RU2395296C1 - Method for making oral proinsulin preparation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to pharmacology and medicine, particularly to endocrinology. A method for making proinsulin preparation consists that in 1-50 % aqueous polyethylene glycol with molar mass 0.4-40 kDa pre-exposed to ionising radiation in amount 1.0-5.0 Mrad in acid medium at pH 2.5 - 4.5 with calcium and/or zinc cations added to final concentration of 5-10 mm, proinsulin is introduced to final concentration of 1-10 mg/ml in the ratio of polyethylene glycol to proinsulin equal (1-500)/1. The mixture is stirred to prepare a homogeneous transparent or slightly opalescent solution.

EFFECT: use of the method allows simplifying process for making proinsulin preparation and ensuring possibility of its application as a hypoglycaemic agent and maintenance of activity at pH 2-3 and room temperature.

4 cl, 1 tbl, 4 ex

Description

The invention relates to pharmacology and medicine, in particular to endocrinology, and can be used to obtain a hypoglycemic preparation based on proinsulin for oral and parenteral use.

Proinsulin is a precursor to the hormone insulin and has a molecular weight of about 9000 Da (Dalt). Proinsulin has an effect on all types of metabolism in the body: it increases the penetration of glucose into the body tissues and its use by them, reduces the glycogen content in the liver and increases its amount in the muscles, increases the intensity of protein synthesis, etc. Proinsulin is formed in the islet beta cells of the pancreas, secreted into the blood as insulin and C-peptide. Peptide C, which is part of proinsulin, has been shown to be a biologically active substance. In particular, the C-peptide prevents the development of diabetes complications. In addition, the C-peptide has an immunomodulatory effect and is involved in the development of insulin and proinsulin tolerance. It was found that the modification of the C-peptide is accompanied by the development of autoimmune diabetes in some mice. Therefore, proinsulin is most suitable for the treatment of patients with diabetes mellitus (DM) of the first type (type 1).

Indeed, the use of intranasal proinsulin to induce oral tolerance in laboratory animals has been shown to be more effective than enteral insulin. However, proinsulin is unstable, it is easily destroyed in the gastrointestinal tract (GIT), and therefore attempts to use it enterally for the induction of immunological tolerance to insulin and proinsulin in humans have ended to no avail (A.Hanninen, L.C. Harrison "Mucosal Tolerance to Prevent Type 1 Diabetes: Can the Outcome Be Improved in Humans? Rev Diabet Stud. 2004; 1 (3): 113-121).

In addition, with parenteral administration of conventional proinsulin, the rapid emergence of antibodies to proinsulin is noted due to its high immunogenicity.

The main route of administration of both insulin and proinsulin into the human body is via subcutaneous or intramuscular injection of the drug. Attempts to administer insulin in the most physiological and oral (oral) route convenient for patients have been unsuccessful, since insulin is readily hydrolyzed by digestive enzymes with a loss of activity. At the same time, there is a theoretical possibility to improve the pharmacokinetics and stability of proinsulin by conjugating it with water-soluble polymers (Katre NV The conjugation of proteins with polyethylene glycol and other polymers. Adv. Drug. Delivery Res. - 1993. - v. - 91-114; Harris JM, Martin NE, Modi M. Pegilation: a novel process for modifying pharmacokinetics. Clin Pharmacokinet. 2001; v. 40. - 539-531).

The benefits of oral proinsulin are obvious since long daily injections can cause various serious complications: they are accompanied by pain syndromes; lead to the development of lipodystrophy, representing not only a cosmetic defect, but also causing a need for increased doses of the hormone; injure the psyche, especially in children; cause stress conditions leading to even more pronounced hyperglycemia, which, in turn, increases the need for a hormone, etc. At the same time, the enteral route of administration of proinsulin promotes the development of enteric tolerance to insulin and proinsulin and the subsidence of the autoimmune process.

A known method of producing a proinsulin preparation suitable for oral use (WO 2004083234, op. September 30, 2004), which consists in first chemically activating an oligomer containing a hydrophilic site and / or lipophilic site, which is used as polyethylene glycol, then contact proinsulin polypeptide with an activated oligomer under conditions sufficient to attach the oligomer to the proinsulin polypeptide and create a conjugate proinsulin polypeptide oligomer, for which comfort solvents such as dimethylsulfoxide (dimethylsulfoxide) and triethyl amine (triethylamine). In addition, the method requires the use of activating chemical agents, such as N-hydroxysuccinimide (N-hydroxysuccinimide), p-nitrophenyl chloroformate (p-nitrophenylchloroformate), 1,3-dicyclohexylcarbodiimide (1,3-dicyclohexylcarbodiimide), hydroxybenzotriazide (hydroxy).

The use of highly toxic reagents for the chemical activation of polymers requires the use of complex and expensive chromatographic purification methods for the purification of synthesis products. In addition, many of these reagents are not produced on an industrial scale and therefore are expensive and inaccessible.

The disadvantages of this method are the technological complexity of obtaining conjugates of proinsulin with an oligomer, the need to use highly toxic chemical agents for activation of polyethylene glycol, which need to be purified by chromatographic methods, as well as the high cost of the preparations obtained. In addition, the inventors described in WO2004083234 did not confirm the presence of hypoglycemic activity in conjugates of proinsulin with polyethylene glycol.

The technical task of the invention is to simplify and reduce the cost of the method of obtaining the proinsulin preparation for oral administration, as well as increasing its therapeutic efficacy in oral administration.

The solution to this problem is achieved by the fact that the proposed method of obtaining a proinsulin preparation for oral use using activated polyethylene glycol, in which according to the invention polyethylene glycol is activated by irradiation with ionizing radiation in an acidic environment in the presence of calcium and / or zinc cations to a final concentration of 5-10 mm, and then mixed with proinsulin, while taking polyethylene glycol with mol. weighing 0.4-40 kDa and a concentration of 1.0-50.0%, in the ratio of proinsulin-polyethylene glycol equal to 1: (1-500) to a final concentration of proinsulin in a mixture of 1-10 mg / ml.

Polyethylene glycol is activated by irradiation with a stream of accelerated electrons or gamma radiation in doses of 1.0-5.0 Mrad.

To acidify the polyethylene glycol, they take predominantly an organic acid, for example, acetic, citric, malic, and maintain the pH 2.5-4.5 upon activation of the polyethylene glycol.

Other organic acids can be used to acidify polyethylene glycol, for example fumaric, succinic, lactic and other acids that are not toxic to the body.

As a water-soluble polymer, in addition to polyethylene glycol, dextrans, polyvinylpyrrolidone, isoprenols, polyacrylamide, polyurethane can also be used.

The proposed method is as follows.

Prepare a 1-50% aqueous solution of polyethylene glycol with a molecular weight of from 400 to 4000 Da. Then the solution is acidified to a pH of 2.5-4.5, calcium and / or zinc cations are added to a final concentration of 5-10 mM and irradiated with high-energy ionizing radiation, mainly gamma radiation or accelerated electron flux in doses that ensure free radical reactions Mostly 1.0-5.0 Mrad. Then, proinsulin is introduced into a solution of radiation-activated polyethylene glycol to a final concentration (for protein) of 1-10 mg / ml (or for proinsulin activity 10-100 IU / ml, respectively), in the ratio of polyethylene glycol: proinsulin equal to (1-500): 1, the mixture is stirred for 10-30 minutes until a uniform, clear or slightly opalescent solution is obtained. The resulting drug is neutralized. When irradiated during the radiation-chemical oxidation of polyethylene glycol, highly active carbonyl groups are formed. The polymer activated in this way forms a water-soluble preparation with proinsulin that effectively reduces the glucose level when administered orally. Due to the high solubility in aqueous solutions, the proinsulin preparation with polyethylene glycol is completely absorbed into the blood without diffusion restrictions.

The technical result obtained is not obvious from the known scientific and technical data on the properties of polyethylene glycol and proinsulin, since as a result of the formation of the drug a complete loss of the specific hypoglycemic activity of proinsulin could occur due to a change in its conformation in the presence of activated polyethylene glycol and its interaction with the insulin cell receptor.

However, it has been experimentally established that the preparation obtained by the proposed method exhibits specific hypoglycemic activity when administered orally.

In addition, it turned out to be unexpected that the presence of calcium and zinc cations upon activation of the polyethylene glycol made it possible to increase the hypoglycemic activity of the drug, the duration of the preservation of hypoglycemic activity, and stability in an acidic environment.

As a source of ionizing radiation, you can use electronic radiation (directed flow of accelerated electrons), gamma radiation, x-ray radiation, laser radiation and ultraviolet radiation. The necessary equipment and ionization techniques are described in the following publications (Gonchar A.M. and Auslender V.L. 1996; Gonchar A.M. and Auslender V.L., 1998, Vereschagin E.I., et al., 2001).

In the most preferred embodiment, it is proposed to act on the polyethylene glycol with a directed stream of accelerated electrons. In the specific case of the method, the polyethylene glycol was irradiated with bremsstrahlung generated by an ILU-6 or ILU-10 accelerator with an electron energy of 2.5 MeV, an absorbed dose of 2 to 10 kGy, and a dose rate of 1.65 kGy / hour.

The invention is illustrated by the following examples of specific preparation of the proposed drug.

Example 1

A 10% aqueous solution of polyethylene glycol (PEG) with a molecular weight of 1500 Da (PEG-1500) is irradiated with a stream of accelerated electrons in a dose of 2.0 Mrad in an acidic medium in the presence of 5 mM calcium cations. The acidification is carried out with a 0.1 M solution of acetic acid to pH 3, and calcium cations are introduced by adding calcium chloride to the solution until the concentration in terms of Ca ^{2+ is} reached up to 5 mM, the pH is checked and, if necessary, titrated. Recombinant proinsulin is added to the irradiated solution of polyethylene glycol to a final concentration of 10 mg in 1 ml (the ratio of polyethylene glycol: proinsulin is 10: 1). The mixture is stirred for 10 minutes to obtain a proinsulin preparation in the form of a slightly opalescent solution. The resulting preparation is neutralized with a 0.1 M NaOH solution. The yield of the finished product is 98%.

Example 2

A 50.0% aqueous solution of polyethylene glycol with a molecular weight of 400 Da is irradiated with inhibitory gamma radiation at a dose of 1.0 Mrad in an acidic environment, pH 2.5 in the presence of zinc cations. For this, acidification is carried out with a 1 M solution of citric acid and a 1 M solution of zinc chloride is added to a solution of polyethylene glycol to a concentration of 10 mM Zn ²⁺ , the pH is checked and, if necessary, triturated with 1 M citric acid. Proinsulin is added to the irradiated solution of polyethylene glycol to a final concentration of 1 mg in 1 ml (the ratio of polyethylene glycol: proinsulin is 500: 1). The mixture is stirred for 30 minutes to obtain a proinsulin preparation in the form of a clear solution. The resulting preparation is neutralized with 0.1 M NaOH. The yield of the finished product is 97%.

Example 3

To compare hypoglycemic activity, a proinsulin preparation was also prepared, mixed with a solution of polyethylene glycol, activated by irradiation with a stream of accelerated electrons in a neutral medium in the absence of divalent metal ions.

For this, a 10% aqueous solution of polyethylene glycol (PEG) with a molecular weight of 1500 Da (PEG-1500) was irradiated with a stream of accelerated electrons in a dose of 2.0 Mrad. Recombinant proinsulin was added to the irradiated solution to a final concentration of 10 mg in 1 ml (the ratio of polyethylene glycol: proinsulin was 10: 1). The mixture was stirred for 10 minutes to obtain a proinsulin preparation in the form of a slightly opalescent solution. The yield of the finished product was 98%.

Example 4. The study of the effect of intragastric administration of a proinsulin preparation, obtained as described in Example 1, on blood glucose in rats in comparison with the original proinsulin (Control) and proinsulin obtained in Example 3.

The experiments were carried out on 60 Wistar male rats weighing 300-350 g. The animals were kept on a standard vivarium diet, with free access to water and food, in accordance with the rules adopted by the European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes . Blood for examination was taken from incisions in the tail. Blood glucose was determined on a SmartScan instrument (Johnson & Johnson company, USA). The proinsulin preparation obtained in example 1 was administered once intragastrically at a dose of 20 mg / kg in one group of animals (experiment 1). The proinsulin preparation obtained in example 3 was also administered once intragastrically at a dose of 20 mg / kg to another group of animals (experiment 2).

Animals of the control group received an equivolume amount of the original proinsulin (control). Glucose was determined before administration of the drug (from 9 to 10 am) and after 1 h, 2 h, 3 h, 5 h, 7 h and 10 h after administration. Statistical processing of the results was carried out using the statistical software package "Statistica for Windows 5.0". The mean values of the indicators and the standard error of the mean were calculated. Intergroup differences were evaluated using Student T-test.

The initial glucose content in the control and experimental groups was 5.0 ± 0.2 mmol / L and 4.8 ± 0.1 mmol / L, respectively, which fit into the physiological norm for this type of laboratory animals (table 1). In rats that received an intragastric drug obtained according to examples 1 and 3 of proinsulin, from 1 to 2 hours inclusive, the blood glucose level was not statistically significant from the values in the control group. 5 hours after administration of the drug, the glucose content in the blood of animals treated with proinsulin prepared according to Example 1 was 4.0 ± 0.1 mmol / L and was significantly lower (25%) compared with the control. These differences persisted even 10 hours after the administration of the proinsulin preparation obtained in Example 1. At the same time, by 10 h, the concentration of glucose in the blood of animals from the control and experimental (experiment 2 — proinsulin prepared according to Example 3) groups leveled off.

Thus, the initial proinsulin did not exert a statistically significant effect on the blood glucose level, the proinsulin preparation obtained in Example 3 (experiment 2) showed a hypoglycemic effect, leveling by 10 hours of observation. At the same time, the proinsulin preparation obtained according to the invention (experiment 1), when administered intragastrically to rats, not only exhibits a hypoglycemic effect, but is also more effective than the preparation obtained according to example 3.

Table 1 Group Initial values Time after administration, h one 2 3 5 7 10 The control 5.0 ± 0.2 5.2 ± 0.1 5.0 ± 0.2 5.1 ± 0.2 5.2 ± 0.1 5.2 ± 0.1 4.9 ± 0.2 Experience 1 4.8 ± 0.3 4.9 ± 0.1 5.0 ± 0.1 4,5 ± 0,2 4.0 ± 0.1 4.0 ± 0.2 4,5 ± 0,2 Experience 2 4.8 ± 0.3 4.9 ± 0.1 5.0 ± 0.1 4.9 ± 0.2 4.1 ± 0.1 4.3 ± 0.2 5.0 ± 0.2 Note: * - p <0.05 compared with control

A comparative assessment of the anaphylactogenic activity of the proinsulin preparation obtained as described in Example 1 and the initial proinsulin was also carried out in accordance with the “Guidelines for the Assessment of Allergic Properties of Pharmacological Substances” [Guide to the experimental (preclinical) study of new pharmacological substances. - M., 2005. - 827 p.].

The reaction of general anaphylaxis (anaphylactic shock) was carried out on 30 guinea pigs weighing 250-300 g (3 groups of 10 animals - 2 doses of the proinsulin preparation obtained in example 1 (therapeutic - 1 mg and 10 therapeutic doses) and control (initial proinsulin) ; the experimental and control preparations were administered subcutaneously and 2 times intramuscularly, allowing injection - intravenously; observation period - 3 weeks;

In the control group, anaphylactic shock with a fatal outcome in 4 out of 10 animals was noted, anaphylactic shock without a fatal outcome in 2 animals. In the groups where the preparation of the proinsulin obtained in Example 1 was administered to animals, anaphylactic shock was not observed in any of the cases.

The study of active skin anaphylaxis was carried out on 30 BALB / c mice (3 groups of 10 animals - 2 doses (therapeutic and 10 therapeutic doses of the preparation obtained in Example 1 of proinsulin) and control (initial proinsulin in a therapeutic dose), experimental and control preparations were administered subcutaneously, Freund's adjuvant was used as adjuvant, and the resolving dose was administered subcutaneously.

In the control group, marked hyperemia and the formation of papules were noted in 5 animals; severe hyperemia and edema in 4 animals. When using the proinsulin preparation obtained according to the invention, two animals showed hyperemia and edema in the group using high (10 therapeutic) doses.

In addition, the proinsulin preparation obtained by the proposed method has the following properties, the appearance of which is due, in particular, to the fact that the activation of polyethylene glycol by ionizing radiation is carried out in an acidic environment in the presence of calcium and / or zinc cations. It was experimentally shown that the proinsulin preparations obtained by the proposed method retained their activity during storage during the day at pH 2-3 even at room temperature.

The inventive method of obtaining a proinsulin preparation for oral administration, in contrast to the prototype method, is simple and economical, since its preparation consists of only two stages, activation of polyethylene glycol by ionizing radiation in an acidic environment and in the presence of calcium and / or zinc cations and mixing it with proinsulin to the desired concentration (activity). For its implementation does not require the use of highly toxic reagents. The proinsulin preparation obtained by the claimed method has therapeutic activity, determined by lowering the concentration of glucose in the blood, and can be used for oral, parenteral and intranasal administration.

In addition, the drug is not allergenic, highly stable, and therefore can be used to induce immunological tolerance to insulin and proinsulin in patients with type 1 diabetes.

Claims (4)

1. A method of obtaining a proinsulin preparation for oral use using activated polyethylene glycol, characterized in that the polyethylene glycol is activated by irradiation with ionizing radiation in an acidic medium in the presence of calcium and / or zinc cations to a final concentration of 5-10 mM, and then mixed with proinsulin, with this take polyethylene glycol mol. weighing 0.4-40 kDa and a concentration of 1.0-50.0%, with a ratio of proinsulin: polyethylene glycol equal to 1: (1-500), to a final concentration of proinsulin in a mixture of 1-10 mg / ml. 2. The method according to claim 1, characterized in that the polyethylene glycol is activated by irradiation with a stream of accelerated electrons or gamma radiation in doses of 1.0-5.0 Mrad. 3. The method according to claim 1, characterized in that for the acidification of polyethylene glycol, they mainly take organic acid, for example, acetic, citric, malic. 4. The method according to claim 1, characterized in that the polyethylene glycol is activated at a pH of 2.5-4.5.