Process for preparing insulin analogs

• US 5,700,662 A
• Filed: 06/06/1995
• Issued: 12/23/1997
• Est. Priority Date: 02/09/1989
• Status: Expired due to Term

• Current Assignee: Eli Lilly and Company
• Original Assignee: Eli Lilly and Company
• Inventors: Chance, Ronald E., Frank, Bruce H., Shields, James E., DiMarchi, Richard D.
• Primary Examiner(s): SCHAIN, HOWARD
• Application Number:
  US08/465,847
• Time in Patent Office: 931 Days
• Field of Search: 514/3, 514/4, 530/303, 435/69.4
• US Class Current: 435/69.4
• 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: A61K 9/0043 Nose
• CPC Class Codes: C07K 14/62 Insulins
• CPC Class Codes: C07K 14/622 at least 1 amino acid in D-form

Abstract

Recombinant processes for preparing insulin analogs modified at position 29 of the B-chain and, optionally, at other positions are claimed. The analogs produced using the claimed processes have modified physico-chemical properties and are useful in treating hyperglycemia.

Claims

1. A process for preparing a therapeutically active insulin analog of the formula;

##STR9## or a pharmaceutically acceptable salt thereof, wherein A₂₁ is Asn, or Ala;

B₁ is Phe, or is absent;

B₂ is Val, or is absent;

B₁₀ is His, or Asp;

B₂₈ is any amino acid;

B₂₉ is L-Pro, D-Pro, L-hydroxyproline, D-hydroxyproline; and

,

B₃₀ is Thr, Ala, or is absent;

said method comprising;

i) transforming a host cell with DNA that encodes a proinsulin-like precursor molecule having the A and B chains of Formula (I);

ii) culturing the recombinant host cell under conditions suitable for expressing the DNA used to transform the host cell and isolating the proinsulin-like precursor molecule encoded by the DNA in step i);

iii) enzymatically cleaving the proinsulin-like precursor molecule to remove the connecting peptide; and

,

iv) recovering the insulin analog.

2. The process of claim 1 wherein B₂₈ is Asp, Val, Leu, Ile, Pro, Arg, His, Lys, Phe, Ala, Gly, norisoleucine, citrulline, or ornithine.

3. The process of claim 1 wherein A₂₁ is Ala, B₁ is Phe, B₂ is Val, B₁₀ is His, B₂₈ is Lys, B₂₉ is Pro, and B₃₀ is Thr.

4. The process of claim 1 wherein A₂₁ is Asn, B₁ is absent, B₂ is Val, B₁₀ is His, B₂₈ is Lys, B₂₉ is Pro, and B₃₀ is Thr.

5. The process of claim 1 wherein A₂₁ is Asn, B₁ is absent, B₂ is Val, B₁₀ is Asp, B₂₈ is Lys, B₂₉ is Pro, and B₃₀ is Thr.

6. The process of claim 1 wherein A₂₁ is Asn, B₁ is absent, B₂ is absent, B₁₀ is His, B₂₈ is Lys, B₂₉ is Pro, and B₃₀ is Thr.

7. The process of claim 1 wherein A₂₁ is Asn, B₁ is absent, B₂ is absent, B₁₀ is Asp, B₂₈ is Lys, B₂₉ is Pro, and B₃₀ is Thr.

8. The process of claim 1 wherein A₂₁ is Asn, B₁ is Phe, B₂ is Val, B₁₀ is His, B₂₈ is Lys, B₂₉ is Pro, and B₃₀ is absent.

9. The process of claim 1 wherein A₂₁ is Asn, B₁ is Phe, B₂ is Val, B₁₀ is His, B₂₈ is Lys, B₂₉ is Pro, and B₃₀ is Ala.

10. A process for preparing a therapeutically active insulin analog of the formula;

##STR10## or a pharmaceutically acceptable salt thereof;

said method comprising;

i) transforming a host cell with DNA that encodes a proinsulin-like precursor molecule having the A and B chains of Formula (II);

ii) culturing the recombinant host cell under conditions suitable for expressing the DNA used to transform the host cell and isolating the proinsulin-like precursor molecule encoded by the DNA in step i);

iii) enzymatically cleaving the proinsulin-like precursor molecule to remove the connecting peptide; and

,

iv) recovering the insulin analog.

11. A process for preparing a therapeutically active insulin analog of the formula;

##STR11## or a pharmaceutically acceptable salt thereof, wherein A₂₁ is Asn, or Ala;

B₁ is Phe, or is absent;

B₂ is Val, or is absent;

B₁₀ is His, or Asp;

B₂₈ is any amino acid;

B₂₉ is L-Pro, D-Pro, L-hydroxyproline, D-hydroxyproline; and

,

B₃₀ is Thr, Ala, or is absent;

said method comprising;

i) transforming a host cell with DNA that individually encodes the A and B chains of Formula (I);

ii) culturing the recombinant host cell under conditions suitable for expressing the DNA used to transform the host cell and isolating the A and B chains of Formula (I) encoded by the DNA in step i);

iii) combining the individual A and B chains to produce a insulin analog of Formula (I); and

,

iv) recovering the insulin analog.

12. The process of claim 11 wherein B₂₈ is Asp, Val, Leu, Ile, Pro, Arg, His, Lys, Phe, Ala, Gly, norisoleucine, citrulline, or ornithine.

13. The process of claim 11 wherein A₂₁ is Ala, B₁ is Phe, B₂ is Val, B₁₀ is His, B₂₈ is Lys, B₂₉ is Pro, and B₃₀ is Thr.

14. The process of claim 11 wherein A₂₁ is Ash, B₁ is absent, B₂ is Val, B₁₀ is His, B₂₈ is Lys, B₂₉ is Pro, and B₃₀ is Thr.

15. The process of claim 11 wherein A₂₁ is Ash, B₁ is absent, B₂ is Val, B₁₀ is Asp, B₂₈ is Lys, B₂₉ is Pro, and B₃₀ is Thr.

16. The process of claim 11 wherein A₂₁ is Asn, B₁ is absent, B₂ is absent, B₁₀ is His, B₂₈ is Lys, B₂₉ is Pro, and B₃₀ is Thr.

17. The process of claim 11 wherein A₂₁ is Ash, B₁ is absent, B₂ is absent, B₁₀ is Asp, B₂₈ is Lys, B₂₉ is Pro, and C₃₀ is Thr.

18. The process of claim 11 wherein A₂₁ is Asn, B₁ is Phe, B₂ is Val, B₁₀ is His, B₂₈ is Lys, B₂₉ is Pro, and B₃₀ is absent.

19. The process of claim 11 wherein A₂₁ is Asn, B₁ is Phe, B₂ is Val, B₁₀ is His, B₂₈ is Lys, B₂₉ is Pro, and B₃₀ is Ala.

20. A process for preparing a therapeutically active insulin analog of the formula;

##STR12## or a pharmaceutically acceptable salt thereof;

said method comprising;

i) transforming a host cell with DNA that encodes a proinsulin-like precursor molecule having the A and B chains of Formula (I);

ii) culturing the recombinant host cell under conditions suitable for expressing the DNA used to transform the host cell and isolating the proinsulin-like precursor molecule encoded by the DNA in step i);

iii) enzymatically cleaving the proinsulin-like precursor molecule to remove the connecting peptide; and

,

iv) recovering the insulin analog.