DD219673A1 - PROCESS FOR PREPARING PREPARATED BIOLOGICALLY ACTIVE PEPTIDES WITH MODIFIED EFFECT - Google Patents

Abstract

A simple process for the production of effect-modified biologically active peptides, in particular insulin, which is based on the immobilization of the active ingredients by embedding, fixation or adsorption using macromolecular gelatinous carrier materials is described. As traeger materials preferably physiologically acceptable and biodegradable compounds of polysaccharide or polyamide / polypeptide type such as agar, agarose, dextrose, starch, collagen or similar biopolymers are used, which can be widely varied in their degree of crosslinking and pore diameter. The introduction of the active ingredient in the matrix can in this case by any physical-chemical operations z. As mixing, penetration, diffusion, adsorption, electrophoresis and other methods. By such immobilization of the hormone is increased durability under physiological conditions of incubation and improvement of the pharmacokinetic properties such. B. Delayed drug delivery or a more favorable Dosierungsmoeglichkeit reached.

Description

Process for the preparation of preparations of biologically active peptides with modified action '. . ·

Field of application of the invention

The invention describes a process for the preparation of preparations of biologically active peptides by immobilization on high molecular weight inert support materials. This results in an increased durability under physiological incubation conditions as well as a targeted improvement.

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selected pharmacological properties such. B. achieved a delay in drug delivery or more favorable dosage options.

Characteristic of the known technical solutions

The fixation of an active ingredient to polymeric matrices is a common method for achieving modified or prolonged pharmacological effects. In principle, this can be achieved by adsorption of the drug on the surface of a carrier, by inclusion, or embedding in the interior of a cross-linked polymer having a suitable pore structure or by covalent anchoring of the active substance to the polymer molecules by means of suitable spacer groups.

For example, normal crystalline insulin has only a short-term effect. It has therefore in the literature numerous .--. given rich attempts to develop by modifying the absorption capacity, the biological half-life or by delaying the release of the hormone, modified-effect preparations, in particular with depot effect. Modification options include, for example, the direct chemical modification of the insulin molecule or of one of the two partial chains, the complexation of insulin with basic proteins or organic bases, and the suspension of crystalline Zn insulins which are prepared under specific conditions. Lente Insuline). Furthermore, attempts to covalently anchor insulin to sepharose or agarose or polyethylene glycol are known from the literature / Cuatrecasas and Hollenberg, Advances in Prot. Chem. 30 »406-429 (1976).

Among the large number of these possibilities, only the Lente insulins have become established as depot preparations in medical practice. Therefore, it is necessary to look for novel ways to specifically alter the pharmacological properties of biologically active peptides. _:

Object of the invention

The aim of the invention is the simple and cost-effective production of preparations of biologically active peptides with improved biological stability and favorable pharmacokinetic properties, preferably depot forms.

Explanation of the essence of the invention

The object of the invention is to develop a process which specifically modifies the biological stability and the pharmacological properties of biologically active peptides, in particular insulin, using macromolecular matrices.

According to the invention the object is achieved in that one carries out an immobilization by inclusion, embedding or adsorption. As matrices, inert, non-toxic, gel-like support materials, which can be obtained in different pore sizes and degrees of crosslinking, are shortlisted. Among them, in particular, the biodegradable polysaccharide or polyamide type members, such as e.g. Agar agarose, agarose, dextrans, starch, polypeptides, collagen or similar compounds or partial degradation products thereof take precedence. In principle, however, other atoxic, physiologically compatible polymers come into consideration. The inclusion, or the fixation or adsorption of the hormone takes place under solution conditions or pH values under which denaturation is largely avoided and for which any methods such as mixing, penetration, diffusion or electrophoresis can be included. The Immobilisierungsformen of the type described can be a stability of Peptidwirkstof fes, against external influences, such as changes in pH and hydrophysierende reach conditions, whereby the full biological activity is maintained over long periods

 At the same time result in this way as a result of changed

Solubility and Diffusion Processes in the Matrix Possibilities for a Dosierbäre drug release, which can be used for the production of depot preparations. Here are, for example, the advantages of the method, darirn that. Insulin using selected buffer systems by electrophoretic methods in polyacrylaraid gels of different degrees of crosslinking bring in such a way that drug concentrations up to 18% can be achieved. The embedding forms thus obtained have the insulin in biologically fully active form and cause a complete and lasting several hours re-delivery. Such Modifizierungsformen possess over the hitherto in the literature / B. K. Davis, Experientia 28, 348 (1972) / described in which insulin was copolymerized in polyacrylamide gels, considerable advantages, since the drug molecule remains chemically unchanged and biologically fully active.

Another possibility of this basic principle is that peptide-like bioactive agents such. B. Insulirt or GnRH in crystalline or dissolved form in agar-agar gels of different consistency under equally non-denaturing conditions are introduced. In this way, there are preparations that make it possible to increase the stability significantly and to control the re-release rate of the hormone.

Embodiments . .

1st example:

Embedding insulin in polyacrylamide matrices by means of polyacrylamide electrophoresis

Reagents: (each made up to 100 ml with distilled water)

Solution A: 1N HCl: 48 ml

./. ν

Tris (hydroxymethyl) amino methane, 36.6 g

N, N, N ', N', tetramethylethylenediamine: 0.23 ml of solution B: freshly recrystallised acrylamide: 30.0 g of freshly recrystallized N, N'-methylenebisacrylamide: 0.8 g

Solution C: Ammonium peroxydisulfate: 0.14 g

Solution D: freshly recrystallized acrylamide: 60.0 g of freshly recrystallized N, N'-methylenebisacrylamide :, Q _t 4 g

Solution E: 'sucrose: 34 g

Glycine:. 2.88 g

Electrophoresis buffer: tris- (hydroxymethyl) -

amino-methane: '0.6 g

Glycine: 2.88 g

used as a 10% aqueous solution for electrophoresis.

To prepare the 7.5% polyacrylamide matrices (= % by weight / total reaction volume), the solutions A, B ₁ C and distilled water in the ratio 1, 2: 4: mixed and 60 minutes in gel tube (70 mm χ 5 mm) poly -

merized. After 60 minutes of pre-electrophoresis, 150 μl insulin solution (250 mg insulin per ml solution E) are applied per gel tube and introduced into the gel by electrophoresis (U = 200 V, 1 = 3 mA / tube, t = 90 minutes). The loaded gels are then pressed through a sieve of appropriate size of mesh directly into absolute acetone, the dehydrated gel is filtered off, washed with acetone and dried over P ₂ ⁰ K at 1 Torr. In animal experiments, the insulin preparations thus obtained show a depot effect.

2nd example:

For the preparation of 15% polyacrylamide matrices, the solutions Vi A, B, C are mixed in the ratio 1: 4: 2 and the procedure is continued as in Example 1.

Due to the small mean pore diameter of the selected matrix, this insulin embedding showed an increased protease effect compared with Example 1.

3rd example:

Production of insulin-loaded dextrans; 5.6 g of dextran (M = 500,000) are stirred into a viscous mass with 4.6 ml of distilled water. 1000 μl of insulin solution (250 mg / ml 0.2 N Na ₂ HPO ₄ solution, pH 9.7) are added to this composition by means of a microsyringe and stirred in. The gel is subjected to predrying in absolute acetone. This is followed by drying on the oil pump at 1 mm Hg and 25 ⁰ C. The cured matrix is crushed in a mortar. In this way, gels of 3 _t 5?

Insulin fraction (based on dry matter) are produced, which showed a depot effect in animal experiments.

4th example:

Preparation of Insulin Intrinsic Forms in Agar-Agar Matrices

5 'and 7.5% agar-agar solutions are prepared by dissolving appropriate amounts of agar-agar in distilled water (90 ^° C.). Oeweilß 3 ml of these solutions is allowed to cool to 40 ⁰ C., and this just before gelling with 50 to 1000 AII insulin solution (250 mg of insulin in 1 ml of 0.2: η Na ₂ HPO ₄ solution; pH 9.7), so that samples with 10 to 60 % by weight insulin fraction (based on dry matter) result. The gelled samples are then comminuted and dried as in Example 1. The embedded forms of insulin and agar-agar obtained in animal experiments, as the preparations obtained in Example 1 and 2, a clear depot effect.

5th example:

There are 7.5% agar-agar solutions obtained by dissolving appropriate amounts of agar AGSR (l, 5 g) in distilled water (18.5 ml) at 90 ⁰ C. Shortly before the Aüsgelieren cooled to 40 ⁰ C solution, the appropriate amount of crystalline insulin (330 mg) was added and stirred. The gelled samples are then comminuted and dried as in Example 1. In this way gels can be made up to 15% by weight of insulin (based on dry matter).

which showed a stronger depot effect in animal experiments than the preparations described under Examples 1, 2, 3 and 4.

6th example:

Preparation of GnRH Embedding Forms in Agar-Agar: 7.5% agar-agar solutions are prepared by dissolving appropriate amounts of agar-agar (450 mg) in distilled water (4.5 ml) at 90 ^° C. Shortly before gelling the agar-agar solutions cooled to 40 ^° C., 1000 μl of GnRH solution (100 mg in 1 ml of distilled water, pH 6.0) are added and stirred. There is a comminution and subsequent drying as stated in Example 1. In this way, gels with 13 % by weight GnRH content (based on dry matter) could be prepared.

Claims (6)

invention claim 1. A process for the preparation of immobilized, biologically active and modified in their pharmacological properties biolO'-gisch active peptides, preferably depot forms, characterized in that the peptide is physically entrapped or embedded in inert macromolecular, gelatinous, atoxic and biodegradable matrices, preferably those of the polysaccharide or polyamide or polypeptide type, with arbitrary porosities and degrees of crosslinking. 2. The method according to item 1, characterized in that This principle is used for the recovery of insulin depot forms. . 3. The method according to item 1, characterized in that the peptide is embedded from a highly concentrated solution by electrophoresis in polymerized polyacrylamide gels. 4. Method according to item 3, characterized in that 5 to 20% polyacrylamide gels (weight% / total reaction volumes) are used for the embedding, 5. The method according to item 1, characterized in that the peptide under non-denaturing pH conditions and temperature conditions are introduced into the forming gel, eg, polysaccharide-type, such as dextran or agar-agar. 6. Method according to item 1, characterized in that the gel loaded with the peptide is shock-dried, e.g. B. by pressing through a sieve in absolute acetone, the dehydrated gel is filtered off with suction, washed with acetone and dried over PpOc at 1 Torr.