WO2013100793A1

WO2013100793A1 - Insulsin derivative with antihyperglycemic activity and method for the production thereof

Info

Publication number: WO2013100793A1
Application number: PCT/RU2011/001061
Authority: WO
Inventors: Артур Викторович МАРТЫНОВ; Борис Славинович ФАРБЕР; Софья Борисовна ФАРБЕР
Original assignee: Martynov Artur Viktorovich; Farber Boris Slavinovich; Farber Sof Ya Borisovna
Priority date: 2011-12-28
Filing date: 2011-12-28
Publication date: 2013-07-04
Also published as: EA023447B1; EA201300207A1; IN2014MN01484A

Abstract

The invention relates to pharmaceutics and medicine and, more specifically, to compositions of a biologically active substance, in particular supramolecular assemblies of acylated insulin oligopeptides, for oral administration in the treatment of diabetes mellitus and complications thereof. The proposed system makes it possible, using a single oral dose, to maintain the level of glucose in the blood at a physiological level for 24 hours (super effect), and the ingredients of the system acquire resistance to enzymatic action, have small dimensions and can be easily absorbed from the intestines. Essence of the invention: The principle active agents are in the form of a supramolecular composition, namely an assembly consisting of acylated oligopeptide products of the enzymatic hydrolysis of insulin. The composition can be produced industrially in sufficient amounts on account of the availability of all of the ingredients of the composition.

Description

SUGAR-LOWERING INSULIN DERIVATIVE

ACTIVITY AND METHOD OF ITS PRODUCTION

Technical field

The invention relates to pharmacy and medicine, namely, to orally, rectally, aerosol and parenterally administered compositions of a biologically active substance, in particular insulin oligopeptides for treating diabetes mellitus, its complications and for enhancing tissue regeneration in burns, diabetic foot syndrome.

State of the art

Diabetes mellitus is one of the most common serious diseases, which is based on an absolute or relative deficiency of the pancreatic hormone - insulin. Insulin therapy (insulin administration from the outside) is the only and traditional method of treating the disease, which makes it possible to compensate for the lack of insulin in the body. The most common way to administer insulin is by subcutaneous injection. This method is inconvenient, it injures the psyche of patients (especially children), causes physical and moral suffering, but most importantly, it can exacerbate the pathology of the disease by itself. The latter is due to the fact that during subcutaneous injection of insulin, the normal concentration of glucose in the blood is achieved due to systematic hyperinsulinemia of peripheral tissues, while the liver (the main site of activity of the endogenous insulin produced in the body) lacks insulin.

The only way to prevent the complications that are inevitably associated with injection insulin therapy, if possible to achieve a complete imitation of the natural route of hormone intake in a living organism, is to simulate the physiological difference in insulin levels in the portal and peripheral circulatory systems. And from this point of view, the oral (by mouth) route of insulin delivery is most favorable.

The main obstacles to the creation of oral forms of insulin are the low resistance of the hormone to the action of proteolytic enzymes of the gastrointestinal tract, as well as the low permeability of insulin through the epithelial the intestinal wall tissue into the bloodstream, due to both low lipophilicity and the size of the hormone macromolecules.

Over the past decades, numerous attempts have been made to create oral forms of insulin, but so far it has not been possible to develop an effective drug that can compete in therapeutic effect with insulin administered by injection. Among the dosage forms of oral preparations, the most attractive and promising is the solid form, since it is the most comfortable and convenient both for use by patients and for storage, in addition, the production technology of such forms is relatively inexpensive and sufficient worked out.

Known solid insulin-containing drug, consisting of a core containing insulin and excipients, and a shell of a biodegradable film-forming polymer material [1]. The preparation is prepared by introducing 1-40 mg of crystalline insulin and 200 mg of a stoichiometric mixture of 5-methoxysalicylic acid and sodium bicarbonate into a hard gelatin capsule or tablet. Then, the capsule (tablet) is coated with a copolymer of hydroxyethyl methacrylate and styrene crosslinked with divinylazobenzene. The membrane is resistant to the action of the environment of the stomach and small intestine, but disintegrates in the large intestine under the influence of microorganisms present there. The disadvantage of this tool is low efficiency and indefinite time to achieve maximum effect. Oral administration to rats of said vehicle containing 1 unit. insulin leads to a decrease in blood glucose concentration by 20% 9 hours after administration. At the same time, subcutaneous administration of a solution of insulin in a dose of 0.1 or 1.0 units. leads to a decrease in blood glucose by 39 and 63%, respectively. The maximum hypoglycemic effect for individual animals is achieved in the period from 1 to 9 hours, and in some animals there is no effect of a decrease in glucose concentration even after 10 hours after drug administration.

A solid insulin-containing drug is known in the form of nanocapsules, which are microparticles with a diameter of about 300 nm, consisting of bio-degradable (due to intestinal microflora) polyalkylcyanoacrylate with insulin immobilized inside it [2]. s

The disadvantages of this tool is low efficiency and indefinite time to achieve maximum effect. So, for example, when it is orally administered to rats at doses of 12.5, 25 and 50 units / kg of insulin, the clinical effect (a 50-60% decrease in blood glucose level) is observed only on the second day after the administration of nanocapsules. There is a need to develop insulin compositions for oral administration that are to some extent protected from the effects of the hostile environment of the stomach. Although numerous attempts have been made to develop such compositions, the applicant does not know a single one that today would be supplied to a large extent on the market. The applicant is only aware of the following proposals. U.S. Patent Known for Describing Oral Administration of Insulin

[3], which uses a system of microemulsions based on the oil / water system, also containing several emulsifiers, and insulin is dissolved in the aqueous phase. However, the marked systems have a number of drawbacks: the onset of the action of orally taken insulin begins after 1–2 hours and does not reach the norm, emulsions tend to stratify, lose activity due to spontaneous proteolysis of insulin due to the absence of protectors, and the insulin used is in no way protected from external factors - enzymes, temperature, light. The authors failed to achieve a drop in glucose levels of 3-5 mg / dL at all, so these systems could not replace parenteral insulin. Thus, it is relevant to create oral preparations that can not only replace prolonged parenteral insulin, but also prolong its effects for at least 24 hours.

SUMMARY OF THE INVENTION An object of the invention is to provide a supramolecular ensemble of excessively negatively charged oligopeptides of an insulin hydrolyzate having sugar-lowering activity when administered orally and dosage forms based on it that can reduce blood glucose to normal and maintain this level for at least a day.

This goal is achieved by the fact that get the pharmaceutical composition, as the main active substances, which contains a quasi-fluid self-acting the system to be organized is the supramolecular ensemble of peptides — products of enzymatic hydrolysis of insulin with trypsin, papain, chymotrypsin, which are then acylated with dicarboxylic acid anhydrides — succinic, acetic, maleic, and additionally contain pharmaceutically acceptable excipients. Such a composition may be incorporated into an ointment, aerosol and used orally.

The content of acylated peptides can range from 0.0001 to 3.0 mass /%.

In the human body, such oligopeptides are resistant to enzymes, protected from further hydrolysis by acylation, and the small size of the peptides allows them to be freely absorbed. In addition, such a system of peptides that were previously intact (insulin molecule), but with altered charges, is capable of self-organization on insulin receptors into an insulin-like supramolecular structure. The negative charge of oligopeptides and their resistance to enzymatic hydrolysis contribute to the 24-hour (or more) prolongation of action and the maintenance of the level of glucose in the blood at the physiological level.

The best embodiment of the invention

Example 1. Synthesis of a quasi-fluid system based on succinylated insulin peptides.

100 mg of crystalline insulin (ES Lilli) was dissolved in 1 ml of 0.1 M hydrochloric acid and then enzymatically hydrolyzed by incubation with proteinase K at room temperature for 1 hour. Then, while stirring the solution, succinic anhydride (7.5 mg), crushed to a powder, is slowly added and 60 minutes are incubated with stirring. The resulting peptides were purified from salts on a Sephadex G-25 chromatographic column, where TRIS-hydrochloride was used as an eluent. Protein yield was controlled by the absorption of the eluate in the ultraviolet region at 280 nm. Desalted peptides were poured into ampoules and lyophilized. The resulting peptides were further used to obtain various dosage forms and study their activity.

Example 2. Obtaining oral forms of succinylated insulin peptides.

The peptides obtained in Example 1 were dissolved in a 0.9% sodium chloride solution to form a solution equivalent to 4.3 mg protein / ml, 2% polysorbate stabilizer was added. Sodium ascorbate, benzalkonium chloride, methyl and propyl parabens can be used as preservatives, and up to 3% PEO-400 to enhance the absorption of peptides. The resulting solution in a 5-fold (with respect to the injectable form) dose was administered to animals orally. Example 3. The study of the sugar-lowering effect of the supramolecular ensemble of insulin peptides (SMAI) when administered orally on a model of alloscan diabetes in rats

The experiments were carried out on sexually mature male rats Wistar, the content, care for animals and removing them from the experiment was carried out in accordance with the "Rules for the work using experimental animals" Experimental animals (n = 40) were divided into 4 groups: K ”- rats receiving 0.1 ml of physiological solution daily (per os) during the experiment per os (n = 10); “SM” - rats that received 0.1 ml of SMAI according to the same scheme (n = 10); “Al” - rats with alloxan diabetes receiving 0.1 ml of physiological saline daily (per os) during the experiment per os (n = 10); “Al + SMAI” - rats with alloxan diabetes receiving 0.1 ml of SMAI according to the same scheme (n = 10). Experimental diabetes mellitus was caused by a single intraperitoneal injection of a diabetogenic dose of alloxan — 17 mg / 100 g body weight. All studies were performed on an empty stomach, rats were given only water. At different periods of the experiment, the blood glucose was measured in the blood of animals using the biosensor glucose level sensor One touch Ultra (USA) and disposable indicator electrodes for it. The data are presented in table 1.

Table 1. - Dynamics of blood glucose in rats with experimental alloxan diabetes after a single oral administration of a supramolecular ensemble of insulin peptides

Notes: * - Statistically significant differences between the group Al + SMAI and Al (p <0.05); ** - Statistically significant differences between the groups Al and K (p <0.05). For statistical processing used the method of univariate analysis of variance and multiple comparisons.

As can be seen from Table 1, the oral administration of a supramolecular ensemble (self-organizing quasi-fluid system) from acylated insulin fragments normalizes the blood glucose level in experimental rats with alloxan diabetes. Already 60 minutes after oral administration of SMAI, the glucose level returns to normal and remains at a level acceptable for diabetic rats for 24 hours. This super effect from the use of oral insulin or its derivatives and the prolongation of the sugar-lowering effect for 24 hours with a single application could not be caused to anyone before.

In order to study the effect of SMAI on maintaining glucose levels in rats with alloxan diabetes, an experiment with glucose loading was carried out.

Example 4. The study of the sugar-lowering effect of the supramolecular ensemble of insulin peptides (SMAI) when administered orally on the model of alloscan diabetes in rats on the background of glucose load

The experiments were performed on sexually mature Wistar male rats. The experimental animals (n = 40) were divided into 4 groups: “K” - rats receiving 0.1 ml of physiological saline daily (per os) during the experiment per os (n = 10); “SM” - rats treated according to the same scheme as 0.1 ml of SMAI (n = 10); “Al” - rats with alloxan diabetes who received 0.1 ml of physiological saline daily (per os) during the experiment per os (n = 10); “Al + SMAI” - rats with alloxan diabetes receiving 0.1 ml of SMAI according to the same scheme (n = 10). Experimental diabetes mellitus was caused by a single intraperitoneal administration of a diabetogenic dose of all ocean — 17 mg / 100 g body weight. Once daily, 1 ml of 5% glucose was intraperitoneally administered to rats and fed 3 times a day. At different periods of the experiment, the blood glucose was measured in the blood of animals using the biosensor glucose level sensor One touch Ultra (USA) and disposable indicator electrodes for it. The data are presented in table 1.

Table 2. - Dynamics of blood glucose in rats with experimental alloxan diabetes after a single oral administration of a supramolecular ensemble of insulin peptides against a background of glucose and food load Experimental Glucose (mmol / L)

group 30 min 60 min 120 min 8 hours 24 hours

Control 9.4 ** ± 2.1

SM 9.2 ± 1.3 8.9 ± 2.1 9.0 ± 1.8 9.4 ± 1.8 9.4 ± 1.8

Al 40.5 ± 3.5 40.0 ± 3.4 40.0 ± 3.3 40.0 ± 3.3 40.3 ± 3.1

Al + SMAI 27.9 * ± 2.1 7.4 * ± 1.4 9.6 * ± 1.6 8.2 * ± 1.5 12.1 * ± 1.4

Notes: * - Statistically significant differences between the group Al + SMAI and Al (p <0.05); ** - Statistically significant differences between the groups Al and K (p <0.05). For statistical processing, the method of univariate analysis of variance and multiple comparisons was used.

As can be seen from table 2, the oral administration of the supramolecular ensemble

(self-organizing quasi-fluid system) from acylated insulin fragments leads to normalization of blood glucose levels in experimental rats with alloxan diabetes. Already 60 minutes after oral administration of SMAI, the glucose level returns to normal (7.4 ± 1.4) and remains at a level acceptable for diabetic rats for 24 hours, regardless of food intake and glucose injections. Against the background of glucose load from the use of oral insulin or its derivatives and prolongation of the sugar-lowering effect for 24 hours with a single use, no one was previously able to obtain this super-effect. Industrial applicability

The invention relates to pharmacy and medicine, namely, to orally, rectally, aerosol and parenterally administered formulations of a biologically active substance, in particular insulin oligopeptides for treating diabetes mellitus, its complications and for enhancing tissue regeneration in case of burns, diabetic foot syndrome. This system allows you to keep blood glucose levels at the physiological level for more than 24 hours with oral use, and the components of the system are resistant to enzymes and are small, easily absorbed from the intestines, through the skin and mucous with any method of application system. Sources of information used:

[1] M. Saffran, G.S. Kumar, C. Savarlar, J.C. Burnham, F. Williams, D.S. Neckers, A new approach to the oral administration of insulin and other peptide drugs, Science, v. 233, p. 1081-1084, 1986.

[2] C. Damge, C. Michel, V. Aprahamian, P. Couveur, J.P. Devissaquet, Nanocapsules as carrier for oral peptide delivery, Journal of Controlled Release, v. 13, p. 233-239, 1990.

[3] US Patent 5824638

Claims

Claim

1. An insulin derivative that has oral hypoglycemic activity and dosage forms based on it, characterized in that the insulin derivative contains a supramolecular ensemble of peptides of the enzymatic insulin hydrolyzate, in which the positive charges of the amino groups of the residues of basic amino acids are replaced by negatively charged carboxyl groups .

2. The insulin derivative of claim 1, wherein pork insulin is used as the insulin.

3. The insulin derivative according to claim 1, characterized in that human genetic engineering insulin is used as the insulin.

4. The derivative of insulin according to claim 1, characterized in that the enzymatic hydrolyzate is obtained by treating insulin with trypsin.

5. The insulin derivative according to claim 1, characterized in that the enzymatic hydrolyzate is obtained by treating the insulin with chymotrypsin.

6. The insulin derivative according to claim 1, characterized in that the enzymatic hydrolyzate is obtained by treating insulin with papain.

7. The insulin derivative according to claim 1, characterized in that the enzymatic hydrolyzate is obtained by treating insulin with proteinase K.

8. The insulin derivative according to claim 1, characterized in that the positive charges of the amino groups of the residues of the basic amino acids are replaced by negatively charged carboxyl groups by acylation with succinic anhydride.

9. The insulin derivative according to claim 1, characterized in that the positive charges of the amino groups of the residues of the basic amino acids are replaced by negatively charged carboxyl groups by acylation with maleic anhydride.

10. The insulin derivative according to claim 1, characterized in that the positive charges of the amino groups of the residues of basic amino acids are replaced by negatively charged carboxyl groups by acylation with acetic anhydride.

11. The insulin derivative according to claim 1, characterized in that the positive charges of the amino groups of the residues of the basic amino acids are replaced by negatively charged carboxyl groups by alkylation with monochloracetic acid.

12. The insulin derivative according to claim 1, characterized in that the peptides, products of the enzymatic hydrolysis of insulin, modify the acylating (alkylating) agent: protein ratio in the range from 0, 1: 1 to 3: 1.

13. The derivative of insulin according to item 12, characterized in that it is made with this ratio of components, mass. %:

The supramolecular ensemble of peptides 0.0001-3.0

the rest is excipients up to 100.

14. The derivative of insulin according to item 13, characterized in that it is manufactured as a liquid dosage form (emulsion, aqueous solution, non-aqueous).

15. The derivative of insulin according to claim 14, characterized in that it contains a hydrophobic base.

16. The insulin derivative of claim 14, characterized in that it comprises a hydrophilic base.

17. The derivative of insulin according to 14, characterized in that it contains a diphilic base.

18. The derivative of insulin according to 14, characterized in that it is made as an aerosol or aerogel for oral administration (application to the cheek from the inside).

19. A method of obtaining a derivative of insulin having a sugar-lowering activity during oral administration and dosage forms based on it, characterized in that a supramolecular ensemble of peptides is taken as an insulin derivative an enzymatic insulin hydrolyzate in which the positive charges of the amino groups of the residues of the basic amino acids are replaced by negatively charged carboxy groups by acylation or alkylation.

20. A method for producing an insulin derivative according to claim 19, characterized in that pig insulin is used as insulin.

21. The method for producing the insulin derivative according to claim 19, characterized in that human genetic engineering insulin is used as the insulin.

22. A method for producing an insulin derivative according to claim 19, characterized in that the enzymatic hydrolyzate is obtained by treating the insulin with trypsin.

23 A method for producing an insulin derivative according to claim 19, characterized in that the enzymatic hydrolyzate is obtained by treating the insulin with chymotrypsin.

24. A method for producing an insulin derivative according to claim 19, wherein the enzymatic hydrolyzate is obtained by treating the insulin with papain.

25. The method of obtaining the insulin derivative according to claim 19, characterized in that the enzymatic hydrolyzate is obtained by treating insulin with proteinase K.

26. The method of producing the insulin derivative according to claim 19, characterized in that the positive charges of the amino groups of the residues of the basic amino acids are replaced by negatively charged carboxyl groups by acylation with succinic anhydride.

27. The method for producing an insulin derivative according to claim 19, characterized in that the positive charges of the amino groups of the residues of the basic amino acids are replaced by negatively charged carboxyl groups by acylation with maleic anhydride.

28. The method for producing an insulin derivative according to claim 19, characterized in that the positive charges of the amino groups of the residues of basic amino acids are replaced by negatively charged carboxy groups by acylation with acetic anhydride.

29. The method for producing an insulin derivative according to claim 19, characterized in that the positive charges of the amino groups of the residues of the basic amino acids are replaced by negatively charged carboxy groups by alkylation with monochloracetic acid.

30. A method for producing an insulin derivative according to claim 19, characterized in that the peptides, products of enzymatic hydrolysis of insulin, are modified with an acylating (alkylating) agent: protein ratio ranging from 0.1: 1 to 3: 1.

31. The method of obtaining the insulin derivative according to claim 30, characterized in that it is manufactured in such a ratio of components, mass. %:

The supramolecular ensemble of peptides 0.0001-3.0

the rest is excipients up to 100.

32. A method for producing an insulin derivative according to claim 31, characterized in that it is manufactured as a liquid dosage form (emulsion, aqueous solution, non-aqueous).

33. The method of obtaining the derivative of insulin according to p, characterized in that it contains a hydrophobic base.

34. The method of obtaining the derivative of insulin according to p, characterized in that it contains a hydrophilic base.

35. The method of obtaining the derivative of insulin according to p, characterized in that it contains a diphilic base.

36. A method for producing an insulin derivative according to claim 32, characterized in that it is performed as an aerosol or an aerogel for oral administration (application to the cheek from the inside).