DD257197A1 - METHOD FOR THE PRODUCTION OF IMMOBILIZED INSULIN PRAEPARATIONS STABILIZED IN ITS BIOLOGICAL ACTIVITY AND MODIFIED IN ITS PHARMACOLOGICAL PROPERTIES - Google Patents

Abstract

The invention relates to a process for the preparation of immobilized insulin preparations modified in their pharmacological properties, preferably for the preparation of parenterally and non-parenterally applicable insulin depot forms, which is also applicable to other peptide hormones. A simple process for the preparation of insulin preparations by complex formation by means of polyamine acids of different structural type is described which leads to delayed drug delivery and long-term stabilization of the hormone as a result of the protective function of the polyamine acids. The use of different kinds of polyamine amino acids allows a wide variation in insulin content and in the pharmacological properties of the complexes.

Description

Field of application of the invention

The invention relates to a process for the preparation of immobilized and modified in their pharmacological properties insulin preparations, preferably for the presentation of parenteral and non-parenteral applicable insulin depot forms, which is also applicable to other peptide hormones.

Characteristic of the known technical solutions

After the introduction of the classic depot forms of insulin (eg surf insulins, Lente insulins) into diabetes therapy, a variety of principles to improve the delay effect or to develop novel forms of application have been investigated. These are u.a. the matrix inclusion of insulin in cross-linked bovine serum albumin, the inclusion in liposomes or the use of phospholipids as a carrier. In order to achieve retardation effects for the insulin delivery or an increase in the stability of the preparation, the chemical fixation of the hormone to polymeric carriers was also processed. Different immobilization principles such as complex formation or covalent bonding were used. Application. As carrier materials were used: polyethylene, polyvinylpyrrolidone and its copolymers with crotonic acid, dextran, Sephadex, agar and other polysaccharides and their crosslinking products and also ethylene vinyl ester polymers.

However, all of these attempts did not yield any substantial success in practical medical use because either insufficient binding capacities or sustained release effects were achieved or carrier materials were used which were not or hardly enzymatically degraded and which also showed a lack of biocompatibility due to high antigenicity.

PAS have been proposed in the patent literature as additives for pharmaceutical preparations in a variety of combinations. The recovery of stabilized insulin sustained release forms of the type described below, however, has not been identified. For example, in the published patent application DE 3200706 A1. 16.8.1982 recommended the use of water-soluble chefatbildasas PAS as additives for improving the absorption properties of drugs in suppositories and suppositories without, however, the specific physical-chemical binding possibilities directly between insulin and PAS recognized and targeted insulin-sustained release forms were exploited.

Object of the invention

The object of the invention is to develop a method which makes it possible to obtain parenterally or non-parenterally administrable prolonged-release forms of insulin.

Explanation of the essence of the invention

The object of the invention is to develop a method which allows the recovery of sustained-release forms of insulin with increased stability using physiologically well tolerated, non-toxic additives and thereby permits available application possibilities.

Eifihdungsgemäß the problem is solved by that are used as carrier materials polyamino acids (in the future called PAS) of different types of structure and different average molecular weight.

PAS form addition products or complexes with each other or with other peptides or proteins due to intermolecular forces such as ion formation, Η-bridges or hydrophobic interactions, a finding which, according to our investigations, is particularly pronounced for the peptide hormone insulin due to stereochemical effects (future insulin / Called PAS complexes). According to the invention, the special binding effect can be achieved in that both components are dissolved under conditions under which insulin is not denatured, but the PAS is converted into a random coil form. In this way, in the subsequent co-precipitation or lyophilization, stable enveloping complex formation of the insulin molecule by the PAS occurs. In addition, according to the present invention, the hormone is released from these complexes with a high degree of delay, thereby resulting in delivery rates of the order of magnitude which provide particular advantages for the use of these complexes as sustained release or depot forms of insulin. At the same time a significant long-term stabilization of the hormone is achieved by such a complex formation. According to the invention further, the retention effect of the average molecular weight and the structure type of the polyamino acid used in the complex is greatly influenced and is regulated in this way within wide limits. Thus, the complexation and thus retardation effect increases significantly with increasing molecular weight and increased polarity of the polyamino acid used, as shown in Fig. 1 on selected in vitro examples. The effects described there were in principle confirmed by corresponding in vivo experiments. In this way, the insulin-dispensing system can be suitably adapted to the specific needs demanded in a practical application. Polyamino acids of different degrees of polymerization are readily accessible by the various routes described in the literature. Likewise, complexes between these and insulin can be obtained in a simple manner by combining appropriately metered quantitative ratios in homogeneous solution under conditions which are not denaturing for the peptide hormone and subsequent precipitation or lyophilization. All polyamino acids used are well tolerated in long-term animal experiments in subcutaneous and intramuscular administration.

embodiments

I.Beispiel:

Preparation of insulin-polyglycine complexes:

1.0 g of polyglycine (M = 2000) are dissolved in 25.0 ml of 85% formic acid and 5.0 ml of insulin solution (120 mg / ml of 85% formic acid) added. Subsequently, the complex is precipitated with stirring with 100 ml of absolute ether and stirred until complete flocculation of the precipitate for about 30 min at 20 ° C on. After centrifuging off the complex, it is washed 5 times with absolute ether and dried in vacuo over P ₂ O ₅ at 1 Torr and 2O ⁰ C.

The insulin-polyglycine complexes obtained in this way contain 38% insulin according to the amino acid analysis.

[a] g ⁵ = -22.8 ° (c = 0.5 / TFA) IR spectra: NH11 620CrTT ¹ NHII 1520 cm ^-1

In vitro measurement of insulin liberation revealed continuous insulin delivery over 30 hours. After subcutaneous administration to rabbits, the blood sugar is lowered to 40% over 10 hours.

2nd example:

Preparation of insulin-poly-L-alanine complexes:

For the preparation of 38% insulin-poly-L-alanine complexes using poly-L-alanine (M = 2000) is carried out as in Example 1.

[a] g ⁵ = -21.3 ° (c = 0.5 / TFA) IR spectra: NHI 1620cm " ¹ NHII 1520cm" ¹ NHV 685cm " ¹ NHA3260cm" ¹

In vitro measurement of insulin liberation revealed continuous insulin delivery over 35 hours. After subcutaneous administration to rabbits, the blood sugar is reduced to 70% over 10 hours.

3rd example:

Preparation of insulin-poly-L-leucine complexes:

Dissolve 1.0g poly-L-leucine (M = 2000) in 25.0ml trifluoroacetic acid and add 5.0ml insulin solution (120mg / ml trifluoroacetic acid). Next proceed as in Example 1.

The insulin-poly-L-leucine complexes thus obtained also contain 38% insulin according to the amino acid analysis.

[a] g ⁵ = + 3.4 ° (C = 0.5 / TFA) IR spectra: NHI 1640cm " ¹ NHII 1530cm" ¹ NHV605cm " ¹

In vitro, continuous insulin liberation takes place over 40 hours.

After subcutaneous administration to rabbits, a reduction in blood sugar to 70% could be achieved over 10 hours.

4, example:

Preparation of Insulin-Poly-L-Iysine Complexes:

1.0 g of poly-L-lysine (M = 4000) are dissolved in 50.0 ml of dist. Dissolved water and added to the solution 0.6 g of insulin. After about 30 min stirring at 20 ⁰ C until complete dissolution of the insulin is e.in freeze-drying.

The resulting insulin-poly-L-lysine complexes are also 38% pure according to amino acid analysis.

[ά] § ⁵ = -34.8 ° (c = 0.5 / 1 N HCl) Ifli spectra: ΟβοΟοητΤ ¹ NHII 1530cm- ¹

In vitro measurement of insulin liberation revealed continuous insulin delivery over 70 hours. After subcutaneous administration to rabbits, a blood sugar reduction to 40% over 12 hours is achieved.

Claims (8)

1. A process for the preparation of immobilized, stabilized in their biological activity and modified in their pharmacological properties fnsulin preparations, parenterally or non-parenterally administered depot forms, characterized in that insulin is converted into atpxische, biologically well-tolerated derivatives, complexes or adducts with polyamino acids. 2. The method according to claim 1, characterized in that this principle is applied for the production of oral insulin depot forms or insulin implants. 3. The method according to claim 1, characterized in that the transfer of insulin into polyamino acid complexes by precipitation from organic solvents. 4; Process according to Claim 1, characterized in that the conversion of the insulin into polyamino acid complexes is carried out by lyophilization from aqueous solutions. 5. The method according to claim 1 and 3, characterized in that insulin is converted by precipitation of formic acid in insulin-polyglycine complexes. 6. The method according to claim 1 and 3, characterized in that insulin is converted by precipitation from trifluoroacetic acid into insulin-polyleucine complexes. 7. The method according to claim 1 and 4, characterized in that insulin is converted by lyophilization of aqueous solutions in insulin-polylysine complexes. . '8th. Process according to claims 1 and 4, characterized in that insulin is converted into insulin-polyglutamic acid complexes by lyophilizing aqueous solutions.