WO2023200363A1

WO2023200363A1 - Method for producing an oral form of an insulin-containing substance

Info

Publication number: WO2023200363A1
Application number: PCT/RU2023/000114
Authority: WO
Inventors: Фания Иршатовна МАГАНОВА; Владимир Петрович ТОКАРЕВ
Original assignee: Фания Иршатовна МАГАНОВА
Priority date: 2022-04-13
Filing date: 2023-04-13
Publication date: 2023-10-19

Abstract

The invention relates to medicine, and more particularly to the chemical and pharmaceutical industry, and to the creation of an oral form of an insulin-containing substance and the delivery thereof for treating complications of type 1 diabetes. Said substance is produced using a method which involves mixing crystalline recombinant insulin, an antioxidant, an additional antioxidant, a non-ionic surfactant, phospholipids and sorbitol in a calculated amount, combining the resulting mixture with an organic solvent by mixing in a reactor under conditions providing for the transition of the organic solvent to a gaseous state, removing the solvent and obtaining a powdered mass in which a phospholipid film is formed in the crystalline structure of the sorbitol, then grinding the powdered mass to produce a fine powder, which is encapsulated with the addition of polyethylene glycol.

Description

METHOD FOR OBTAINING ORAL FORM OF A SUBSTANCE CONTAINING INSULIN Technical field

The invention relates to medicine, namely to the chemical and pharmaceutical industry, to the creation of an oral form of the insulin substance and its delivery for the prevention of complications of type 1 diabetes.

Prior Art

Recently, a lot of information has appeared about the results of studies that have demonstrated the effectiveness and safety of oral insulin. It is assumed that this form of insulin use indicates the safety of the drug and is due to its intestinal form. Absorbed in the intestines, 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 its greater effectiveness.

There are known successful clinical trials of the drug ORMD-0801 (Oramed Pharmaceuticals), which can become a commercially available oral insulin drug.

However, the forms of insulin used (oral or injectable) also depend on the type of disease and, accordingly, the composition of the drug. Because In type 1 diabetes, the body does not produce insulin; such diabetes is treated by administering insulin through injections. It is believed that oral medications are not suitable for treating type 1 diabetes because insulin will be broken down in the digestive system.

To improve the effectiveness of the oral form of insulin (slow down its breakdown in the digestive tract), they try to speed up the absorption of insulin through the membrane of the small intestine.

Publications are known from the prior art that disclose various proposed compositions of the oral form of insulin and methods for their preparation. So in publication CN 11297269 dated June 18, 2021, cl. A61K 38/28 discloses a composition that is prepared from insulin and/or insulin analogs and a pharmaceutical composition for promoting absorption in the small intestine, wherein the pharmaceutical composition for promoting absorption in the small intestine consists of sodium lauryl sulfate, chitosan and sodium citrate. According to the pharmaceutical composition, a compound adjuvant is prepared to promote absorption in the small intestine, and after combining the compound adjuvant with insulin and/or insulin analogs, the absorption of the effective component in the small intestine can be improved.

Also known is the publication CN 110974943 dated 04/10/2020, class. A61K 38/28, which states that the medicinal product contains: (a) an inner core layer containing insulin or a biologically active derivative thereof as an active ingredient; (b) an adhesive outer core layer formed on the inner core layer; (c) a protective coating layer formed on the outer core layer; and (d) a localized drug release coating layer formed on the coating backing layer, wherein the coating backing layer is selected from one or more of ethylcellulose, Eudragit RS (Reed-Solomon), or wax; and the localized drug release coating layer contains Eudragit L100 and Eudragit S100, which are mixed in a mass ratio of (0-1):(1-4) and used as a substrate material for the localized drug release coating.

Nanostructured compositions for oral delivery of insulin and methods for their preparation are known, disclosed in RF patent 2753018 dated 08/11/2021, international publication W02019148810 dated 08/08/2019 class. A61K 38/28; A61K47/36, etc. The main disadvantage of these known forms of insulin is the complexity of the technology for preparing such a composition and the possibility of use not for all types of diabetes. As mentioned earlier, in case of disruption of the pancreas, or to be more precise, its beta cells, insulin is not produced, so type 1 diabetes occurs.

If the cells and tissues of the body do not respond to insulin, often due to obesity or improper secretion of the hormone, type 2 diabetes begins to develop.

The essence of the declared decision

The technical problem to be solved by the claimed group of inventions is associated with eliminating the shortcomings of known analogues.

The technical result of the use of the invention is the creation of an oral form of a substance, including insulin, of targeted action, suitable for the prevention of complications of type 1 diabetes.

The specified technical result is achieved through a method for producing an oral form of a substance containing insulin, in which crystalline recombinant insulin, an antioxidant, an additional antioxidant, a nonionic surfactant, phospholipids and sorbitol in a calculated amount are mixed, the resulting mixture is combined with an organic solvent by stirring in a reactor under conditions of transition of the organic solvent into a gaseous state, it is removed and a powder mass is obtained, in which a phospholipid film is formed in the crystalline structure of sorbitol, then the powder mass is crushed to obtain a fine powder, which is encapsulated with the addition of polyethylene glycol.

In this case, a substance from the group: beta-carotene, astaxanthin, dehydroquercetin, abisil, etc. is used as an antioxidant. This antioxidant serves to interrupt the body’s autoimmune reaction to pancreatic beta cells, leading them over time to apoptosis and blocking insulin production.

As an additional antioxidant, a substance from the group is used: tocopherol acetate, ascorbic acid, etc. Additional antioxidant serves to protect antioxidant molecules from oxidation, since the latter is a very active substance in redox reactions.

The presence of an antioxidant and insulin in the substance allows it to perform two functions, namely, to deliver insulin molecules to the body's cells and, over time, restore the functioning of the beta cells of the pancreas, thereby promoting the release of insulin.

Tween-60 or Tween-80 is used as a nonionic surfactant.

Chloroform or diethyl alcohol is used as an organic solvent, in which crystalline beta-carotene and the outer cell membrane of vegetable oils are dissolved to release phospholipids. Vegetable oils such as flaxseed oil, black cumin oil, sunflower oil, etc. are used as a source of phospholipids. In this case, the antioxidant and phospholipids are taken in quantities that ensure their joint solubility in an organic solvent.

To mix these components, use equipment designed for this purpose (reactor, for example, Mixing brand, vacuum pump). Under selected conditions, for example, a temperature not higher than +50°C, a stirring time of, for example, no more than 3 hours, the organic solvent passes into a different state of aggregation - gaseous, which makes it possible to remove it using a vacuum pump, followed by condensation and collection in its original form for reuse.

The sorbitol used has a crystalline structure, which, as a result of the technological process, is filled with the resulting phospholipids in the form of a film lining all the pores of the structure.

The resulting powder mass is crushed to obtain a fine powder in a hammer mill with a 160-200 micron sieve. A given powder dispersion is important for the encapsulation process and its rapid dissolution in the small intestine upon contact with the liquid phase (water) to form liposomes. Insulin is a hypoglycemic polypeptide hormone with a molecular weight of about 6000, which is produced in the beta cells of the islets of Langerhans in the pancreas. It has a multifaceted effect on metabolism in almost all tissues. The main effect of insulin is to regulate carbohydrate metabolism, in particular the utilization of glucose in the body. Insulin increases the permeability of plasma membranes to glucose and other macronutrients, activates key enzymes of glycolysis, stimulates the formation of glycogen from glucose in the liver and muscles from glycogen glucose, and enhances the synthesis of fats and proteins.

Impaired insulin secretion due to the destruction of beta cells - absolute insulin deficiency - is a key element in the pathogenesis of type 1 diabetes mellitus. Impaired action of insulin on tissue - relative insulin deficiency - plays an important role in the development of type 2 diabetes mellitus.

Purified, it is an amorphous powder, colorless, soluble in acidified or alkaline water, in weak alcohol or glycerin, insoluble in ether, chloroform, absolute alcohol, completely soluble in sodium phosphate solutions.

Once insulin enters the portal bloodstream, it first reaches the liver, where approximately half of it is inactivated. Thus, portal insulinemia is much higher than in peripheral blood, which seems necessary to stop the flow of glucose into the liver.

The problem with using exogenous insulin is that "insulin is inactive when administered orally" because it is broken down by proteolytic digestive enzymes; hence the need for parenteral administration and the inability to truly mimic the endocrine pancreas because physiologically insulin first reaches the liver, being diluted in the peripheral circulation.

The claimed invention makes it possible to obtain an oral form of insulin substance suitable for the prevention of complications of type 1 diabetes. The authors of this invention proposed the composition of a substance obtained by the specified method, which provides the specified technical result, and which includes crystalline recombinant insulin, an antioxidant, an additional antioxidant, an ionic surfactant, sorbitol with a phospholipid film formed in its crystalline structure, in an effective amount , which is effective in preventing complications of type 1 diabetes.

By adding polyethylene glycol (PEG 400) to the substance, it can be encapsulated and used for oral administration.

As a special case of implementing the composition of the proposed substance containing insulin, there can be a substance that includes the following substances in their ratio, mg/g:

• Crystalline recombinant insulin - 8-10

• Beta-carotene - 20-30

• Alpha tocopherol acetate - 20-25

• Ascorbic acid - 10-13

• Phospholipids - 80-100

• Twin-80 - 20-25

• Sorbitol - 842-797

Quantitative characteristics of the constituent components were obtained experimentally.

The specified substance is placed in hard gelatin enteric capsules No. 3 (encapsulation process). This type of capsule is necessary to deliver the substance to the small intestine. Here, the gelatin capsule shell dissolves and after contact of the phospholipid film with the liquid phase, lipid vesicles-liposomes are formed. Liposomes (phospholipid vesicles) have long been studied as a promising dosage form that significantly changes the distribution of medicinal substance and protecting it from the effects of enzymes. These are microspheres ranging in size from 25 nm to 1 μm, which serve as containers for medicinal substances, with polar compounds located in the internal aqueous volume, and hydrophobic ones soluble in the membrane. The properties of liposomes and their behavior are determined primarily by the presence of a closed membrane shell. Despite its molecular thickness (about 4 nm), the lipid bilayer is characterized by exceptional mechanical strength and flexibility. In the liquid crystalline state of the bilayer, its components have high molecular mobility, so that in general the membrane behaves as a fairly liquid, fluid phase. Thanks to this, liposomes maintain their integrity under various damaging influences, and their membrane has the ability to self-heal structural defects that arise in it. 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 disrupted and liposomes can break down into smaller particles. The membrane of liposomes consists of natural phospholipids, which determines many of their attractive qualities. They are non-toxic, biodegradable, under certain conditions they can be absorbed by cells, their membrane can merge with the cell membrane, which provides intracellular delivery of their contents.

The effectiveness of liposomal drugs is largely determined by the amount of drug included in them. However, numerous technologies for the production of liposomal drugs ensure that no more than 50% of the active principle (drug substance) is included in liposomes. The influence of the method of obtaining a liposomal preparation, the ratio of its components and excipients on the degree of inclusion of substances in liposomes was studied. It has been established that the most optimal method to ensure the inclusion of the maximum amount of medicinal substances in liposomes is the method of producing dry proliposomal preparations, upon contact of which with the liquid phase (water) liposomes are formed. In contrast to methods known from the prior art, the claimed invention makes it possible to obtain effective liposomal preparations of fat-soluble substances (100% inclusion). The high degree of incorporation of molecules of medicinal substances into liposomes allows for a high degree of bioavailability of the substance, which is determined by the difference between the volume of the administered substance and its volume in the target organ. Dry proliposomal substance is a fine powder enclosed in a hard gelatin capsule No. 3, enteric, which contains a sorbent (sorbitol), in the crystal structure of which a phospholipid film (4 nm thick) is formed. When taking such a capsule, it passes through the gastrointestinal tract, reaching the small intestine. Here the gelatin shell of the capsule is destroyed, upon contact with the liquid phase, the phospholipid film collapses with the spontaneous formation of liposomes (hundreds of thousands of particles with a size of minus 8-9 degrees). Liposomes have a high degree of capture of hydrophilic (water-soluble) or hydrophobic molecules (insoluble in water) drug substances, and the degree of capture depends on their molecular weight. For beta-carotene it is 6-8 molecules, and for insulin 2 molecules. Next, liposomes loaded with molecules of a drug substance containing insulin enter the liver through the portal vein and then into the systemic circulation.

To overcome the macrophage system, a protective film of polyethylene glycol is used, for example PEG-400, which is added to the substance at the encapsulation stage. In this way, and due to the dynamics of blood circulation, a high degree of bioavailability of the substance is achieved.

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

The use of the claimed substance is shown in the Example, which is one of the possible cases of use and does not limit such use. Example.

To confirm the effectiveness of the use of the claimed substance in the prevention of complications of type 1 diabetes, two groups of mice were used:

- first group - female mice of the balb/c line, 11 weeks old (18 individuals);

- second group (control group) - female balb/c mice aged 11 weeks (12 individuals).

Using the claimed method, a substance of the following composition was prepared, mg per 1 capsule weighing 0.17 g:

Crystalline recombinant insulin - 2 mg

Beta-carotene - 6 mg

Alpha tocopherol acetate - 5 mg

Ascorbic acid - 2.5 mg phospholipids obtained from flax seeds - 20 mg

Twin-80 - 3 mg

Polyethylene glycol PEG 400 - 1.5 mg

Sorbitol - 130 mg,

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

2 minutes after preparing the solution, it was administered orally using a 2 ml syringe without a needle to individuals of the first group. The volume of the administered substance was 150 μl, which contained 0.75 IU of insulin.

The control group (of 12 individuals) was administered mQ orally in an appropriate volume.

Before administration of the substance, as well as at various time intervals after administration, the level of glucose in the blood of animals was measured by taking it from the tail artery, using a TaiDoc 4239 veterinary glucose analyzer.

The blood glucose level in balb/c mice after oral administration of a solution of proliposomal insulin is shown in Fig. 1. 75 minutes after administration of the solution, the animals developed signs of hypoglycemic coma (loss of consciousness, lack of ability to move, rapid heartbeat), which was confirmed by a decrease in blood glucose levels (3.0 mM - with the norm being 4.8-6.5 mM). Animals with symptoms of hypoglycemia were administered orally a 1 M glucose solution. All animals survived the experiment and felt normal.

Thus, it was confirmed that the active substance - insulin was delivered to the cells of the body, i.e. The pharmacological effect of the proposed substance has been confirmed. This experiment allows us to offer the claimed oral insulin substance as both a preventive and therapeutic agent.

The substance in the form of a capsule, prepared by the claimed method, in a dose (15.6 IU) of modified insulin, was administered orally to an experimental animal. Male outbred Sprague Dawley rats aged 10 weeks were used in the experiment. The weight of the experimental animals was 387 g and 393 g. The rats were kept one at a time in individually ventilated cages (IVC) of the OptiRAT system (Animal Care Systems) at a temperature of 22-24°C, relative humidity 45% with a light cycle day/night 12/12. Dust-free birch sawdust was used as bedding material. Before the experiment, the animals were kept under standard conditions with free access to water and food. Before the experiment, the animals were completely deprived of food for 12 hours, but had free access to water.

Capsules with a length of 11.2 = 1 = 0.5 mm and a diameter of 5.0 ± 0.4 mm were pre-lubricated with vegetable oil, placed on the root of the animal’s tongue, and the animal was helped to swallow it using a probe for intragastric administration of liquid drugs. The control animal was administered a similar capsule without the drug.

After administration of the capsules, the condition of the animals was observed for 3 hours. To assess the dynamics of glucose levels in the blood of animals, blood was taken every 10 minutes. Glucose levels were assessed in blood from the tail artery using a glucometer. Already 10 minutes after administration of the capsule form of the drug, a decrease in blood glucose levels was noted. After 20 minutes, stabilization of glucose levels was observed, and after 50 minutes, a trend towards an increase in glucose values was recorded. At the same time, in the control animal during this period it changed little. The noted changes were recorded in the range of 5-6.5 mM, which may be due to minor stress in the animal from the procedure of introducing the capsule and drawing blood. Indeed, after 60 minutes, the control animal’s glucose level returned to its original values. Subsequently, some fluctuations in the level of glucose in the blood of animals (control and drug) were observed, which were apparently associated with the anxiety of the animals from the blood collection procedure and lack of access to food.

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

Throughout the experiment, the animals did not show significant deviations in activity and behavior. The animals were alive after the experiment and showed no abnormalities in their vital processes.

Claims

CLAIM

1. A method for obtaining an oral form of a substance containing insulin, in which crystalline recombinant insulin, an antioxidant, an additional antioxidant, a nonionic surfactant, phospholipids and sorbitol in a calculated amount are mixed, the resulting mixture is combined with an organic solvent by stirring in a reactor under conditions of transition of the organic solvent into gaseous state, remove it and obtain a powder mass in which a phospholipid film is formed in the crystalline structure of sorbitol, then the powder mass is crushed to obtain a fine powder with a particle size of 160-200 microns, which is encapsulated with the addition of polyethylene glycol.

2. The method according to claim 1, characterized in that a substance from the group: beta-carotene, astaxanthin, dehydroquercetin, abisil is used as an antioxidant.

3. The method according to claim 1, characterized in that a substance from the group: tocopherol acetate, ascorbic acid is used as an additional antioxidant.

4. The method according to claim 1, characterized in that a substance from the group: Tween-80, Tween-60 is used as a nonionic surfactant.

5. The method according to claim 1, characterized in that the organic solvent is a solvent in which phospholipids and antioxidants are dissolved.

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 flaxseed oil, black cumin oil or sunflower oil are used as plant raw materials for phospholipids.

8. The method according to claim 1, characterized in that the fine powder is obtained by grinding in a hammer mill with a mesh of 160-200 microns.

9. The method according to claim 1, characterized in that antioxidants and phospholipids are taken in quantities that ensure their joint solubility in an organic solvent.

10. A substance for the prevention of complications of type 1 diabetes, obtained by the method according to claim 1, including crystalline recombinant insulin, an antioxidant, an additional antioxidant, a surfactant, sorbitol with a phospholipid film formed in its crystalline structure, in an effective amount.

11. The substance according to claim 10, characterized in that it additionally includes polyethylene glycol.

12. Substance according to any one of paragraphs. 10-11, characterized in that it is formulated for oral administration.