Modified physiologically active proteins and medicinal compositions containing the same

• US 6,620,916 B1
• Filed: 09/25/1998
• Issued: 09/16/2003
• Est. Priority Date: 09/26/1996
• Status: Expired due to Fees

• Current Assignee: Ajinomoto Co., Inc.
• Original Assignee: Ajinomoto Co., Inc.
• Inventors: Hayashi, Eiko, Tabata, Tomoyuki, Sato, Haruya, Suzuki, Manabu, Takahara, Yoshiyuki, Yatagai, Masanobu, Ejima, Chieko
• Primary Examiner(s): Low, Christopher S. F.
• Assistant Examiner(s): Lukton, David
• Application Number:
  US09/068,987
• Time in Patent Office: 1,817 Days
• Field of Search: 530/324, 530/395, 530/399, 530/402, 530/351, 435/41, 435/68.1
• US Class Current: 530/402
• 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 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: C07H 15/04 attached to an oxygen atom of the saccharide radical
• CPC Class Codes: C07H 15/08 Polyoxyalkylene derivatives
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 14/55 IL-2
• CPC Class Codes: C07K 14/56 IFN-alpha

Abstract

The present invention relates to a modified physiologically active protein, modified physiologically active proteins produced by the process and pharmaceutical compositions containing the same.

Claims

1. A process for producing a modified physiologically active protein comprising:

(a) reacting a physiologically active protein with a terminal amino group of a branched chain ligand, in the presence of transglutaminase obtained from a microorganism, for a time and under conditions effective to form an amide bond between the γ

-carboxamide group of a glutamine residue in said protein and the terminal amino group in said branched chain ligand;

wherein the physiologically active protein has a molecular weight of 1-200 kD and contains at least one glutamine residue;

the branched chain ligand contains a galactose (Gal) or N-acetyl galactosamine (GalNac) group and an amino acid derivative containing an amino group, and said ligand bears a terminal amino group which becomes a substrate for the transglutaminase; and

(b) isolating the modified physiologically active protein, of which binding affinity for an asialoglycoprotein receptor present on the surface of a hepatocyte is lower than the binding affinity of an asialoorosomucoid for said receptor, with the proviso that said branched chain ligand is other than that of formula (V) or (XVIII)

2. The process for producing the modified physiologically active protein of claim 1, wherein the branched-chain ligand is a galactose (Gal) or N-acetylgalactosamine (GalNAc)-containing amino acid derivative represented by

3. The process of claim 2, wherein AA represents two amino acids that are bonded together in an amide linkage and wherein the amide linkage is between the alpha-amino group of one amino acid and the alpha-carboxyl group of the other amino acid.

4. The process of claim 2, wherein AA represents two amino acids, each independently selected from glutamic acid and aspartic acid, and wherein there is an amide bond between the alpha-amino group of one amino acid and the side chain carboxyl group of the other amino acid.

5. The process of claim 2, wherein the branched chain ligand is a ligand represented by one of a formula selected from the group consisting of (II), (III), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), and (XV):

wherein

X represents R—

(OCH₂CH₂)_p—

NH—

or R—

O(CH₂)_q—

NH—

in which R represents galactose or N-acetylgalactosamine, p is an integer of from 1 to 6, and q is an integer of from 1 to 18, and

Y represents —

CO(CH₂)_nNH₂ in which n is an integer of from 1 to 8;

Z represents R(OCH₂CH₂)_pCO—

or R—

O(CH₂)_qCO—

in which R represents galactose or N-acetylgalactosamine, p is an integer of from 1 to 6, and q is an integer of from 1 to 18;

W represents —

NH(CH₂)_nNH₂ in which n is an integer of from 1 to 8;

Q represents R(OCH₂CH₂)_p—

or R—

O(CH₂)_q—

in which R represents galactose or N-acetylgalactosamine, p is an integer of from 1 to 6, and q is an integer of from 1 to 18.

6. A modified physiologically active protein produced by the process of claim 1, wherein said physiologically active protein is a cytokine.

7. The modified physiologically active protein of claim 6, wherein the protein has a liver accumulation property.

8. The modified physiologically active protein of claim 6, wherein the physiologically active protein is interleukin-2 (IL-2) or interferon-α

(IFN-α

).

9. The modified physiologically active protein of claim 6, wherein the branched-chain ligand is a galactose (Gal) or N-acetylgalactosamine (GalNAc)-containing amino acid derivative represented by formula (I)

10. The modified physiologically active protein of claim 9, wherein the group AA of the branched-chain ligand represented by formula (I) is one or two amino acids independently selected from the group consisting of glutamic acid and aspartic acid.

11. The modified physiologically active protein of claim 9, wherein AA represents two amino acids that are bonded together in an amide linkage and wherein the amide linkage is between the alpha-amino group of one amino acid and the alpha-carboxyl group of the other amino acid.

12. The modified physiologically active protein of claim 9, wherein AA represents two amino acids, each independently selected from the group consisting of glutamic acid and aspartic acid, and wherein there is an amide bond between the alpha-amino group of one amino acid and the side chain carboxyl group of the other amino acid.

13. The modified physiologically active protein of claim 9, wherein the branched chain ligand is a ligand represented by one of a formula selected from the group consisting of (II), (III), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), and (XV):

wherein

X represents R—

(OCH₂CH₂)_p—

NH—

or R—

O—

(CH₂)_q—

NH—

in which R represents galactose or N-acetylgalactosamine, p is an integer of from 1 to 6, and q is an integer of from 1 to 18;

Y represents —

CO(CH₂)_nNH₂ in which n is an integer of from 1 to 8;

Z represents R(OCH₂CH₂)pCO—

or R—

O(CH₂)_qCO—

in which R represents galactose or N-acetylgalactosamine, p is an integer of from 1 to 6, and q is an integer of from 1 to 18;

W represents —

NH(CH₂)_nNH₂ in which n is an integer of from 1 to 8; and

Q represents R(OCH₂CH₂)_p—

or R—

O(CH₂)_q—

in which R represents galactose or N-acetylgalactosamine, p is an integer of from 1 to 6, and q is an integer of from 1 to 18.

14. A pharmaceutical composition comprising the modified physiologically active protein of claim 6, and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition of claim 14, wherein the physiologically active protein is interleukin-2 (IL-2) or interferon-α

(IFN-α

).

16. The pharmaceutical composition of claim 14, wherein the branched-chain ligand is a galactose (Gal) or N-acetylgalactosamine (GalNAc)-containing amino acid derivative represented by formula (I)

17. The pharmaceutical composition of claim 16, wherein AA represents two amino acids that are bonded together in an amide linkage and wherein the amide linkage is between the alpha-amino group of one amino acid and the alpha-carboxyl group of the other amino acid.

18. The pharmaceutical composition of claim 16, wherein AA represents two amino acids, each independently selected from glutamic acid and aspartic acid, and wherein there is an amide bond between the alpha-amino group of one amino acid and the side chain carboxyl group of the other amino acid.

19. The pharmaceutical composition of claim 16, wherein the group AA of the branched-chain ligand of formula (I) is one or two amino acids independently selected from the group consisting of glutamic acid and aspartic acid.

20. The pharmaceutical composition of claim 16 wherein the branched chain ligand is a ligand represented by one of a formula selected from the group consisting of (II), (III), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), and (XV):

wherein

X represents R—

(OCH₂CH₂)_p—

NH—

or R—

O(CH₂)_q—

NH—

in which R represents galactose or N-acetylgalactosamine, p is an integer of from 1 to 6, and q is an integer of from 1 to 18;

Y represents —

CO(CH₂)_nNH₂ in which n is an integer of from 1 to 8;

Z represents R(OCH₂CH₂)_pCO—

or R—

O(CH₂)_qCO—

in which R represents galactose or N-acetylgalactosamine, p is an integer of from 1 to 6, and q is an integer of from 1 to 18, and

W represents —

NH(CH₂)_nNH₂ in which n is an integer of from 1 to 8; and

Q represents R(OCH₂CH₂)_p—

or R—

O(CH₂)_q—

in which R represents galactose or N-acetylgalactosamine, p is an integer of from 1 to 6, and q is an integer of from 1 to 18.

21. A process for producing a modified physiologically active protein comprising reacting a physiologically active protein with a terminal amino group of a first ligand, in the presence of a first transglutaminase, for a time and under conditions effective to form an amide bond between the γ

-carboxamide group of a glutamine residue in said physiologically active protein and the terminal amino group of said first ligand;

(a) reacting said modified protein with an amino group of a second ligand in the presence of a second transglutaminase, for a time and under conditions effective to form an amide bond between the γ

-carboxamide group of a glutamine residue in said protein and the amino group in said second ligand,

wherein the first transglutaminase is obtained from a microorganism, and the second transglutaminase is obtained from a source other than a microorganism, the first ligand is a branched-chain ligand which contains a galactose (Gal) or N-acetyl galactosamine (GalNAc) group and an amino acid derivative containing an amino group, and said first ligand bears a terminal amino group which becomes a substrate for the transglutaminase, and the second ligand is structurally distinct from the first ligand,

the physiologically active protein has a molecular weight of 1-200 kD and contains at least one glutamine residue, and

(b) isolating the modified physiologically active protein, of which binding affinity for an asialoglycoprotein receptor present on the surface of a hepatocyte is lower than the binding affinity of an asialoorosomucoid for said receptor, with the proviso that said branched chain ligand is other than that of formula (V) or (XVIII)

22. The process of claim 21, wherein the second ligand is a polyethylene glycol derivative, and the transglutaminase obtained from a source other than a microorganism is a transglutaminase obtained from an animal.

23. A modified physiologically active protein produced by the process of claim 21, wherein said physiologically active protein is a cytokine.

24. The modified physiologically active protein of claim 21, wherein the physiologically active protein modified with the branched chain ligand is further modified with a transglutaminase other than a transglutaminase obtained from a microorganism, using a ligand other than the branched-chain ligand of claim 21.

25. The modified physiologically active protein of claim 24, wherein the ligand other than the branched-chain ligand is a polyethylene glycol derivative, and the transglutaminase obtained from a source other than a microorganism is a transglutaminase obtained from an animal.

26. A pharmaceutical composition comprising the modified, physiologically active protein of claim 23 and a pharmaceutically acceptable carrier.