [http://www.vidal.ru/poisk_preparatov/ferroplex_14861.htm].

The closest analogue of the invention is a solution for intravenous or subcutaneous administration of Epocrine containing epoetin alpha (recombinant human erythropoietin), albumin in the form of a solution of albumin for infusion of 10%, sodium citrate, pentasesquihydrate or sodium citrate dihydrate, sodium chloride, citric acid monohydrate and water for injection [Antianemic drugs Epocrine, solution [Electronic resource] Access mode: https://health.mail.ru/drug/epokrin/. Date of appeal: February 26, 2015].

The objective of the invention is to expand the arsenal of drugs with reparative action in case of anemia of various origins.

EFFECT: obtaining a new oral medicine in the form of hard gelatin capsules with liposomes, which has reparative activity in hemorrhagic anemia, which provides the maximum intake of active substances from the gastrointestinal tract into the bloodstream, in combination with antioxidant, antispasmodic, anti-inflammatory, regenerating and antibacterial action .

The specified technical result is achieved in that the agent having reparative activity in hemorrhagic anemia, containing 10% aqueous solution of albumin as an active substance and auxiliary substances, according to the invention, a 10% aqueous solution of albumin and 10% alcohol extract of propolis are included in liposomes obtained by dissolution egg lecithin in a mixture of diethyl ether and chloroform in the ratio 2: 1, at a temperature of 37 ° C, followed by distillation of the organic layer to a dry residue, adding 10% aqueous solution creates albumin and processing the mixture sonicated twice the frequency of 22 kHz and a power of 500 W, to the resulting emulsion was added 10% alcoholic extract of propolis, wherein the liposomes encapsulated for oral administration in the following ratio liposomes g per 1 capsule:

Aqueous solution of albumin 10% 1,0 Propolis extract alcohol 10% 0.2 Egg Lecithin 0.22

In this case, ethyl alcohol of 70% is used as a solvent to obtain a 10% alcohol extract of propolis; use number 0 capsules having a diameter of 7.65 mm, a length of 21.7 mm, a filling volume of 0.68 ml, obtained in the following ratio of components, g per 1 capsule:

gelatin 0.0423 glycerol 0.0184 titanium dioxide 0,00025 purified water 0,0351

The method of obtaining liposomes with 10% albumin and propolis extract 10% alcohol:

5 g of egg lecithin (egg lecithin according to TU 6-09-0001-87 D) is dissolved in a mixture of diethyl ether and chloroform (2: 1) with constant stirring on a shaker for 10 minutes. The organic layer was distilled off on a rotary evaporator under vacuum in a water bath at 37 ° C almost to a dry residue, and a 10% 10 ml albumin solution was introduced into the resulting mixture. The resulting mixture was treated with ultrasound with a frequency of 22 kHz and a power of 500 W (apparatus UZDN-2T) for 30 seconds 1-5 times (to select the optimal switching method). To the emulsion formed, 2 ml of propolis extract was added and shaken for 1 hour on a shaker (laboratory shaker S - 3.02L, type of movement - orbital rotation, speed 300 rpm). The emulsion flask was attached to a rotary evaporator, and when the flask was rotated, the pressure was gradually reduced so that the organic solvent did not boil, which was completely removed. The flask was removed from the evaporator and shaken until a homogeneous suspension structure was formed on the shaker.

The obtained liposomes are enclosed in capsules for oral administration, for example, number 0, weight 0.096 g, having a diameter of 7.65 mm, length 21.7 mm, filling volume 0.68 ml, obtained in the following ratio of components, g per 1 capsule:

gelatin 0.0423 glycerol 0.0184 titanium dioxide 0,00025 purified water 0,0351

The invention is illustrated by the following materials: the figure shows a diagram of the content of ceruloplasmin (CP) in the blood of rats, where 1 is a group of intact animals; 2 - group of control animals; 3 - a group of animals that received an albumin solution intraperitoneally; 4 - a group of animals that received a liposomal dosage form with albumin orally; 5 - a group of animals receiving a liposome dosage form with albumin and propolis extract orally; table 1 - determination of the effectiveness of the degree of inclusion of albumin in various concentrations; table 2 - the results of the determination of TAC - products; table 3 shows the determination of hemoglobin after acute hemorrhagic anemia.

The first stage of the study was to study the degree of inclusion of albumin in the liposome form. It was found that for optimal inclusion of albumin, double treatment with ultrasound is sufficient, and after repeated treatment, albumin is destroyed (table 1).

During storage of liposomes and preparations based on them, lipid oxidation is observed with the formation of malondialdehyde and lipid hydroperoxides, and the content of unsaturated groups in lipids also increases. Such changes directly affect the stability of the liposomal membrane and can lead to a decrease in the time of preservation of the integrity of the membrane and the destruction of the liposomal preparation. The introduction of stabilizing additives into the preparation can both inhibit the oxidation process and accelerate it. The resulting samples were incubated at a temperature of 37 ° C. On days 1, 2, 6, 10, samples were taken in a volume of 1 ml, which were stored in a freezer at -20 ° C before experiments. The following parameters were determined in the samples: the content of TBA-active products, the content of unsaturated compounds, and the change in the pH of the extra liposomal medium.

Determination of TBA-active products

450 μl of a solution of thiobarbituric acid (2.5 mg / ml) in 2% phosphoric acid was added to 50 μl of a suspension of liposomes. The resulting solution was incubated at 100 ° C for 1 hour, and then, after adding 500 μl of 96% ethanol, a spectrum was recorded in the range 450–650 nm. The optical density at 460, 500 and 532 nm, which are characteristic of the absorption maxima of the corresponding TBA-KS (carbonyl compounds), table 2.

Determination of ferroxidase activity of albumin.

The determination was carried out on white rats males weighing 200 - 250 g

There were 10 animals in each group. A study was conducted of the effect of the liposome form with albumin and propolis extract on the relief of the effects of experimental hemorrhagic anemia in rats. After six hemorrhages, rats developed acute anemia, which manifested itself in a decrease in hemoglobin concentration to 59.8% of the initial level, and the level of serum iron decreased by 3 times. For 5 days, albumin for infusion was administered intraperitoneally to rats of the control group at a dose of 10 mg / day, and experimental animals received intragastric liposomes with the same amount of albumin, as well as albumin and propolis extract. The determination of ceruloplasmin was monitored according to the Revin method in the Kamyshnikov modification based on the oxidation of p-phenylenediamine with the participation of ceruloplasmin [Kolb V.G., Kamyshnikov B.C. Determination of the activity of ceruloplasmin in serum by the modified Revin method. Handbook of clinical chemistry. - 2nd ed. - Minsk, 1982. - S. 290-292].

When studying the pharmacological effect of liposomes with albumin and propolis extract in conditions of acute hemorrhagic anemia, it was found that, starting from the 7th day after the administration of the drug, the hemoglobin level increases to 89.3% (with the introduction of the infusion solution) and 86.4% with the introduction of liposomes with albumin and 87.5% - with the introduction of liposomes with albumin and propolis extract. In the control group, on the 7th day, the hemoglobin level was 64.5%, and on 14 - 72.9% (table 3).

When determining the level of ceruloplasmin, it was found that serum ferroxidase activity, decreased as a result of acute blood loss, was restored to the initial level only in rats treated with albumin both intraperitoneally and in the form of liposomes (Figure 1). The effective use of this dosage form in acute blood loss to restore the level of hemoglobin and ceruloplasmin has been shown.

Dry albumin placental VFS 42-1104-81.

A solution of albumin 10% [FS 42-122-04].

Propolis TU GOST 28886-90. The introduction of alcohol propolis extract in combination with albumin into the capsules with liposomes increases antimicrobial activity, tissue regeneration, and contributes to the manifestation of the analgesic, hemostatic, immunomodulating, reparative effect of the dosage form.

In the available scientific, medical and patent literature, information about the popularity of capsules with liposomes containing, as active substances, an aqueous solution of albumin 10% and alcohol extract of propolis 10%, and egg lecithin with a certain ratio of components was not found as excipients of the liposome base. Thus, the claimed invention meets the criterion of "novelty."

As a result of experimental studies, it was found that the proposed capsules with liposomes have high bioavailability and a number of advantages: they have a satisfactory appearance, are homogeneous. The composition of the capsules containing liposomes promotes the rapid and further prolonged release of the active substances: albumin and propolis extract. Giving the drug the form of capsules with liposomes ensures the convenience and hygiene of use, storage stability. Due to the introduction of albumin in the composition of the drug in combination with propolis extract, liposome capsules have a wide range of biological and therapeutic activity: they have a reparative, combined with antioxidant, antispasmodic, anti-inflammatory, regenerative and antibacterial effect. In addition, capsules with liposomes have a uniform release of active substances and a pronounced prolonged action, convenient for oral administration by patients on their own. In addition to the manifestation of known properties, each component of the claimed composition, in the aggregate, enhances its effect, which is explained, obviously, by the effect of their synergism. Thus, the claimed invention meets the criterion of "inventive step".

Example 1. A white capsule (number 0, diameter 7.65 mm, length 21.7 mm, filling volume 0.68 ml, weight 0.096 g) was obtained in the following ratio of components, g: gelatin - 0.0423; glycerol - 0.0184; titanium dioxide - 0,00025; purified water - 0.0351. The composition of the liposomal dosage form in one capsule, g: aqueous solution of albumin 10% - 1.0; alcohol propolis extract 10% - 0.2; egg lecithin - 0.22.

The method of obtaining liposomes with albumin and propolis extract 10% alcohol: 5 g of egg lecithin (egg lecithin according to TU 6-09-0001-87 D) is dissolved in a mixture of diethyl ether and chloroform (2: 1) with constant stirring on a shaker for 10 minutes. The organic layer was distilled off on a rotary evaporator under vacuum in a water bath at 37 ° C almost to a dry residue, and a 10% 10 ml albumin solution was introduced into the resulting mixture. The resulting mixture was treated with ultrasound with a frequency of 22 kHz and a power of 500 W (apparatus UZDN-2T) for 30 seconds 2 times. To the emulsion formed, 2 ml of propolis extract was added and shaken for 1 hour on a shaker (laboratory shaker S - 3.02L, type of movement - orbital rotation, speed 300 rpm). The emulsion flask was attached to a rotary evaporator, and when the flask was rotated, the pressure was gradually reduced so that the organic solvent did not boil, which was completely removed. The flask was removed from the evaporator and shaken until a homogeneous suspension structure was formed on the shaker. A white capsule is used for filling (number 0, diameter 7.65 mm, length 21.7 mm, filling volume 0.68 ml, weight 0.096 g). The prepared capsules for oral administration are filled with liposome mass, after which the quality of the capsules with liposomes is evaluated by their appearance, uniformity, solubility. The resulting capsules with liposomes of the correct form, solid and uniform. The effectiveness of the degree of inclusion of albumin in various concentrations was determined, the number of sonication treatments for 30 seconds - 2 times, the efficiency of inclusion of albumin 91.2% (table 1). Table 2 presents the results of the determination of TBA products; table 3 shows the determination of the level of hemoglobin after acute hemorrhagic anemia; the figure 1 presents a diagram - the content of CP in the blood of rats. It was found that animals treated with albumin intraperitoneally and orally statistically significantly restored hemoglobin levels compared with the control group not receiving treatment. On the 7th day in animals treated with albumin both intraperitoneally and orally, the level of CP increased by 100% compared to the control. On the 14th day, the level of CP increased by more than 200% (compared with the control). Moreover, the most effective increase was observed when taking a liposomal dosage form with albumin and propolis extract.

Example 2. A white capsule (number 0, diameter 7.65 mm, length 21.7 mm, filling volume 0.68 ml, weight 0.096 g) was obtained in the following ratio of components, g: gelatin - 0.0423; glycerol - 0.0184; titanium dioxide - 0,00025; purified water - 0.0351. The composition of the liposomal dosage form in one capsule, g: aqueous solution of albumin 10% - 1.0; alcohol propolis extract 10% - 0.2; egg lecithin - 0.22.

The method of obtaining liposomes with albumin and propolis extract 10% alcohol: 5 g of egg lecithin (egg lecithin according to TU 6-09-0001-87 D) is dissolved in a mixture of diethyl ether and chloroform (2: 1) with constant stirring on a shaker for 10 minutes. The organic layer was distilled off on a rotary evaporator under vacuum in a water bath at 37 ° C almost to a dry residue, and a 10% 10 ml albumin solution was introduced into the resulting mixture. The resulting mixture was treated with ultrasound with a frequency of 22 kHz and a power of 500 W (apparatus UZDN-2T) for 30 seconds 1 time. To the emulsion formed, 2 ml of propolis extract was added and shaken for 1 hour on a shaker (laboratory shaker S - 3.02L, type of movement - orbital rotation, speed 300 rpm). The emulsion flask was attached to a rotary evaporator, and when the flask was rotated, the pressure was gradually reduced so that the organic solvent did not boil, which was completely removed. The flask was removed from the evaporator and shaken until a homogeneous suspension structure was formed on the shaker. A white capsule is used for filling (number 0, diameter 7.65 mm, length 21.7 mm, filling volume 0.68 ml, weight 0.096 g). The prepared capsules for oral administration are filled with liposome mass, after which the quality of the capsules with liposomes is evaluated by their appearance, uniformity, solubility. The resulting capsules with liposomes of the correct form, solid and uniform. The effectiveness of the degree of inclusion of albumin in various concentrations was determined, the number of sonication with ultrasound was 30 seconds 1 time, the efficiency of inclusion of albumin was 75.6% (table 1).

Example 3. A white capsule (number 0, diameter 7.65 mm, length 21.7 mm, filling volume 0.68 ml, weight 0.096 g) was obtained in the following ratio of components, g: gelatin - 0.0423; glycerol - 0.0184; titanium dioxide - 0,00025; purified water - 0.0351. The composition of the liposomal dosage form in one capsule, g: aqueous solution of albumin 10% - 1.0; alcohol propolis extract 10% - 0.2; egg lecithin - 0.22.

The method of obtaining liposomes with albumin and propolis extract 10% alcohol: 5 g of egg lecithin (egg lecithin according to TU 6-09-0001-87 D) is dissolved in a mixture of diethyl ether and chloroform (2: 1) with constant stirring on a shaker for 10 minutes. The organic layer was distilled off on a rotary evaporator under vacuum in a water bath at 37 ° C almost to a dry residue, and a 10% 10 ml albumin solution was introduced into the resulting mixture. The resulting mixture was treated with ultrasound with a frequency of 22 kHz and a power of 500 W (apparatus UZDN-2T) for 30 seconds 10 times. To the emulsion formed, 2 ml of propolis extract was added and shaken for 1 hour on a shaker (laboratory shaker S - 3.02L, type of movement - orbital rotation, speed 300 rpm). The emulsion flask was attached to a rotary evaporator, and when the flask was rotated, the pressure was gradually reduced so that the organic solvent did not boil, which was completely removed. The flask was removed from the evaporator and shaken until a homogeneous suspension structure was formed on the shaker. A white capsule is used for filling (number 0, diameter 7.65 mm, length 21.7 mm, filling volume 0.68 ml, weight 0.096 g). The prepared capsules for oral administration are filled with liposome mass, after which the quality of the capsules with liposomes is evaluated by their appearance, uniformity, solubility. The resulting capsules with liposomes of the correct form, solid and uniform.

Determine the degree of inclusion of albumin (table 1). It was found that with 10-fold and repeated sonication (more than 5 times), partial denaturation of the protein occurs and the liposomes separate when standing (the protein precipitates). An additional line of precipitation was determined by immunoelectrophoresis, which indicates the heterogeneity of albumin.

From the above examples it is seen that the composition described in example 1 is optimal from the point of view of ease of use, high release of active substances and at the same time the effect of prolongation.

Obtaining the proposed capsules with liposomes is easily feasible, and when using them, the indicated technical result is achieved. Thus, the claimed invention meets the criterion of "industrial applicability".

Clinical trials have shown that the number of released drugs with a given ratio of components in one capsule with liposomes is the same in the studied patients. This enables the doctor in practical medicine, depending on the form and severity of the disease, to recommend to patients the necessary number of capsules with liposomes for self-treatment, when taken orally, which makes the claimed capsules with liposomes a promising dosage form in the complex treatment of diseases associated with anemia of various origins .

Claims (3)

1. An agent having reparative activity in hemorrhagic anemia containing 10% aqueous albumin solution as an active substance and excipients, characterized in that 10% aqueous albumin solution and 10% alcohol extract of propolis are included in liposomes obtained by dissolving egg lecithin in mixtures of diethyl ether and chloroform in the ratio 2: 1, at a temperature of 37 ° C, followed by distillation of the organic layer to a dry residue, addition of a 10% aqueous solution of albumin and two-fold treatment of the mixture with ultra sound frequency of 22 KHz and 500 watts, to the resulting emulsion was added 10% alcoholic extract of propolis, wherein the liposomes encapsulated for oral administration, the liposomes at the following ratio of components, 1 gram per capsule:
Aqueous solution of albumin 10% 1,0 Propolis extract alcohol 10% 0.2 Egg Lecithin 0.22 2. The tool according to claim 1, characterized in that 70% ethyl alcohol is used as a solvent to obtain a 10% alcoholic extract of propolis. 3. The tool according to p. 1, characterized in that use number 0 capsules having a diameter of 7.65 mm, a length of 21.7 mm, a filling volume of 0.68 ml, obtained in the following ratio of components, g per 1 capsule:
gelatin 0.0423 glycerol 0.0184 titanium dioxide 0,00025 purified water 0,0351

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