Insulin derivatives, a process for the preparation thereof, the use thereof, and a pharmaceutical formulation containing them

• US 5,506,202 A
• Filed: 05/16/1995
• Issued: 04/09/1996
• Est. Priority Date: 12/29/1988
• Status: Expired due to Term

• Current Assignee: Hoechst Aktiengesellschaft (Sanofi )
• Original Assignee: Hoechst Aktiengesellschaft (Sanofi )
• Inventors: Geisen, Karl, Riess, Gunther J., Vertesy, Laszlo, Sauber, Klaus
• Primary Examiner(s): Schain, Howard E.
• Assistant Examiner(s): Prickril, Benet
• Application Number:
  US08/442,296
• Time in Patent Office: 329 Days
• Field of Search: 530/303, 514/3, 514/4, 435/68.1, 435/69.4
• US Class Current: 514/6.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 3/08 for glucose homeostasis pancreatic hormones A61P5/48
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C07K 14/62 Insulins

Abstract

New insulin derivatives, a process for their preparation and use, and a pharmaceutical formulation containing them are disclosed. The derivatives contain the basic amino acid arginine at the amino-terminal position of the insulin A-chain, and various amino acid substitutions at the carboxyl terminus of the insulin B-chain. The compounds are suitable for the treatment of diabetes mellitus, have a delayed profile of action and are very well tolerated.

Claims

1. An essentially purified insulin derivative of the formula II ##STR8## or a physiologically tolerated salt thereof, wherein a) R³⁰ and R³¹ together are OH;

or

b) R³⁰ is a residue of a neutral, genetically encodable L-amino acid, and R³¹ is OH or a physiologically acceptable organic group having 0 to 3 α

-amino acids that are neutral or basic naturally occurring L-amino acids selected from the group consisting of Gly, Ala, Ser, Thr, Val, Leu, Ile, Asn, Gln, Cys, Met, Tyr, Phe, Pro, Hyp, Arg, Lys, Hyl, Orn, Cit, and His, provided that R³⁰ is not Ala at the same time that R³¹ is OH, and further provided that the A- and B-chains are not the sequences of bovine insulin.

2. An insulin derivative and the physiologically tolerated salts thereof as claimed in claim 1, wherein in formula II R³⁰ and R³¹ together are OH.

3. An insulin derivative and the physiologically tolerated salts thereof as claimed in claim 1, wherein for case b) in formula II R³⁰ is a radical of Ala, Thr, or Ser and R³¹ is OH, Arg-OH, or Arg-Arg-OH.

4. An insulin derivative and the physiologically tolerated salts thereof as claimed in claim 1, wherein the A chain and the B chain (B1-B29) in formula II are the sequences of human- or, pork or beef insulin.

5. An insulin derivative as claimed in claim 1, which has an isoelectric point between 5.5 and 9.0.

6. A pharmaceutical formulation that contains an effective amount of at least one insulin derivative, or its physiologically tolerated salts, of the formula II as claimed in claim 1 in dissolved, amorphous, crystalline, or amorphous and crystalline form.

7. A pharmaceutical formulation as claimed in claim 6, which contains an effective amount of at least one of the following insulin derivatives covered by formula II (a/b):

Ar^A0, des-B30-human insulin

Arg^A0 -human insulin,

Arg^A0, Arg^B31 -OH-human insulin and

Arg^A0, Arg^B31, ArgB32-OH-human insulin and the physiologically tolerated salts thereof.

8. A pharmaceutical formulation as claimed in claim 6 or 7 as solution or suspension for injection having a pH of between about 3.0 and 9.0, preferably about 5.0 and 8.5.

9. A method for treating a patient suffering from diabetes mellitus, which comprises administering to said patient a pharmaceutical formulation as claimed in claim 6.

10. An insulin derivative and the physiologically tolerated salts thereof as claimed in claim 3, wherein R³⁰ is a radical of Thr.

11. A process for the preparation of an insulin derivative of the formula II, or its physiologically tolerated salts, as defined in claim 1, which comprises contacting with lysyl endopeptidase an insulin product of the formula III or IV ##STR9## wherein X is a residue of identical or different genetically encodable L-amino acids,

Y is Lys or Arg,

n is 0 or an integer from 1 to 60,

R₁ and R₂ denote hydrogen, a residue of a natural amino acid, or a peptide residue of 1 to 90 natural amino acids wherein the amino or carboxyl groups of the natural amino acids are protected or unprotected, and

the A and B(1-29) chains have the sequence of human or pork insulin,

to cleave the bonds at the C-terminal end of the lysyl residues, thereby forming an Arg^A0 -de-B30-insulin derivative of formula II wherein R³⁰ and R³¹ are together OH.

12. The process of claim 11, wherein R₁ and R₂ denote a peptide residue of 70 to 80 natural amino acids wherein the amino or carboxyl groups of the natural amino acids are protected or unprotected.

13. The process of claim 11, wherein when Y is Arg, the insulin product of the formula III or IV is also mixed with trypsin or a trypsin-like endopeptidase to eliminate the moiety R₁ -Y from the B chain.

14. The process of claim 11, further comprising:

a) reacting the Arg^A0 -des-B30- insulin derivative of formula II wherein R³⁰ and R³¹ are together OH, in the presence of lysyl endopeptidase, trypsin, or a trypsin-like endopeptidase, with a compound of the formula V

space="preserve" listing-type="equation">H--R.sup.30 --R.sup.31 (V),

wherein

R³⁰ is a residue of a neutral, genetically encodable L-amino acid, and R³¹ is OH or a physiolgically acceptable organic group having 0 to 3 α

-amino acids that are neutral or basic naturally occurring L-amino acids selected from the group consisting of Gly, Ala, Set, Thr, Val, Leu, Ils, Asn, Gln, Cys, Met, Tyr, Phe, Pro, Hyp, Arg, Lys, Hyl, Orn, Cit, and His, provided that R³⁰ is not Ala at the same time that R³¹ is OH, and further provided that the A- and B-chains are not the sequences of bovine insulin,

and wherein free COOH, CH, SH, NH₂, guanidino, or imidazole functionalities, when present, are protected by protective groups or are unprotected; and

b) eliminating said protective groups to form an insulin derivative of formula II ##STR10## or a physiologically tolerated salt thereof.

15. A process for the preparation of an insulin derivative of the formula II, or its physiologically acceptable salts, as defined in claim 1, which comprises

a) reacting an Arg^A0 -des-octapeptide (B23-30)-insulin derivative of the formula VI ##STR11## in the presence of trypsin or a trypsin-like endopeptidase, with compound of the formula VII

16. A process for the preparation of an insulin derivative of the formula II, or its physiologically acceptable salts, as defined in claim 1, which comprises

a) reacting an insulin derivative of the formula II'"'"' ##STR13## wherein R³⁰ and R³¹ are together OH;

or

R³⁰ is the residue of a neutral, genetically encodable L-amino acid, and R³¹ is OH or a physiologically acceptable organic group having 0 to 3 α

-amino acids that are neutral or basic naturally occurring L-amino selected from the group consisting of Gly, Ala, Ser, Thr, Val, Leu, Ile, Asn, Gln, Cys, Met, Tyr, Phe, Pro, Hyp, Arg, Lys, Hyl, Orn, Cit, and His, provided that R³⁰ is not Ala at the same time that R³¹ is OH, and further provided that the A- and B-chains are not the sequences of bovine insulin, and

wherein reactive amino groups other than the amino group of Gyl^A1 are protected by protective groups, and the amino group of Gyl^A1 is unprotected, with arginine having its amino groups protected by protective groups and its carboxyl group free or in the form of an acid halide or an azide, and

b) eliminating said protective groups to form an insulin derivative of formula II, ##STR14## or a physiologically tolerated salt thereof.

17. A pharmaceutical formulation as claimed in claim 6, wherein the insulin derivatives or their physiologically tolerated salts are in amorphous or crystalline form.

18. pharmaceutical formulation as claimed in claim 8, wherein the solution or suspension has a pH of between about 5.0 and 8.5.