CN119031906A - Process for preparing oral forms of substances containing insulin - Google Patents

Abstract

The present invention relates to medicine, and more particularly to the chemical and pharmaceutical industries, and to the preparation of an oral form of a substance containing insulin and its delivery for the treatment of complications of type I diabetes. The substance is prepared using a method comprising mixing crystalline recombinant insulin, an antioxidant, an additional antioxidant, a nonionic surfactant, a phospholipid and sorbitol in calculated amounts, by mixing in a reactor under conditions that convert the organic solvent to a gaseous state, combining the resulting mixture with an organic solvent, removing the solvent, and obtaining a powdered substance in which a phospholipid membrane is formed in the crystal structure of sorbitol, and then grinding the powdered substance to prepare a fine powder, which is encapsulated with the addition of polyethylene glycol.

Description

Process for preparing oral forms of substances containing insulin

Technical Field

The present invention relates to medicine, that is to the chemical and pharmaceutical industries, to the development of oral forms of insulin substances and their delivery for the prevention of complications of type 1 diabetes.

Prior art

Recently, a lot of information has emerged about the results of studies demonstrating the efficacy and safety of oral forms of insulin. It is assumed that this form of insulin application indicates the safety of the drug and is related to its enteric form. After being absorbed in the intestine, insulin enters the liver, which is similar to the natural transport of insulin in the body. Therefore, the oral form of insulin can be considered more physiological than the injection form, which contributes to a better effectiveness of its use.

It is known that clinical trials of ORMD-0801 (Oramed Pharmaceuticals) were successful and it may become a commercially available oral insulin drug.

However, the form of insulin used (oral or injected) also depends on the type of disease, and therefore also on the composition of the drug. Since type 1 diabetes results in the body not producing insulin, this type of diabetes is treated with insulin injections. Oral medications are not considered suitable for the treatment of type 1 diabetes because insulin is broken down in the digestive system.

In order to increase the efficacy of oral forms of insulin (by slowing its breakdown in the digestive tract), attempts have been made to accelerate the absorption of insulin across the intestinal membrane.

Publications known from the prior art disclose various proposed compositions for oral forms of insulin and processes for their preparation.

For example, in the publication case CN 11297269 with a publication date of June 18, 2021 and a classification number of A61K 38/28, a composition prepared from insulin and/or insulin analogs and a pharmaceutical composition for stimulating absorption in the small intestine are disclosed. The pharmaceutical composition for promoting absorption in the small intestine is composed of sodium lauryl sulfate, chitosan and sodium citrate. According to the pharmaceutical composition for stimulating absorption in the small intestine, a composite adjuvant is prepared, and after combining the composite adjuvant with insulin and/or insulin analogs, the absorption of the active ingredient in the small intestine can be improved.

In addition, publication CN 110974943, dated April 10, 2020, with classification number A61K 38/28, indicates that the pharmaceutical product includes: (a) an inner core layer containing insulin or a biologically active derivative thereof as an active substance; (b) a sticky outer core layer formed in the inner core layer; (c) a protective coating layer formed in the outer core layer; and (d) a coating layer with local drug release formed in the backing layer of the coating, the backing layer of the coating layer being selected from one or more of the following: ethylcellulose, Eudragit RS (Reed-Solomon) or wax; and the coating for local drug release contains Eudragit L100 and Eudragit S100, which are mixed in a weight ratio of (0-1):(1-4) and used as a matrix material for the coating for local drug release. Nanostructured compositions for oral insulin delivery and methods for their preparation are disclosed in Russian Patent 2753018 on August 11, 2021, International Publication WO2019148810 on August 8, 2019, Classification Numbers A61K38/28; A61K47/36, etc. The main disadvantage of these known forms of insulin is that the technology for preparing such compositions is complex and that they may not be used for all types of diabetes. As mentioned earlier, if the pancreas, or more accurately its beta cells, are dysfunctional, no insulin is produced, which is why type 1 diabetes occurs. In the absence of a response to insulin by cells and tissues in the body (usually due to obesity or improper hormone secretion), type 2 diabetes develops.

The nature of the claimed solution

The group of claimed inventions aims to solve the technical problems associated with eliminating the limitations of known analogues.

The result of using the technology of the present invention is the preparation of oral forms of substances (including insulin) with targeted effects, which are suitable for preventing complications of type 1 diabetes.

The above technical result is achieved by a process for preparing an oral form of a substance containing insulin, wherein crystalline recombinant insulin, an antioxidant, an additional antioxidant, a nonionic surfactant, a phospholipid and sorbitol are mixed in calculated amounts, by mixing in a reactor under conditions that convert the organic solvent to a gaseous state, combining the resulting mixture with an organic solvent, removing the solvent, and obtaining a powdered substance in which a phospholipid membrane is formed in the crystal structure of sorbitol, and then grinding the powdered substance to obtain a finely divided powder, which is encapsulated with the addition of polyethylene glycol.

In this case, substances from the group of beta-carotene, astaxanthin, dihydroquercetin, abisil, etc. are used as antioxidants. The designated antioxidants serve to interrupt the body's autoimmune reaction to the beta cells of the pancreas, which ultimately leads to beta cell apoptosis and blocks the production of insulin.

As additional antioxidants, substances from the group of tocopherol acetate, ascorbic acid, etc. are used. Additional antioxidants serve to protect the antioxidant molecules from oxidation, since the latter are very active substances in redox reactions.

The presence of antioxidants and insulin in said substance makes it possible to perform two functions, namely, to deliver the insulin molecule to the cells in the body and, over time, to restore the function of the beta cells of the pancreas, thereby promoting the secretion of insulin.

Tween-60 or Tween-80 was used as the nonionic surfactant.

Chloroform or diethanol is used as an organic solvent, in which the outer membrane of crystalline β-carotene and vegetable oil cells are dissolved to release phospholipids. Vegetable oils such as linseed oil, black cumin oil, sunflower oil, etc. are used as sources of phospholipids. In this case, antioxidants and phospholipids are taken in amounts to ensure their co-solubility in the organic solvent.

To mix these components, equipment designed for this purpose is used (reactor (e.g. Mixing brand), vacuum pump). Under selected conditions, e.g. at a temperature not exceeding +50° C., with a mixing time of e.g. not exceeding 3 hours, the organic solvent gradually changes into a different state of aggregation - a gaseous state, which allows it to be removed by a vacuum pump and then condensed and collected in its original form for reuse.

The sorbitol used has a crystalline structure which, after the process, is filled with the phospholipids obtained in the form of a membrane which lines all the pores of the structure.

The obtained powdered material was ground in a hammer mill using a 160-200 μm sieve to obtain finely divided powder. Specified dispersion of the powder is important for the encapsulation process and its rapid dissolution in the small intestine after contact with the liquid phase (water) to form liposomes.

Insulin is a hypoglycemic polypeptide hormone with a molecular weight of approximately 6000, which is formed in the beta cells of the pancreatic islets of Langerhans. It has a multifaceted effect on metabolism in almost all tissues. The main role of insulin is to regulate carbohydrate metabolism, especially the handling of glucose in the body. Insulin increases the permeability of the plasma membrane to glucose and other macronutrients, activates key glycolytic enzymes, promotes the formation of glycogen from glucose in the liver and in the muscles, and increases the synthesis of fat and protein.

Impaired insulin secretion due to beta cell destruction, i.e., absolute insulin deficiency, is a key factor in the pathogenesis of type 1 diabetes. Impaired insulin action on tissues, i.e., relative insulin deficiency, plays an important role in the development of type 2 diabetes.

After purification, it is an amorphous powder, colorless, soluble in acidic or alkaline water, weak alcohol or glycerol, insoluble in ether, chloroform, anhydrous ethanol, and completely soluble in sodium phosphate solution.

After entering the portal bloodstream, insulin first reaches the liver, where about half of it is inactivated. Therefore, portal insulinemia is much higher than peripheral blood, which seems to be necessary to prevent glucose from flowing into the liver.

The problem with using exogenous insulin is related to the fact that insulin is inactive when administered orally, as it is broken down by proteolytic digestive enzymes; thus parenteral administration is required, given that it is not possible to truly simulate the endocrine pancreas, as physiologically insulin first reaches the liver and is diluted in the peripheral circulation.

The claimed invention makes it possible to obtain an oral form of an insulin substance suitable for preventing complications of type 1 diabetes.

The inventors of the present invention have proposed a composition of matter obtained by the method, which ensures the technical results, and the composition includes an effective amount of crystalline recombinant insulin, an antioxidant, another antioxidant, an ionic surfactant, and sorbitol that forms a phospholipid membrane in its crystal structure, and the composition is beneficial for preventing complications of type 1 diabetes.

When polyethylene glycol (PEG 400) is added to the material, insulin can be encapsulated and used for oral administration.

A specific embodiment of the proposed insulin-containing composition of matter may be a composition comprising the following substances in their proportions (mg/g):

·Crystallized recombinant insulin-8-10

β-Carotene -20-30

·α-Tocopheryl acetate-20-25

·Ascorbic acid -10-13

Phospholipids-80-100

· Twain - 80-20-25

·Sorbitol-842-797

Quantitative characterization of the constituent components was obtained experimentally.

The specified material is placed in hard gelatin enteric capsule N 3 (encapsulation process). This type of capsule is necessary for delivering the material to the small intestine. The gelatin capsule shell dissolves here, and forms lipid vesicles-liposomes after the phospholipid membrane contacts the liquid phase. Liposomes (phospholipid vesicles) have been studied for a long time as a promising dosage form, which significantly changes the distribution of drug substances and protects drug substances from the influence of enzymes. The size range of these microspheres is 25nm to 1 μm, which serves as a container for drug substances, polar compounds are located in the internal water volume, and hydrophobic compounds are freely dissolved in the membrane.

The properties of liposomes and their behavior are first determined by the presence of a closed membrane shell. Despite the molecular thickness (about 4nm), the lipid bilayer is still characterized by excellent mechanical strength and flexibility. In the liquid crystal state of the bilayer, its components have a high degree of molecular mobility, so that the membrane usually acts as a fairly liquid, fluid phase. Therefore, liposomes maintain integrity under various damaging effects, and their membranes have the ability to self-repair their structural defects. Liposomes change size and shape in response to changes in the osmotic concentration of the external aqueous solution. Under severe osmotic stress, the integrity of the bilayer can be destroyed and the liposomes can be broken down into smaller particles. The membrane of the liposome is composed of natural phospholipids, which determines many of their attractive properties. They are non-toxic, biodegradable, can be absorbed by cells under certain conditions, and their membranes can fuse with cell membranes, thereby achieving intracellular delivery of their contents.

The effectiveness of liposomal drugs is largely determined by the amount of drug substance contained therein. However, many technologies for preparing liposomal drugs can provide that no more than 50% of active substances (drug substances) are contained in liposomes. The effects of the method for obtaining liposomal drugs, the proportion of their components and excipients on the degree of inclusion of substances in liposomes have been studied. It has been determined that the best method to ensure that the maximum amount of drug substance is contained in the liposomes is a method for obtaining dry proliposomal drugs, which form liposomes when contacted with a liquid phase (water). Unlike the methods known in the prior art, the claimed invention allows the preparation of effective liposomal drugs (100% inclusion) of fat-soluble substances. The high inclusion of molecules of drug substances in liposomes ensures a high bioavailability of the substance, which is determined by the difference between the volume of the substance administered and its volume in the target organ. The dried proliposomal substance is a finely divided powder encapsulated in a hard gelatin enteric capsule N 3, the substance containing an adsorbent (sorbitol) forming a phospholipid membrane (4nm thick) in the crystal structure of the adsorbent. When such a capsule is taken, it passes through the gastrointestinal tract and reaches the small intestine. Here, the destruction of the gelatin shell of the capsule occurs, and when in contact with the liquid phase, the phospholipid membrane collapses and liposomes (hundreds of thousands of particles of size 10 to the negative 8-9 power) are spontaneously formed. Liposomes have a high degree of capture of hydrophilic (water soluble) or hydrophobic (water insoluble) molecules of the drug substance, and the degree of capture depends on their molecular weight. For beta-carotene, it is 6-8 molecules, and for insulin, it is 2 molecules. The liposomes loaded with molecules of the drug substance containing insulin then enter the liver through the portal vein and then enter the systemic circulation.

In order to overcome the influence of the macrophage system, a protective film of polyethylene glycol, such as PEG-400, is used, which is added to the substance during the encapsulation phase. In this way, a high bioavailability of the substance is achieved due to the dynamics of the blood circulation.

The substance is provided as a food supplement for the prevention of complications of type 1 diabetes, as well as for its treatment.

The use of the claimed substances is shown in the examples, which are one of the possible cases of application and do not limit such application.

Example

In order to demonstrate the efficacy of the claimed substances in preventing complications of type 1 diabetes, two groups of mice were used:

- Group 1 - female balb/c mice, 11 weeks old (18 animals);

- Second group (control group) - female balb/c mice, 11 weeks old (12 animals).

The claimed process is used to prepare a substance of the following composition (in mg), each capsule weighing 0.17 g:

Crystalline Recombinant Insulin - 2mg

Beta-carotene - 6mg

Alpha-Tocopheryl Acetate - 5mg

Ascorbic acid - 2.5mg

Phospholipids from flaxseed - 20 mg

Tween-80-3mg

Polyethylene glycol PEG 400-1.5mg

Sorbitol - 130mg,

The substance was dissolved in 3 or 1 ml of autoclaved mQ (deionized water). A homogenous solution was obtained by vortexing.

Two minutes after the preparation of the solution, the solution was orally administered to the animals of the first group using a 2 ml needle-free syringe. The volume of the substance administered was 150 μl, which contained 0.75 IU of insulin.

The control group (12 animals) was orally administered with an appropriate volume of mQ.

The blood glucose levels of the animals were measured before administration of the substances and at different time intervals thereafter by sampling from the tail artery using a TaiDoc 4239 veterinary glucose analyzer.

Blood glucose levels in balb/c mice after oral administration of a solution in the proliposomal form of insulin are shown in FIG1 .

75 minutes after the administration of the solution, the animals showed signs of hypoglycemic coma (loss of consciousness, inability to move, increased heart rate), which was confirmed by a drop in blood glucose levels (3.0 mM - limit 4.8-6.5 mM). Animals with symptoms of hypoglycemia were orally administered a 1 M glucose solution. All animals survived the experiment and felt normal.

Therefore, it is confirmed that the active substance insulin is delivered to the cells in the body, that is, the pharmacological action of the proposed substance is proved. This experiment enables the claimed oral insulin substance to be provided as a preventive and therapeutic agent. The substance in the form of capsules prepared in the claimed manner is orally administered to experimental animals with an adjusted dose of insulin (15.6IU). The experiment involves male outbred Sprague Dawley rats at 10 weeks of age. The body weight of the experimental animals is 387g and 393g. Rats are raised in independent ventilation cages (IVC) of OptiRAT system (Animal Care Systems) one by one, at a temperature of 22-24°C, a relative humidity of 45%, and a day/night photoperiod of 12/12. Dust-free birch wood chips are used as bedding. Before the experiment, animals were raised under standard conditions with free access to water and food. Before the experiment, animals were completely fasted 12 hours ago, but kept free access to water.

Capsules 11.2±0.5 mm in length and 5.0±0.4 mm in diameter were pre-lubricated with vegetable oil and placed on the tongue base of the animals. A probe was used to help the animals swallow the capsules for intragastric administration of liquid medication. Control animals were given similar capsules without medication.

After the introduction of the capsules, the condition of the animals was observed for 3 hours. In order to evaluate the dynamics of glucose levels in the blood of the animals, blood was taken every 10 minutes. Blood glucose levels were assessed from the tail artery using a glucometer.

After 10 minutes of introducing the capsule form of medicine, notice the decline of blood sugar level.After 20 minutes, observe the stability of glucose level, and after 50 minutes, record the trend that glucose value increases.Meanwhile, little change in control animals during this period.The change noted in record is in the scope of 5-6.5mM, which may be relevant with the slight stress from the process of introducing capsule and drawing blood in animals.In fact, after 60 minutes, the glucose level in control animals has returned to its original value.Afterwards, observe some fluctuations in the blood sugar level of animal (control and medicine), which is obviously relevant with the anxiety from the process of drawing blood of animal and lack of food.

After 3 hours of the experiment, the animals had free access to food.

Throughout the experiment, the animals did not show any obvious deviations in activity and behavior. After the experiment, the animals survived and did not show any abnormalities in the course of life.

Claims (12)

1. A method for preparing an oral form of a substance containing insulin, wherein crystalline recombinant insulin, an antioxidant, an additional antioxidant, a nonionic surfactant, a phospholipid and sorbitol are mixed in calculated amounts, the resulting mixture is combined with an organic solvent by mixing in a reactor under conditions that convert the organic solvent to a gaseous state, the solvent is removed, and a powdered substance is obtained in which a phospholipid membrane is formed in the crystalline structure of sorbitol, and the powdered substance is then ground to obtain a finely divided powder with a particle size of 160-200 μm, which powder is encapsulated with the addition of polyethylene glycol. 2. The method according to claim 1, characterized in that a substance from the group of beta-carotene, astaxanthin, dihydroquercetin and abisil is used as antioxidant. 3. The method according to claim 1, characterized in that a substance from the group of tocopherol acetate and ascorbic acid is used as an additional antioxidant. 4. The method according to claim 1, characterized in that a substance from the group of Tween-80 and Tween-60 is used as non-ionic surfactant. 5. The method according to claim 1, characterized in that a solvent in which the phospholipid and the antioxidant are dissolved is used as the organic solvent. 6. The method according to claim 1, characterized in that phospholipids obtained from vegetable oils are used as phospholipids. 7. The method according to claim 6, characterized in that linseed oil, black cumin oil or sunflower oil is used as plant-based raw material for the phospholipids. 8. Process according to claim 1, characterised in that the finely divided powder is obtained by grinding in a hammer mill with a mesh of 160-200 μm. 9. The method according to claim 1, characterized in that the antioxidant and the phospholipid are taken in an amount to ensure their co-solubility in the organic solvent. 10. A material for preventing complications of type 1 diabetes prepared by the method of claim 1, comprising an effective amount of crystalline recombinant insulin, an antioxidant, another antioxidant, a surfactant, and sorbitol forming a phospholipid membrane in its crystalline structure. 11. Substance according to claim 10, characterised in that it additionally comprises polyethylene glycol. 12. Substance according to any one of claims 10-11, characterised in that it is formulated for oral administration.