DK149778B - ANALOGY PROCEDURE FOR PREPARING DES-PHEB1 HUMAN INSULIN OR ITS DERIVATIVES - Google Patents

Abstract

1. Claims for Contracting States : BE, LI, DE, FR, GB, IT, NL, SE Insulin of the formula I see diagramm : EP0046979,P10,F1 in which X denotes Thr or OH and Y denotes Asp or Asn. 1. Claims for Contracting States : AT A process for the preparation of an insulin of the formula I, see diagramm : EP0046979,P12,F1 in which X denotes Thr or OH and Y denotes Asp or Asn, which comprises a) converting human insulin, desamido**A21 -human insulin, des-Thr**B30 -human insulin, desamido**A21 -des-Thr**B30 -human insulin, des-Ala**B30 -swine insulin or desamido**A21 - des-Ala**B30 swine insulin into the N**alpha AI -N**vepsiln B29 -bis-Boc compound and subjecting this to Edman degradation, b) treating des-Phe**BI -human insulin or des-Phe**BI -des-Ala**B20 -swine insulin with acid, or c) treating a compound of the formula I in which X denotes Ala with carboxypeptidase A.

Description

in 149778

The invention relates to an analogous process for the preparation of novel des-Phe human insulin or novel derivatives thereof of the general formula

Gly (Al> 5 Ile Val (B2)

Val Asn

Glu Gin

Gin His S — Cys Leu 10 · ·

Cys-S- S-Cys

Thr Gly

»I

Ser Ser

Ile His (I) S— Cys Leu 15 * «

Looks Val »9

Leu Glu

Tyr Aia

Gin Leu

»I

Leu Tyr 20 r t

Glu Leu

Asn Val t »

Tyr S-Cys

Cys-S 'Gly Y (A21) Glu 25,

Arg

Gly

Phe

Phe 30 Τ | Γ

Thr

Pro

Light (B29) t

X

Wherein Y is Thr or OH and Y is Asp or

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Asn, and this method is characterized by a) converting human insulin, A21 desamido human insulin, 5 B30 des-Thr human insulin, A21 B30 desamido -des-Thr human insulin, B30 des-Ala insulin pig or Ά21 B30 desamido - des-Ala porcine insulin to the corresponding NaAl-N £ B29-bis-tert.-butyloxycarbonyl compound and subjected to an Edman degradation, or b) in cases where Y is Asp

B1 des-Phe-human insulin or B1 B30 des-Phe -des-Ala-pig insulin with acid or

B1 c) in cases where X is OH, treat des-Phe swine insulin or desamidoA21-des-PheB1 swine insulin with carboxypeptidase A.

These novel compounds of formula (I), except for a small change in the amino acid sequence, have the same sequence as the human insulin.

Insulins with lack of B-phenylalanine, e.g.

Bl -phenylalanine swine insulin, is already known, cf.

For example, DE Patent Specification No. 2,005,658, but des -phenylala-

However, human nin insulin (des-Phe human insulin) has not been described so far.

Since human insulin can now be produced in several ways as a starting product for experimental purposes, it has been possible for the first time to recognize the beneficial

B1 properties of des-Phe-human insulin (formula I with X = Thr and Y = Asn).

The compounds of the process according to the invention exhibit surprising properties both in terms of improved stability against human insulin against denaturation and in relation to

In particular, des-Phe-porcine insulin improved solubility, and these properties are both important in connection with the ability to deliver insulin by pumps. Furthermore, the compounds are characterized by a low antigenicity, ie. that they to a lesser extent induce antibody formation. In the long-term treatment with insulins, the production of antibodies, as is well known, plays a critical role, and this production can even be induced by homologous insulin. Thus, it is e.g. It has been observed that human insulin also manages to produce antibodies in humans.

A sensitive test model for the production of antibodies is goats which, in the presence of Freund's adjuvant, form antibodies against swine insulin and human insulin very quickly. According to Diabetes, 2T_ (1978), page 14, images 4, 15, the binding capacity of antibodies to the heterologous B1 des-Phe insulin from pigs is lower, and antibody formation occurs more slowly than with unchanged pig insulin or even with homologous sheep insulin. In this test model, the compounds prepared according to the invention, in particular the compounds with X = OH, cause a further slower antibody formation and the binding capacity relative to the capacity B1 for des-Phe insulin from pigs is reduced by an additional 15%.

Similar to human insulin, the compounds of this invention in the treatment of humans in relation to swine insulin also have the advantage of having a better glucose tolerance. This characteristic does not seem to change

Bl at the cleavage of Phe.

In the present compounds with X = Thr, the solubility relative to the compounds containing: in-B1 30 contains Phe is generally considerably increased, so that highly concentrated solutions such as e.g. are needed for insulin pumps.

bl

In addition, the des-Phe human insulin (formula I with X = Thr) in particular exhibits a rapid onset and long-lasting effect.

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If, besides PheB1, ThrB3 ° (formula I with X = OH) is also enzymatically cleaved from the human insulin, all of the mentioned beneficial properties of the des-PheB ^ human insulin are still obtained. The same is true for the compound which, instead of AsnÅ21 with AspA21, carries an additional carb oxyl group (formula I with X = OH and Y = Asp). In this case, there is even a further increase in solubility, which is primarily observed in the neutral and weakly basic range.

10 The particular economic advantage of the compounds of formula I with X = OH lies in the fact that, in the preparation, the human insulin that is still very difficult to access is not necessarily used at present, but can be used as pig insulin. Swine insulin differs from human insulin only with respect to amino acid BJ (Ala instead of

Thr) and, after the cleavage of this amino acid, is identical to B30 with des-Thr human insulin.

B30

The cleavage of Ala from swine insulin has already been described in U.S. Patent No. 3,364,116.

However, with respect to its immunological properties, Des-AlaB30 swine insulin differs slightly from that of swine insulin. In contrast, the further removal of Phe triggers structural changes in the insulin molecule, which are responsible for the beneficial biological properties.

For the preparation of the present compounds with X - Thr and Y = Asn, human insulin is used which, by analogy with German Patent Specification No. 2,005,658 via the NaA1-N2 B2-bis-tert-butyloxycarbonyl derivative, is converted into the corresponding running des-Phe compound.

For the preparation of the compound of formula I, B30 where X = OH, and Y = Asn, starting from des-Ala-porcine insulin, prepared e.g. according to Hoppe-Seyler's, Z.

Physiol, -Chem. 359 (1978), page 799, by enzymatic cleavage of AlaB3 * \ or from the des-Thr human insulin des-AlaB3 +, respectively, and Phe cleaved by analogy with German U.S. Patent No. 2,005,658. You can

Also reverse the sequence of steps and assume des-Phe - B30 swine insulin and cleave Ala enzymatically.

If Y is to be equal to Asp, the starting compounds are first treated in a known manner at a pH of 2-3 with aqueous acid and purified by ion exchange chromato-B1 B30 graphy. Phe and Ala are then removed as described above.

The acid treatment of the 10 corresponding compounds in which Y is Asn can also be carried out. This has the advantage that at the final purification by ion exchange chromatography a very uniform product is obtained. Thus, one can only settle for 1-2 purification operations, while in the first-mentioned method, a further purification step is usually required.

The novel human insulin analogs of the present invention are used for the treatment of diabetes mellitus and, because of their very good solubility at a pH of 7-7.5, can also be used in an insulin pump at a concentration of approx. 10%. The dosage depends on how the patient is adjusted and roughly corresponds to the dose of porcine insulin, but in patients with elevated antibody titers, however, may be significantly lower.

The compounds of formula I according to the invention can also be processed into pharmaceutical preparations *

The process according to the invention is illustrated in more detail in the following examples.

30 Examples.

The intermediate and final products are characterized by paper and gel electrophoresis at a pH of 2 and 8.3, by amino acid analysis, thin layer chromatography and HPLC in the known systems.

35 The thin-layer chromatography is performed on silica gel plates from Merck. The running agent is isoamyl alcohol: pyridine: water in the ratio 35:35:30.

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Example 1

bl

Des-Phe-human insulin a) 1 g of human insulin is taken up in 70 ml of 80% aqueous dimethyl acetamide. 1.45 g of tert-butyloxycarbonyl azide and 3.3 ml of 1 N sodium bicarbonate solution are added and stirred for 5 hours at 35 ° C. The solution is then concentrated at a bath temperature not exceeding 50 ° C. The residue is triturated with ether and digested with 10 ml of 2% acetic acid. The yield is 942 mg of N, N-di-tert-butyloxycarbonyl-human insulin.

B) The compound prepared is dissolved in 4 ml of 95% pyridine. 0.03 ml of phenyl isothiocyanate is added and stirred for 4 hours at room temperature. Then, in vacuo, concentrate at a bath temperature not exceeding 50 ° C to a small volume and precipitate the compound with ether. Yield: 830 mg of di-tert-butyloxycarbonyl-Na-phenyl thiocarbamoyl-human insulin.

c) Store 820 mg of the compound of b) for 1 hour in 8.5 ml of trifluoroacetic acid at room temperature. By adding 100 ml of ether, 725 mg of 20-des-phenylalanine-human insulin is precipitated, which in a known manner can be crystallized at a pH of 5.0 to 5.5. The compound is uniform in the thin-layer chromatogram and, by the gel electrophoresis at pH = 8, behaves as a human insulin with a slight deviation in the running distance conditioned by the degradation of Phe.

Phe: calculated: 2.00. Found: 2.02.

Example 2 • T_ 'qJ B30

Des-Phe -des-Thr-human insulin B30 a) 1.0 g of des-Ala-pig insulin, prepared according to 30

Hoppe-Seyler's, Z. Physiol.Chem., 359 (1978), page 799, is reacted analogously to Example 1a-c. 710 mg of the title compound is obtained.

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Yield: 623 mg. Crystallization at pH = 5.4.

Phe: calculated: 2.00. Found: 1.99

B1 b) 500 mg of des-Phe porcine insulin, prepared analogously to Example 1 from porcine insulin, is dissolved in 100 ml of 0.1 M 5 ammonium hydrogen carbonate buffer at a pH of 8.2.

Carboxypeptidase A 3 mg is added which is kept for 15 hours at room temperature and then lyophilized. To remove salts, 1% acetic acid is chromatographed on 1% acetic acid over a SephadexG 15 column.

For further purification, chromatograph in 0.05% isopropanol with 0.05 M tris buffer at a pH of 8 and a NaCl gradient ranging from 0 to 0.25 M in a 2.5 x 50 cm column. of DEAE-Sephadex A-25. The eluate is peeled off by dialysis against distilled water and lyophilized.

Yield: 276 mg.

The compound is uniform in the thin layer chromatogram. The running distance of the gel electrophoresis at pH = 8 is somewhat higher than that of human insulin.

Phe: calculated: 2. Found: 1.98. Ala: calculated 1. Found: 1.01.

20

Example 3

Des-Phe ^ - [Asp ^^] - human insulin A21 a) 0.5 g of [Asp] human insulin, prepared by 3 days storage of human insulin in aqueous trifluoroacetic acid at a pH of 2 ^ lyophilization and purification by chromatography on DEAE-Sephadex A-25 analogous to Example 2b) is reacted analogously to Examples 1a-c). After chromatography analogous to Example 2a) 324 mg of the title compound are obtained.

30 Phe: calculated: 2. Found: 2.02.

When characterized by gel electrophoresis at a pH of 8, the running distance corresponds to the running distance of desamidoinsulin (slight deviation due to the lack of phenylalanine).

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B1 b) 0.5 g of des-Phe-human insulin, prepared according to Example 1, is stored as described under a) in aqueous trifluoroacetic acid and then purified on DEAE-Sephadex5 ^ A-25.

Crystallize in a known manner at a pH of 5.0 5 and obtain 365 mg of the title compound identical to that prepared in (a).

Example 4

Pes-PheB ^ -des-ThrB ^ Q- [ÅspA ^] - human insulin OT o * 5 Λ 0.5 g of des-Phe -des-Tnr-human insulin, prepared according to Example 2, is treated with acid according to Example 3b) and cleaned.

Yield: 344 mg.

Phe: calculated: 2. Found: 1.99. Calculated: 1. Found: 1.02.

Further characterization is done by gel electrophoresis. at a pH of 8 analog to Example 3a).

The compound is uniform in the thin layer chromatogram. The running distance of the gel electrophoresis at pH = 8 is clearly higher than that of human insulin and corresponds roughly 20 to desamido-swine insulin.

Claims (2)

5 Gly (A1) Ile Val (B2) Val Asn Glu Gin Gin His u S-Cys Leu • i Cys-SS-Cys Thr Gly I t Ser Ser Ile His 15 * · S-Cys Leu Ser Val U) Leu Glu I t Tyr Ala V f Gin Leu 20 »* Leu Tyr» f Glu Leu »» Asn Val ft Tyr S-Cys Cys-Gly 25. i Y (A2D Glu Arg Gly Phe Phe 30, Tyr Thr Pro Lys (B29) 35 X