RU2740060C1 - Dosage form for treating and preventing ulcerative colitis, containing pegylated liposomal form of erythropoietin in form of suppositories, having anti-inflammatory and immunomodulatory effect - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present invention refers to medicine, namely to a method for preparing a medicinal agent for treating and preventing ulcerative colitis in the form of suppositories containing PEGylated liposomal form of erythropoietin (EPO), characterized in that the suppository base is prepared by melting polyoxyethylene glycol 4000, the emulsifying agent T-2, Kolliphor and Cremophor RH 40 at a temperature of not more than 70 °C, adding polyoxyethylene glycol 1500 and polyoxyethylene glycol 400 and stirring obtained base, introducing a liposomal emulsion containing EPO and a lipid film from a mixture of lecithin and polyoxyethylene glycol 2000, and suppositories are prepared from the suppository mass by pouring, comprising: Erythropoietin - 2000 ME, Polyoxyethylene glycol 4000 - 0.75 g, Polyoxyethylene glycol 1500 - 0.5 g, Polyoxyethylene glycol 400 - 0.5 g, Polyoxyethylene glycol 2000 - 0.25 g, Lecithin - 0.05 g, Emulsifier T2 - 0.1 g, Cremophor RH 40 - 1.25 g, Kolliphor - 0.75 g.

EFFECT: present invention provides a new domestic drug preparation for treating ulcerative colitis, which is a PEGylated liposomal form of EPO and containing PEG 4000, PEG 1500, PEG 400, T2, Cremophor RH as auxiliary substances, a Kolliphor for preparing a dosage form in the form of rectal suppositories for local treatment of ulcers of the large and small intestine, characterized by ease of use, as well as the presence of anti-inflammatory and immunomodulatory effect.

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Description

The invention relates to medicine and pharmacy, and in particular to a method for producing a drug for the treatment of ulcerative colitis (UC), representing a PEGylated liposomal form of erythropoietin (EPO) and containing PEG 4000, PEG 1500, PEG 400, Emulsifier T2, Cremophor CO as auxiliary substances , Colifor for obtaining a dosage form in the form of rectal suppositories for the local treatment of ulcers of the large and small intestines. When implementing the method, a mass is prepared containing a PEGylated liposomal form of EPO, equivalent to 2000 ME, evenly distributed in a mixture of PEG 1500, PEG 400, T2, Cremophor CO, Colifor at a certain ratio of components and dosed by weight when receiving suppositories by pouring. The technical result is a new method for the treatment of ulcerative colitis due to the therapeutic pleiotropic effect of EPO.

Discussion

The invention relates to medicine, in particular to pharmacy, for the development of an agent with a liposomal dosage form with PEGylated EPO, and can be used for the local treatment of ulcers of the large and small intestines in UC.

Currently, anti-inflammatory and / or immunosuppressive therapy regimens are used for the treatment of UC in the acute stage, including corticosteroid hormones, 5-aminosalicylic acid (5-ASA) preparations, immunosuppressants, biological therapy drugs considered as alternative therapy for those who are resistant to standard treatment and steroid-dependent forms of YAK. 5-ASA drugs are used mainly for the induction and maintenance of remission of mild and moderate forms of UC and CD, while glucocorticosteroids (GCS) have been and remain the main agents for the treatment of patients with moderate and severe disease or in case of ineffectiveness of 5-ASA. The method of administration, the dose of the drug depend on the severity of the course of the disease and the localization of the inflammatory process. Apply hydrocortisone, methylprednisolone, or prednisolone.

A known method for the treatment of UC using steroid hormones, which have an active effect on water-salt, protein, carbohydrate and fat metabolism. The pharmacotherapeutic effect of steroid drugs in UC is also based on their anti-allergic and anti-inflammatory properties. Steroid hormones are often used to treat severe and moderate forms of UC and are prescribed in fractional doses up to 30 mg per day (prednisolone).

A known method of treating UC with non-steroidal anti-inflammatory drugs - salazosulfanilamide drugs (sulfasalazine, salazopyridazine, salazodimethoxin), azo derivatives of sulfapyridine and 5-aminosalicylic acid (5-ASA). A known method for the treatment of ulcerative colitis in case of mild exacerbation, the distal location of the process, the absence of secondary infection In this case, treatment is limited to one of the salazosulfonamide drugs, for example salazopyridazine, 0.5 g 6 times a day for 2 weeks, and then 4 times a day until a persistent effect (Abdulkhakov S.R. Nonspecific ulcerative colitis: modern approaches to diagnosis and treatment / Abdulhakov S.R., Abdulhakov R.A. - cyberleninka. 2009). The disadvantages of this method of treatment are the ineffectiveness of its use in moderate and severe forms of UC, as well as the toxicity of salazosulfanilamide preparations due to the presence of sulfapyridine in their composition.

There is a known method of treating UC with non-steroidal anti-inflammatory drugs, which consist of one active substance - 5-ASA (mesalazine). Such drugs (salofalk, mezacol, salosinal, etc.) do not contain sulfapyridine and, therefore, are devoid of its side properties. For the treatment of moderate forms of UC, salofalk and its analogs are used in the form of tablets of 0.5 g 4 times a day. For distal forms (proctitis and proctosigmoiditis), suppositories and enemas are used (Abdulhakov S.R. Nonspecific ulcerative colitis: modern approaches to diagnosis and treatment / Abdulhakov S.R., Abdulhakov P.A. - cyberleninka. 2009). The disadvantages of the known method of treatment are a decrease in blood clotting and increased bleeding, which is undesirable in the presence of a significant eroded surface in the intestine, inhibition of resistance to infections, toxic effects on the liver and kidneys. However, the main disadvantage of the known method of treatment is the lack of influence of the drugs used on the energy metabolism of the affected colonocytes, which slows down the processes of regeneration of ulcerated areas of the intestinal mucosa.

Various compounds can be used as active substances in the composition of suppositories for local therapy: antibiotics, antioxidants, immunomodulators, drugs and vitamins. Endogenous regulators of homeostasis, in particular EPO, are the subject of close study. Its wide range of pleiotropic effects has attracted great interest from many researchers in recent years. EPO and its biotechnological derivatives are highly suitable for local and systemic use for the specific initiation and regeneration of the affected mucous membrane of the large and small intestines in ulcerative colitis and can promote healing. Through inhibition of GSK-3β, EPO inhibits cell death and promotes angiogenesis, which leads to rapid wound regeneration. It has also been proven that it prevents the release of pro-inflammatory cytokines IL-2, IL-6, IL-8, IFN-γ, TNF-α, inhibiting leukocyte and monocyte-macrophage infiltration in the lesion focus. In addition, attention is drawn to its antioxidant properties, which in ulcerative colitis can contribute to limiting the zone of secondary alteration (KimY J, 2010; LawlerPR, 2010; McPherson RJ, 2010; DangJ, 2010; Osikov MB et al., 2012, 2012, 2013, 2015, 2017).

The closest analogue of the presented invention today are Salofalk rectal suppositories for the complex treatment of nonspecific UC and Crohn's disease, containing the active ingredient Mesalazine and a suppository-solid fat base. The drug has a local anti-inflammatory, antibacterial and antioxidant effect. The main disadvantage of Salofalk suppositories is a number of side effects such as headaches, dizziness, pancreatitis, renal and liver failure, allergic and fibrotic reactions from the lungs. The objective of the invention is to expand the arsenal of drugs for the treatment of ulcers of the small and large intestine in modern gastroenterology in diseases of ulcerative colitis.

EFFECT: obtaining a new domestic drug for the treatment of ulcerative colitis, which is a PEGylated liposomal form of EPO and contains PEG 4000, PEG 1500, PEG 400, T2, Cremophor CO, Colifor as auxiliary substances for obtaining a dosage form in the form of rectal suppositories for local treatment of ulcers of the large and small intestines, characterized by ease of use, as well as the presence of anti-inflammatory and immunomodulatory effects.

This problem is solved by the fact that suppositories with EPO for the treatment of ulcers of the large and small intestines, containing EPO in the form of liposomes as an active substance, and a mixture of polyethylene oxides of molecular weight 400, 1500 and 4000, T2, Cremophor CO and Colifor, as an active substance the ratio of the components of the mass, g, to obtain the mass:

Liposomal emulsion equivalent to EPO at 2000ME

PEG 1500 0.5 PEG 400 0.5 Emulsifier T2 0.1 Colifor 0.55 Cremophor CO 0.5 Lecithin 0.05 PEG 4000 0.75 PEG 2000 0.25

All components that make up the product are approved for medical use.

The base was selected experimentally in the process of studying the release of the substance from the bases with a preliminary identification of the compatibility of erythropoietin with the proposed bases. It has been established that suppositories based on a mixture of polyethylene oxides of molecular weight 4000, 1500 and 400 have a good antioxidant and reparative effect. Egg lecithin according to TU 6-09-0001-87 D. Refers to esters of amino alcohol choline and diglyceride phosphoric acids, due to which it allows to obtain stable emulsions in oil-water systems. It is also a food additive (code E322) and is widely used in the food industry.

Polyoxyethylene glycol (PEG) 4000 TU 2483-166-05757587-2000. It is used as an auxiliary substance in the production of medicines in the pharmaceutical industry. It is most commonly used as a softener, thickener, binder and pour-relief agent. Has no emulsifying effect.

Polyoxyethylene glycol (PEG) 2000. Waxy substance. Let's dissolve in many organic solvents: benzene, carbon tetrachloride, acetonitrile. It is used as solvents, stabilizers, regulators of humidity and viscosity, as well as stabilizers of emulsions and emulsifiers - they help to combine substances that normally do not combine. In the food industry it is registered as a food additive. It is included in the composition of laxatives, is used as a filler and binding ingredient for suppositories, tablets, ointments, foams. Polyoxyethylene glycol (PEG) 1500 TU 2483-167-05757587-2000. Represents waxy scales or dense mass of white or slightly gray color. It is widely used in the cosmetic and pharmaceutical industries as a binder and is also used in many cutting and hydraulic fluids. PEG 1500 is used as an emulsifier, dispersant, detergent, antistatic agent in the textile industry. It is often used in the production of polyurethane foams, powder metallurgy, in ceramics, and also as a base ingredient in the development of medicines, such as a hydrophilic base for toothpastes, creams, a component of lotions, deodorants, shampoos.

Polyoxyethylene glycol (PEG) 400 TU 2483-007-71150986-2006. It is used in the pharmaceutical industry as an auxiliary substance in the production of medicines. It is used as a softener, thickener, binder, glidant and ease-of-flow agent. It can be used as a solubilizer, does not have an emulsifying effect.

Emulsifier T-2 (Russia) TU 18-17-05-76. It is used as a stabilizer, plasticizer and emulsifier, gives the mass sufficient elasticity.

Kolifor (Germany) 9003-11-6. Lutrol® F 68. Available in granular form with an average particle size of about 1000 microns, it is a hydrophilic polymer, a block copolymer of polyoxyethylene polyoxypropylene. One of the physical ways to increase the solubility of an active ingredient is to increase its surface area by micronization. Another way is the use of solubilizers, such as surfactant polymers - poloxamers.

Cremophor SO-40 BASF (Germany). It is a nonionic surfactant and is a raw material for the production of cosmetic products. It can also be used as a solubilizer, which has the ability to enhance the pharmacological action of drugs, which allows you to reduce their doses, eliminates the incompatibility of ingredients in the compositions. It is absolutely resistant to hard water and has a solubilizing ability, and also has a relatively low toxicity.

Shaping the drug into rectal suppositories provides storage stability and ease of use. In addition to the manifestation of the known properties, each component of the claimed composition in the aggregate enhances its effect, which is explained by the effect of their synergism. As a result of the experimental studies, it was found that the proposed suppositories have a number of advantages and high bioavailability, are homogeneous and have a satisfactory appearance. The therapeutic effect of the active substances appears 6 minutes after administration; complete dissolution after 12 minutes. To date, suppositories are very promising for use in gastroenterology in the treatment of ulcers of the small and large intestines.

Example 1. Composition for 1 suppository, weighing 3 g: EPO 2000 ME; PEG 4000 - 0.75 g; PEG 2000 - 0.25 g; PEG 1500 - 0.5 g; PEG 400 - 0.5 g; Emulsifier T-2 - 0.1 g; Colifor - 0.75 g; Cremophor CO 40 - 1.25 g; Lecithin - 0.05 g.

Obtaining liposomes. An exact weighed portion (about 0.05 g) of lecithin was mixed with PEG 2000 and dissolved in 7 ml of chloroform. Then the resulting lipid solution was transferred into a round-bottom flask with a capacity of 150 ml and the organic solvent was evaporated on a rotary evaporator under vacuum (0.7 mbar) and a temperature not higher than 35 ± 20C until a thin lipid film was formed, which was then dried under vacuum for an hour (no organic the solvent was monitored by GLC in accordance with the General Pharmacopoeia Monograph 1.1.0008.15 "Residual organic solvents"). Next, the lipid film was hydrated with 3 ml of 125 mM ammonium sulfate solution with constant stirring until the film completely disappeared from the walls of the flask and a white emulsion (dispersion of large multilayer liposomes) was formed. EPO was dissolved in 5 ml of isotonic sodium chloride solution and introduced into the lipid film with constant stirring at a speed of 120 rpm for 1 hour.

Getting suppositories. PEG 4000, emulsifier T-2, colifor and Cremophor CO are melted in a beaker in a water bath at a temperature not exceeding 70 ° C. PEG 1500 and 400 are added to the molten mixture and mixed. The EPO liposomal emulsion is introduced into the semi-cooled base, stirring continuously with a glass rod. Metal suppository forms are wiped with a gauze swab moistened with vaseline oil, cooled in a refrigerator at a temperature of 3-5 ° C for 5-6 minutes and the prepared suppository mass is poured at a temperature of 38-40 ° C. The molds are placed in the refrigerator for 7-10 minutes, after which the frozen suppositories are removed.

Example 2. Composition for 1 suppository, weighing 4 g: EPO 2000 ME; PEG 2000 - 0.40 g; PEG 4000 - 0.90 g; PEG 1500-0.7; PEG 400-0.7; Emulsifier T-2 - 0.1 g; Colifor - 0.90 g; Cremophor CO 40 -1.40 g; Lecithin - 0.05 g. Obtaining liposomes. See example 1.

Getting suppositories. PEG 1500, emulsifier T-2, lutrol F68 and Cremophor CO are melted in a beaker in a water bath at a temperature not exceeding 70 ° C. PEG 4000 and 400 are added to the molten mixture and mixed. The EPO liposomal emulsion is introduced into the semi-cooled base, stirring continuously with a glass rod. Metal suppository forms are wiped with a gauze swab moistened with vaseline oil, cooled in a refrigerator at a temperature of 3-5 ° C for 5-6 minutes and the prepared suppository mass is poured at a temperature of 38-40 ° C. The molds are placed in the refrigerator for 7-10 minutes, after which the frozen suppositories are removed. The obtained suppositories are of the correct shape, firm and homogeneous.

Example 3. Composition for 1 suppository, weighing 4 g: EPO 2000 ME, PEG 1500 - 0.3 g; PEG 4000 - 0.7 g; Emulsifier T-2 - 0.1 g; Colifor - 0.70 g; Lecithin - 0.05; Cremophor SO-40 - 1.25 g.

Obtaining liposomes. See example 1.

Getting suppositories. PEG 1500, PEG 4000, emulsifier T-2, lutrol F68, and Cremophor CO-40 are melted in a beaker in a water bath at a temperature not exceeding 70 ° C. To remove air without stirring, incubate for 30 minutes at a temperature of 45 ° C. EPO is introduced into the suppository mass, stirring continuously with a glass rod. Metal suppository forms are wiped with a gauze swab moistened with vaseline oil, cooled in a refrigerator at a temperature of 3-5 ° C for 5-6 minutes and the prepared suppository mass is poured at a temperature of 38-40 ° C for 1-2 seconds. The molds are placed in the refrigerator for 7-10 minutes, after which the frozen suppositories are removed. The quality of suppositories is assessed by their appearance, elasticity, uniformity, and solubility. The obtained suppositories are of the correct shape, firm and homogeneous.

Example 4. Composition for 1 suppository, weighing 4 g: EPO 2000 ME; PEG 4000 0.6 g; PEG 1500 - 0.4 g; Emulsifier T-2 - 0.1 g; Colifor - 0.75 g; Cremophor CO - 40 - 1.20 g; Lecithin - 0.05 g.

Liposomes and medical suppositories are made in a similar manner. The resulting suppositories are uniform, regular in shape.

Example 5. Composition for 1 suppository, weighing 4 g: EPO 200 ME, PEG 1500 - 0.65 g; PEG4000 - 0.3 g; Emulsifier T - 2 - 0.1 g; Lecithin - 0.05 g, Lutrol F68 - 0.75 g; Colifor-40 - 1.25 g.

Liposomes and medical suppositories are made in a similar manner. The resulting suppositories are uniform, regular in shape. Evaluation of the release of EPO from suppositories was evaluated in the "Dissolution" test in a buffer solution with a pH of 6.8. Found that the most uniform release of EPO was observed from the composition described in example 1 (Fig. 1).

The effectiveness of the dosage form was evaluated under the conditions of experimental UC simulation. The modeling was carried out in two stages using a 3% oxazolone solution (Sigma Aldrich, USA). The work was performed on 49 Wistar rats weighing 210-230 g divided into 3 groups: I (n = 7) - intact animals; II (n = 21) - animals with UC; III (n = 21) - animals with UC under conditions of using rectal suppositories containing PEGylated liposomal form of EPO. Rectal suppositories of 0.3 g containing PEGylated liposomal form of EPO at a dose of 200 IU of the drug were used 2 times a day. Disease activity index (DAI) (Best index - 1976) was assessed by changes in body weight, stool consistency and the presence of rectal bleeding on days 3, 5 and 7.

The data were statistically processed using the Statistica 10.0 for Windows program. Hematological parameters were determined using a BC-2800Vet veterinary hematological analyzer (Mindray, China).

It was revealed that in animals with UC, starting from 3 days, there was an increase in the frequency of defecations, an unformed, mushy consistency of stools, as well as the appearance of blood in the feces. On days 5 and 7, a decrease in body weight was added to the indicated signs, which averaged 10%. All this led to a statistically significant increase in the disease activity index (DAI) on days 3, 5 and 7 of the experiment (p <0.05). By day 7, there was an increase in the severity of symptoms (Figure 2). In the blood, the number of leukocytes and lymphocytes increases, and the volumetric hemoglobin content decreases (Figs. 3, 4, 5).

Under the conditions of using rectal suppositories containing PEGylated liposomal form of EPO in experimental UC, there was a decrease in the frequency of defecations, restoration of stool consistency and a decrease in blood impurity in feces, which led to a decrease in body weight loss, which was 5%. All this was reflected in a statistically significant decrease in the DAI index on days 3.5 and 7 of the experiment (p <0.05) (Fig. 2).

In the blood, there was a decrease in the number of leukocytes, lymphocytes on days 3 and 7 and an increase in the volumetric hemoglobin content for the entire day of the experiment (Figs. 3, 4, 5).

Claims (10)

A method of obtaining a medicinal product for the treatment and prevention of ulcerative colitis in the form of suppositories containing a PEGylated liposomal form of erythropoietin (EPO), characterized by the fact that a suppository base is prepared by melting Polyoxyethylene glycol 4000, emulsifier T-2, colifor and cremophor CO 40 at a temperature not exceeding 70 ° C, adding Polyoxyethylene glycol 1500 and Polyoxyethylene glycol 400 and mixing the resulting base, introducing into it a liposomal emulsion containing EPO and a lipid film from a mixture of lecithin with Polyoxyethylene glycol 2000, and from the suppository mass by pouring, suppositories are prepared, including: Erythropoietin - 2000 ME Polyoxyethylene glycol 4000 - 0.75 g Polyoxyethylene glycol 1500 - 0.5 g Polyoxyethylene glycol 400 - 0.5 g Polyoxyethylene glycol 2000 - 0.25 g Lecithin - 0.05 g Emulsifier T2 - 0.1 g Cremophor SO 40 - 1.25 g Colifor - 0.75 g.

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